miR-182 targets CHL1 and controls tumor growth and invasion in papillary thyroid carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Hongling; Fang, Jin; Zhang, Jichen

2014-07-18

Highlights: • miR-182 and CHL1 expression patterns are negatively correlated. • CHL1 is a direct target of miR-182 in PTC cells. • miR-182 suppression inhibits PTC cell growth and invasion. • CHL1 is involved in miR-182-mediated cell behavior. - Abstract: In this study, we investigated the role and underlying mechanism of action of miR-182 in papillary thyroid carcinoma (PTC). Bioinformatics analysis revealed close homolog of LI (CHL1) as a potential target of miR-182. Upregulation of miR-182 was significantly correlated with CHL1 downregulation in human PTC tissues and cell lines. miR-182 suppressed the expression of CHL1 mRNA through direct targeting ofmore » the 3′-untranslated region (3′-UTR). Downregulation of miR-182 suppressed growth and invasion of PTC cells. Silencing of CHL1 counteracted the effects of miR-182 suppression, while its overexpression mimicked these effects. Our data collectively indicate that miR-182 in PTC promotes cell proliferation and invasion through direct suppression of CHL1, supporting the potential utility of miR-182 inhibition as a novel therapeutic strategy against PTC.« less

Perl Embedded in PTC's Pro/ENGINEER, Version 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

2003-12-22

Pro-PERL (AKA Pro/PERL) is a Perl extension to the PTC Pro/TOOLKIT API to the PTC Pro/ENGINEER CAD application including an embedded interpreter. It can be used to automate and customize Pro/ENGINEER, create Vendor Neutral Archive (VNA) format files and re-create CAD models from the VNA files. This has applications in sanitizing classified CAD models created in a classified environment for transfer to an open environment, creating template models for modification to finished models by non-expert users, and transfer of design intent data to other modeling technologies.

Tasting Phenylthiocarbamide (PTC): A New Integrative Genetics Lab with an Old Flavor

ERIC Educational Resources Information Center

Merritt, Robert B.; Bierwert, Lou Ann; Slatko, Barton; Weiner, Michael P.; Ingram, Jessica; Sciarra, Kristianna; Weiner, Evan

2008-01-01

First reported in the early 1930s, variation in the ability to taste phenylthiocarbamide (PTC) has since become one of the most widely studied of all human genetic traits. Guo and Reed (2001) provide an excellent review of work on this polymorphism prior to the identification and sequencing of the PTC gene by Kim et al. (2003), and Wooding (2006)…

DNA sequence analysis of the composite plasmid pTC conferring virulence and antimicrobial resistance for porcine enterotoxigenic Escherichia coli.

PubMed

Fekete, Péter Z; Brzuszkiewicz, Elzbieta; Blum-Oehler, Gabriele; Olasz, Ferenc; Szabó, Mónika; Gottschalk, Gerhard; Hacker, Jörg; Nagy, Béla

2012-01-01

In this study the plasmid pTC, a 90 kb self-conjugative virulence plasmid of the porcine enterotoxigenic Escherichia coli (ETEC) strain EC2173 encoding the STa and STb heat-stable enterotoxins and tetracycline resistance, has been sequenced in two steps. As a result we identified five main distinct regions of pTC: (i) the maintenance region responsible for the extreme stability of the plasmid, (ii) the TSL (toxin-specific locus comprising the estA and estB genes) which is unique and characteristic for pTC, (iii) a Tn10 transposon, encoding tetracycline resistance, (iv) the tra (plasmid transfer) region, and (v) the colE1-like origin of replication. It is concluded that pTC is a self-transmissible composite plasmid harbouring antibiotic resistance and virulence genes. pTC belongs to a group of large conjugative E. coli plasmids represented by NR1 with a widespread tra backbone which might have evolved from a common ancestor. This is the first report of a completely sequenced animal ETEC virulence plasmid containing an antimicrobial resistance locus, thereby representing a selection advantage for spread of pathogenicity in the presence of antimicrobials leading to increased disease potential. Copyright © 2011. Published by Elsevier GmbH.

[Dose-Response Dependences for Frequency of RET/PTC Gene Rearrangements in Papillary Thyroid Carcinoma after Irradiation. Simple Pooling Analysis of Molecular Epidemiological Data].

PubMed

Koterov, A N; Ushenkova, L N; Biryukov, A P

2016-01-01

On the basis of all possible publications on the theme included in the previously formed base of sources on molecular epidemiology of RET/PTC rearrangements in thyroid papillary carcinoma a pooled analysis ("simple pooling data") on determination of the dose-effect dependences for RET/PTC frequency in radiogenic carcinomas of various irradiated groups was performed. (They are groups subjected to radiotherapeutic exposure, residents near the Chernobyl nuclear power plant (CNPP) and victims of nuclear bombing). The tendency to Pearson linear correlation (r = 0.746; p = 0.148) between the frequency of RET/PTC and the estimated dose on thyroid in the regions affected by the CNPP accident was revealed. But this tendency was recognized to be random owing to abnormally low values of the indicator for the most contaminated Gomel region. The method tentatively called "case-control" showed reliable differences in thyroid dose values for carcinomas with RET/PTC and without those. The versatility of changes was found: the lack of RET/PTC for radiotherapeutic impacts was associated with higher doses, whereas in case of the CNPP accident and for nuclear bombing victims it was the opposite. Probably, in the first case the "cellular cleaning" phenomenon after exposure to very high doses took place. Search of direct Pearson correlations between average/median thyroid doses on groups and RET/PTC frequency in carcinomas of these groups showed a high reliability for the dose-effect dependences- at the continuous dose scale (for RET/PTC in total and RET/PTC1 respectively: r = 0.830; p = 0.002 and r = 0.906; p = 0.0003); while there was no significant correlation received for RET/PTC3. When using the weighting least square regression analysis (proceeding from the number of carcinomas in samples), the specified regularities remained. Attempts to influence the strength of correlation by exception ofthe data of all the samples connected with the accident on the CNPP did not significantly

Transcriptional activity of TGFβ1 and its receptors genes in thyroid gland.

PubMed

Kajdaniuk, Dariusz; Marek, Anna; Marek, Bogdan; Mazurek, Urszula; Fila-Daniłow, Anna; Foltyn, Wanda; Morawiec-Szymonik, Elżbieta; Siemińśka, Lucyna; Nowak, Mariusz; Głogowska-Szeląg, Joanna; Niedziołka-Zielonka, Danuta; Seemann, Michał; Kos-Kudła, Beata

2016-01-01

Determination of gene-candidates' profile expression responsible for fibrosis, immunosuppression, angiogenesis, and neoplasia processes in the pathogenesis of thyroid gland disease. Sixty-three patients underwent thyroidectomy: 27 with non-toxic nodular goitre (NG), 22 with toxic nodular goitre (TNG), six with papillary cancer (PTC), and eight with Graves' disease (GD). In thyroid tissues, transcriptional activity of TGFbeta1 and its receptors TGFbetaRI, TGFbetaRII, and TGFbetaRIII genes were assessed using RT-qPCR (Reverse Transcriptase Quantitative Polymerase Chain Reaction). Molecular analysis was performed in tissues derived from GD and from the tumour centre (PTC, NG, TNG) and from peripheral parts of the removed lobe without histopathological lesions (tissue control). Control tissue for analysis performed in GD was an unchanged tissue derived from peripheral parts of the removed lobe of patients surgically treated for a single benign tumour. Strict regulation observed among transcriptional activity of TGFb1 and their receptor TGFbetaRI-III genes in control tissues is disturbed in all pathological tissues - it is completely disturbed in PTC and GD, and partially in NG and TNG. Additionally, higher transcriptional activity of TGFb1 gene in PTC in comparison with benign tissues (NG, GD) and lower expression of mRNA TGFbRII (than in TNG, GD) and mRNA TGFbetaRIII than in all studied benign tissues (NG, TNG, GD) suggests a pathogenetic importance of this cytokine and its receptors in PTC development. In GD tissue, higher transcriptional activity of TGFbetaRII and TGFbetaRIII genes as compared to other pathological tissues was observed, indicating a participation of the receptors in the pathomechanism of autoimmune thyroid disease (AITD). TGFbeta1 blood concentrations do not reflect pathological processes taking place in thyroid gland. (Endokrynol Pol 2016; 67 (4): 375-382).

Establishment of a non-tumorigenic papillary thyroid cell line (FB-2) carrying the RET/PTC1 rearrangement.

PubMed

Basolo, Fulvio; Giannini, Riccardo; Toniolo, Antonio; Casalone, Rosario; Nikiforova, Marina; Pacini, Furio; Elisei, Rossella; Miccoli, Paolo; Berti, Piero; Faviana, Pinuccia; Fiore, Lisa; Monaco, Carmen; Pierantoni, Giovanna Maria; Fedele, Monica; Nikiforov, Yuri E; Santoro, Massimo; Fusco, Alfredo

2002-02-10

A novel human thyroid papillary carcinoma cell line (FB-2) has been established and characterized. FB-2 cells harbor the RET/PTC1 chimeric oncogene in which the RET kinase domain is fused to the H4 gene. FB-2 cells neither formed colonies in semisolid media nor induced tumors after heterotransplant into severe combined immunodeficient mice. However, HMGI(Y), HMGI-C and c-myc genes, which are associated to thyroid cell transformation, were abundantly expressed in FB-2 cells but not in normal thyroid cells. FB-2 cells only partially retained the differentiated thyroid phenotype. In fact, the PAX-8 gene, which codes for a transcriptional factor required for thyroid cell differentiation, was expressed, while thyroglobulin, TSH-receptor and thyroperoxidase genes were not. Moreover, FB-2 cells produced high levels of interleukin (IL)-6 and IL-8. Copyright 2001 Wiley-Liss, Inc.

Applicability of plasmid calibrant pTC1507 in quantification of TC1507 maize: an interlaboratory study.

PubMed

Meng, Yanan; Liu, Xin; Wang, Shu; Zhang, Dabing; Yang, Litao

2012-01-11

To enforce the labeling regulations of genetically modified organisms (GMOs), the application of DNA plasmids as calibrants is becoming essential for the practical quantification of GMOs. This study reports the construction of plasmid pTC1507 for a quantification assay of genetically modified (GM) maize TC1507 and the collaborative ring trial in international validation of its applicability as a plasmid calibrant. pTC1507 includes one event-specific sequence of TC1507 maize and one unique sequence of maize endogenous gene zSSIIb. A total of eight GMO detection laboratories worldwide were invited to join the validation process, and test results were returned from all eight participants. Statistical analysis of the returned results showed that real-time PCR assays using pTC1507 as calibrant in both GM event-specific and endogenous gene quantifications had high PCR efficiency (ranging from 0.80 to 1.15) and good linearity (ranging from 0.9921 to 0.9998). In a quantification assay of five blind samples, the bias between the test values and true values ranged from 2.6 to 24.9%. All results indicated that the developed pTC1507 plasmid is applicable for the quantitative analysis of TC1507 maize and can be used as a suitable substitute for dried powder certified reference materials (CRMs).

49 CFR 236.1015 - PTC Safety Plan content requirements and PTC System Certification.

Code of Federal Regulations, 2010 CFR

2010-10-01

... established and can maintain a quality control system for PTC system design and manufacturing acceptable to... an explanation of the design principles and assumptions; (3) A risk assessment of the as-built PTC... the specific procedures and test equipment necessary to ensure the safe and proper installation...

MiR-34a targets GAS1 to promote cell proliferation and inhibit apoptosis in papillary thyroid carcinoma via PI3K/Akt/Bad pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Yanfei; Qin, Huadong; Cui, Yunfu, E-mail: yfma77@126.com

Highlights: •MiR-34a is up- and GAS1 is down-regulated in papillary thyroid carcinoma. •GAS1 is a direct target for miR-34a. •MiR-34a promotes PTC cells proliferation and inhibits apoptosis through PI3K/Akt/Bad pathway. -- Abstract: MicroRNAs (miRNAs) are fundamental regulators of cell proliferation, differentiation, and apoptosis, and are implicated in tumorigenesis of many cancers. MiR-34a is best known as a tumor suppressor through repression of growth factors and oncogenes. Growth arrest specific1 (GAS1) protein is a tumor suppressor that inhibits cancer cell proliferation and induces apoptosis through inhibition of RET receptor tyrosine kinase. Both miR-34a and GAS1 are frequently down-regulated in various tumors.more » However, it has been reported that while GAS1 is down-regulated in papillary thyroid carcinoma (PTC), miR-34a is up-regulated in this specific type of cancer, although their potential roles in PTC tumorigenesis have not been examined to date. A computational search revealed that miR-34a putatively binds to the 3′-UTR of GAS1 gene. In the present study, we confirmed previous findings that miR-34a is up-regulated and GAS1 down-regulated in PTC tissues. Further studies indicated that GAS1 is directly targeted by miR-34a. Overexpression of miR-34a promoted PTC cell proliferation and colony formation and inhibited apoptosis, whereas knockdown of miR-34a showed the opposite effects. Silencing of GAS1 had similar growth-promoting effects as overexpression of miR-34a. Furthermore, miR-34a overexpression led to activation of PI3K/Akt/Bad signaling pathway in PTC cells, and depletion of Akt reversed the pro-growth, anti-apoptotic effects of miR-34a. Taken together, our results demonstrate that miR-34a regulates GAS1 expression to promote proliferation and suppress apoptosis in PTC cells via PI3K/Akt/Bad pathway. MiR-34a functions as an oncogene in PTC.« less

Identification of HMX1 target genes: A predictive promoter model approach

PubMed Central

Boulling, Arnaud; Wicht, Linda

2013-01-01

Purpose A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway’s function, we sought to identify the target genes. Methods We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently. Results The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay. Conclusions Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM

The drug target genes show higher evolutionary conservation than non-target genes.

PubMed

Lv, Wenhua; Xu, Yongdeng; Guo, Yiying; Yu, Ziqi; Feng, Guanglong; Liu, Panpan; Luan, Meiwei; Zhu, Hongjie; Liu, Guiyou; Zhang, Mingming; Lv, Hongchao; Duan, Lian; Shang, Zhenwei; Li, Jin; Jiang, Yongshuai; Zhang, Ruijie

2016-01-26

Although evidence indicates that drug target genes share some common evolutionary features, there have been few studies analyzing evolutionary features of drug targets from an overall level. Therefore, we conducted an analysis which aimed to investigate the evolutionary characteristics of drug target genes. We compared the evolutionary conservation between human drug target genes and non-target genes by combining both the evolutionary features and network topological properties in human protein-protein interaction network. The evolution rate, conservation score and the percentage of orthologous genes of 21 species were included in our study. Meanwhile, four topological features including the average shortest path length, betweenness centrality, clustering coefficient and degree were considered for comparison analysis. Then we got four results as following: compared with non-drug target genes, 1) drug target genes had lower evolutionary rates; 2) drug target genes had higher conservation scores; 3) drug target genes had higher percentages of orthologous genes and 4) drug target genes had a tighter network structure including higher degrees, betweenness centrality, clustering coefficients and lower average shortest path lengths. These results demonstrate that drug target genes are more evolutionarily conserved than non-drug target genes. We hope that our study will provide valuable information for other researchers who are interested in evolutionary conservation of drug targets.

A Greek family with a follicular variant of familial papillary thyroid carcinoma: TCO, MNG1, fPTC/PRN, and NMTC1 excluded as susceptibility loci.

PubMed

Tsilchorozidou, Tasoula; Vafiadou, Eleni; Yovos, John G; Romeo, Giovani; McKay, James; Lesueur, Fabienne; Bonora, Elena

2005-12-01

The familial form of nonmedullary thyroid carcinoma (FNMTC) has been recognized as a distinct clinical entity and is characterized by multifocality and a more severe phenotype than its sporadic counterpart. The majority of FNMTC pedigrees are small in size, show variable modes of inheritance, and may present with a variety of additional benign thyroid disorders. The existence of marked phenotypic differences between FNMTC families suggests that there is genetic heterogeneity. Recent studies have mapped a susceptibility locus for FNMTC at 2q21. This locus appears particular relevant to families with at least one case of the follicular variant of papillary thyroid cancer (fvPTC). We describe the clinical and pathologic characteristics of a large three-generation fPTC kindred, with two of the four PTC patients presented with the follicular variant of PTC. It is of interest the occurrence of PTC in three siblings within a period of 3 years. In addition, multinodular goiter (MNG) was diagnosed in seven individuals, lymphocytic thyroiditis in four, while one diagnosed with a benign adenoma. From the PTC patients, one had MNG and fvPTC, one MNG, lymphocytic thyroiditis and papillary pattern of PTC, one lymphocytic thyroiditis and fvPTC, and one MNG and papillary pattern of PTC. The inheritance pattern was autosomal dominant with incomplete penetrance and women were affected more frequently than men. Considering all PTC-affected individuals, the limit of detection (LOD) score we got for this family on 2q21 was 0.5. The low LOD score is caused by a PTC patient who does not share the affected haplotype, suggesting that maybe a new locus for PTC predisposition is present in this kindred. Linkage analysis also excluded TCO, MNG, and fPTC/PRN as susceptibility loci to FNMTC in this family.

Temperature characteristics for PTC material heating diesel fuel

NASA Astrophysics Data System (ADS)

Gu, Lefeng; Li, Xiaolu; Wang, Jun; Li, Ying; Li, Ming

2010-08-01

This paper gives a way which utilizes the PTC (Positive Temperature Coefficient) material to preheat diesel fuel in the injector in order to improve the cold starting and emissions of engine. A new injector is also designed. In order to understand the preheating process in this new injector, a dynamic temperature testing system combined with the MSP430F149 data acquisition system is developed for PTC material heating diesel fuel. Especially, the corresponding software and hardware circuits are explained. The temperature of diesel fuel preheating by PTC ceramics is measured under different voltages and distances, which Curie point is 75 °C. Diesel fuel is heated by self-defined temperature around the Curie point of PTC ceramics. The diesel fuel temperature rises rapidly in 2 minutes of the beginning, then can reach 60 °C within 5 minutes as its distance is 5mm away from the surface of PTC ceramics. However, there are a lot of fundamental studies and technology to be resolved in order to apply PTC material in the injector successfully.

Identification of downstream metastasis-associated target genes regulated by LSD1 in colon cancer cells.

PubMed

Chen, Jiang; Ding, Jie; Wang, Ziwei; Zhu, Jian; Wang, Xuejian; Du, Jiyi

2017-03-21

This study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer. We obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1. A total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1. LSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon

Anaplastic lymphoma kinase (ALK) gene rearrangements in radiation-related human papillary thyroid carcinoma after the Chernobyl accident.

PubMed

Arndt, Annette; Steinestel, Konrad; Rump, Alexis; Sroya, Manveer; Bogdanova, Tetiana; Kovgan, Leonila; Port, Matthias; Abend, Michael; Eder, Stefan

2018-04-06

Childhood radiation exposure has been associated with increased papillary thyroid carcinoma (PTC) risk. The role of anaplastic lymphoma kinase (ALK) gene rearrangements in radiation-related PTC remains unclear, but STRN-ALK fusions have recently been detected in PTCs from radiation exposed persons after Chernobyl using targeted next-generation sequencing and RNA-seq. We investigated ALK and RET gene rearrangements as well as known driver point mutations in PTC tumours from 77 radiation-exposed patients (mean age at surgery 22.4 years) and PTC tumours from 19 non-exposed individuals after the Chernobyl accident. ALK rearrangements were detected by fluorescence in situ hybridisation (FISH) and confirmed with immunohistochemistry (IHC); point mutations in the BRAF and RAS genes were detected by DNA pyrosequencing. Among the 77 tumours from exposed persons, we identified 7 ALK rearrangements and none in the unexposed group. When combining ALK and RET rearrangements, we found 24 in the exposed (31.2%) compared to two (10.5%) in the unexposed group. Odds ratios increased significantly in a dose-dependent manner up to 6.2 (95%CI: 1.1, 34.7; p = 0.039) at Iodine-131 thyroid doses >500 mGy. In total, 27 cases carried point mutations of BRAF or RAS genes, yet logistic regression analysis failed to identify significant dose association. To our knowledge we are the first to describe ALK rearrangements in post-Chernobyl PTC samples using routine methods such as FISH and IHC. Our findings further support the hypothesis that gene rearrangements, but not oncogenic driver mutations, are associated with ionizing radiation-related tumour risk. IHC may represent an effective method for ALK-screening in PTCs with known radiation aetiology, which is of clinical value since oncogenic ALK activation might represent a valuable target for small molecule inhibitors. © 2018 The Authors The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and

Rearranged anaplastic lymphoma kinase (ALK) gene in adult-onset papillary thyroid cancer amongst atomic bomb survivors.

PubMed

Hamatani, Kiyohiro; Mukai, Mayumi; Takahashi, Keiko; Hayashi, Yuzo; Nakachi, Kei; Kusunoki, Yoichiro

2012-11-01

We previously noted that among atomic bomb survivors (ABS), the relative frequency of cases of adult papillary thyroid cancer (PTC) with chromosomal rearrangements (mainly RET/PTC) was significantly greater in those with relatively higher radiation exposure than those with lower radiation exposure. In contrast, the frequency of PTC cases with point mutations (mainly BRAF(V600E)) was significantly lower in patients with relatively higher radiation exposure than those with lower radiation exposure. We also found that among ABS, the frequency of PTC cases with no detectable gene alterations in RET, neurotrophic tyrosine kinase receptor 1 (NTRK1), BRAF, or RAS was significantly higher in patients with relatively higher radiation exposure than those with lower radiation exposure. However, in ABS with PTC, the relationship between the presence of the anaplastic lymphoma kinase (ALK) gene fused with other gene partners and radiation exposure has received little study. In this study, we tested the hypothesis that the relative frequency of rearranged ALK in ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, would be greater in those having relatively higher radiation exposures. The 105 subjects in the study were drawn from the Life Span Study cohort of ABS of Hiroshima and Nagasaki who were diagnosed with PTC between 1956 and 1993. Seventy-nine were exposed (>0 mGy), and 26 were not exposed to A-bomb radiation. In the 25 ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, we examined archival, formalin-fixed, paraffin-embedded PTC specimens for rearrangement of ALK using reverse transcription-polymerase chain reaction and 5' rapid amplification of cDNA ends (5' RACE). We found rearranged ALK in 10 of 19 radiation-exposed PTC cases, but none among 6 patients with PTC with no radiation exposure. In addition, solid/trabecular-like architecture in PTC was closely associated with ALK rearrangements, being observed in

CaPTC Biennial Meetings

Cancer.gov

CaPTC hosts the 'Biennial Science of Global Prostate Cancer Disparities in Black Men' conference to address the growing global public health problem of prostate cancer among Black men in industrialized and developing countries.

Integrative analysis of RUNX1 downstream pathways and target genes

PubMed Central

Michaud, Joëlle; Simpson, Ken M; Escher, Robert; Buchet-Poyau, Karine; Beissbarth, Tim; Carmichael, Catherine; Ritchie, Matthew E; Schütz, Frédéric; Cannon, Ping; Liu, Marjorie; Shen, Xiaofeng; Ito, Yoshiaki; Raskind, Wendy H; Horwitz, Marshall S; Osato, Motomi; Turner, David R; Speed, Terence P; Kavallaris, Maria; Smyth, Gordon K; Scott, Hamish S

2008-01-01

Background The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. Results Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. Conclusion This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both

Gene signature of the post-Chernobyl papillary thyroid cancer.

PubMed

Handkiewicz-Junak, Daria; Swierniak, Michal; Rusinek, Dagmara; Oczko-Wojciechowska, Małgorzata; Dom, Genevieve; Maenhaut, Carine; Unger, Kristian; Detours, Vincent; Bogdanova, Tetiana; Thomas, Geraldine; Likhtarov, Ilya; Jaksik, Roman; Kowalska, Malgorzata; Chmielik, Ewa; Jarzab, Michal; Swierniak, Andrzej; Jarzab, Barbara

2016-07-01

Following the nuclear accidents in Chernobyl and later in Fukushima, the nuclear community has been faced with important issues concerning how to search for and diagnose biological consequences of low-dose internal radiation contamination. Although after the Chernobyl accident an increase in childhood papillary thyroid cancer (PTC) was observed, it is still not clear whether the molecular biology of PTCs associated with low-dose radiation exposure differs from that of sporadic PTC. We investigated tissue samples from 65 children/young adults with PTC using DNA microarray (Affymetrix, Human Genome U133 2.0 Plus) with the aim of identifying molecular differences between radiation-induced (exposed to Chernobyl radiation, ECR) and sporadic PTC. All participants were resident in the same region so that confounding factors related to genetics or environment were minimized. There were small but significant differences in the gene expression profiles between ECR and non-ECR PTC (global test, p < 0.01), with 300 differently expressed probe sets (p < 0.001) corresponding to 239 genes. Multifactorial analysis of variance showed that besides radiation exposure history, the BRAF mutation exhibited independent effects on the PTC expression profile; the histological subset and patient age at diagnosis had negligible effects. Ten genes (PPME1, HDAC11, SOCS7, CIC, THRA, ERBB2, PPP1R9A, HDGF, RAD51AP1, and CDK1) from the 19 investigated with quantitative RT-PCR were confirmed as being associated with radiation exposure in an independent, validation set of samples. Significant, but subtle, differences in gene expression in the post-Chernobyl PTC are associated with previous low-dose radiation exposure.

Rearranged Anaplastic Lymphoma Kinase (ALK) Gene in Adult-Onset Papillary Thyroid Cancer Amongst Atomic Bomb Survivors

PubMed Central

Mukai, Mayumi; Takahashi, Keiko; Hayashi, Yuzo; Nakachi, Kei; Kusunoki, Yoichiro

2012-01-01

Background We previously noted that among atomic bomb survivors (ABS), the relative frequency of cases of adult papillary thyroid cancer (PTC) with chromosomal rearrangements (mainly RET/PTC) was significantly greater in those with relatively higher radiation exposure than those with lower radiation exposure. In contrast, the frequency of PTC cases with point mutations (mainly BRAFV600E) was significantly lower in patients with relatively higher radiation exposure than those with lower radiation exposure. We also found that among ABS, the frequency of PTC cases with no detectable gene alterations in RET, neurotrophic tyrosine kinase receptor 1 (NTRK1), BRAF, or RAS was significantly higher in patients with relatively higher radiation exposure than those with lower radiation exposure. However, in ABS with PTC, the relationship between the presence of the anaplastic lymphoma kinase (ALK) gene fused with other gene partners and radiation exposure has received little study. In this study, we tested the hypothesis that the relative frequency of rearranged ALK in ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, would be greater in those having relatively higher radiation exposures. Methods The 105 subjects in the study were drawn from the Life Span Study cohort of ABS of Hiroshima and Nagasaki who were diagnosed with PTC between 1956 and 1993. Seventy-nine were exposed (>0 mGy), and 26 were not exposed to A-bomb radiation. In the 25 ABS with PTC, and with no detectable gene alterations in RET, NTRK1, BRAF, or RAS, we examined archival, formalin-fixed, paraffin-embedded PTC specimens for rearrangement of ALK using reverse transcription–polymerase chain reaction and 5′ rapid amplification of cDNA ends (5′ RACE). Results We found rearranged ALK in 10 of 19 radiation-exposed PTC cases, but none among 6 patients with PTC with no radiation exposure. In addition, solid/trabecular-like architecture in PTC was closely associated with ALK

Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines.

PubMed

Gulec, Cagri; Coban, Neslihan; Ozsait-Selcuk, Bilge; Sirma-Ekmekci, Sema; Yildirim, Ozlem; Erginel-Unaltuna, Nihan

2017-04-01

ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses. Copyright © 2017 Elsevier Inc. All rights reserved.

Selected single-nucleotide polymorphisms in FOXE1, SERPINA5, FTO, EVPL, TICAM1 and SCARB1 are associated with papillary and follicular thyroid cancer risk: replication study in a German population

PubMed Central

Sigurdson, Alice J.; Brenner, Alina V.; Roach, James A.; Goudeva, Lilia; Müller, Jörg A.; Nerlich, Kai; Reiners, Christoph; Schwab, Robert; Pfeiffer, Liliane; Waldenberger, Melanie; Braganza, Melissa; Xu, Li; Sturgis, Erich M.; Yeager, Meredith; Chanock, Stephen J.; Pfeiffer, Ruth M.; Abend, Michael; Port, Matthias

2016-01-01

Several single-nucleotide polymorphisms (SNPs) have been associated with papillary and follicular thyroid cancer (PTC and FTC, respectively) risk, but few have replicated. After analyzing 17525 tag SNPs in 1129 candidate genes, we found associations with PTC risk in SERPINA5, FTO, HEMGN (near FOXE1) and other genes. Here, we report results from a replication effort in a large independent PTC/FTC case–control study conducted in Germany. We evaluated the best tagging SNPs from our previous PTC study and additionally included SNPs in or near FOXE1 and NKX2-1 genes, known susceptibility loci for thyroid cancer. We genotyped 422 PTC and 130 FTC cases and 752 controls recruited from three German clinical centers. We used polytomous logistic regression to simultaneously estimate PTC and FTC associations for 79 SNPs based on log-additive models. We assessed effect modification by body mass index (BMI), gender and age for all SNPs, and selected SNP by SNP interactions. We confirmed associations with PTC and SNPs in FOXE1/HEMGN, SERPINA5 (rs2069974), FTO (rs8047395), EVPL (rs2071194), TICAM1 (rs8120) and SCARB1 (rs11057820) genes. We found associations with SNPs in FOXE1, SERPINA5, FTO, TICAM1 and HSPA6 and FTC. We found two significant interactions between FTO (rs8047395) and BMI (P = 0.0321) and between TICAM1 (rs8120) and FOXE1 (rs10984377) (P = 0.0006). Besides the known associations with FOXE1 SNPs, we confirmed additional PTC SNP associations reported previously. We also found several new associations with FTC risk and noteworthy interactions. We conclude that multiple variants and host factors might interact in complex ways to increase risk of PTC and FTC. PMID:27207655

Selected single-nucleotide polymorphisms in FOXE1, SERPINA5, FTO, EVPL, TICAM1 and SCARB1 are associated with papillary and follicular thyroid cancer risk: replication study in a German population.

PubMed

Sigurdson, Alice J; Brenner, Alina V; Roach, James A; Goudeva, Lilia; Müller, Jörg A; Nerlich, Kai; Reiners, Christoph; Schwab, Robert; Pfeiffer, Liliane; Waldenberger, Melanie; Braganza, Melissa; Xu, Li; Sturgis, Erich M; Yeager, Meredith; Chanock, Stephen J; Pfeiffer, Ruth M; Abend, Michael; Port, Matthias

2016-07-01

Several single-nucleotide polymorphisms (SNPs) have been associated with papillary and follicular thyroid cancer (PTC and FTC, respectively) risk, but few have replicated. After analyzing 17525 tag SNPs in 1129 candidate genes, we found associations with PTC risk in SERPINA5, FTO, HEMGN (near FOXE1) and other genes. Here, we report results from a replication effort in a large independent PTC/FTC case-control study conducted in Germany. We evaluated the best tagging SNPs from our previous PTC study and additionally included SNPs in or near FOXE1 and NKX2-1 genes, known susceptibility loci for thyroid cancer. We genotyped 422 PTC and 130 FTC cases and 752 controls recruited from three German clinical centers. We used polytomous logistic regression to simultaneously estimate PTC and FTC associations for 79 SNPs based on log-additive models. We assessed effect modification by body mass index (BMI), gender and age for all SNPs, and selected SNP by SNP interactions. We confirmed associations with PTC and SNPs in FOXE1/HEMGN, SERPINA5 (rs2069974), FTO (rs8047395), EVPL (rs2071194), TICAM1 (rs8120) and SCARB1 (rs11057820) genes. We found associations with SNPs in FOXE1, SERPINA5, FTO, TICAM1 and HSPA6 and FTC. We found two significant interactions between FTO (rs8047395) and BMI (P = 0.0321) and between TICAM1 (rs8120) and FOXE1 (rs10984377) (P = 0.0006). Besides the known associations with FOXE1 SNPs, we confirmed additional PTC SNP associations reported previously. We also found several new associations with FTC risk and noteworthy interactions. We conclude that multiple variants and host factors might interact in complex ways to increase risk of PTC and FTC. Published by Oxford University Press 2016.

Diagnostic Limitation of Fine-Needle Aspiration (FNA) on Indeterminate Thyroid Nodules Can Be Partially Overcome by Preoperative Molecular Analysis: Assessment of RET/PTC1 Rearrangement in BRAF and RAS Wild-Type Routine Air-Dried FNA Specimens.

PubMed

Ko, Young Sin; Hwang, Tae Sook; Kim, Ja Yeon; Choi, Yoon-La; Lee, Seung Eun; Han, Hye Seung; Kim, Wan Seop; Kim, Suk Kyeong; Park, Kyoung Sik

2017-04-12

Molecular markers are helpful diagnostic tools, particularly for cytologically indeterminate thyroid nodules. Preoperative RET/PTC1 rearrangement analysis in BRAF and RAS wild-type indeterminate thyroid nodules would permit the formulation of an unambiguous surgical plan. Cycle threshold values according to the cell count for detection of the RET/PTC1 rearrangement by real-time reverse transcription-polymerase chain reaction (RT-PCR) using fresh and routine air-dried TPC1 cells were evaluated. The correlation of RET/PTC1 rearrangement between fine-needle aspiration (FNA) and paired formalin-fixed paraffin-embedded (FFPE) specimens was analyzed. RET/PTC1 rearrangements of 76 resected BRAF and RAS wild-type classical PTCs were also analyzed. Results of RT-PCR and the Nanostring were compared. When 100 fresh and air-dried TPC1 cells were used, expression of RET/PTC1 rearrangement was detectable after 35 and 33 PCR cycles, respectively. The results of RET/PTC1 rearrangement in 10 FNA and paired FFPE papillary thyroid carcinoma (PTC) specimens showed complete correlation. Twenty-nine (38.2%) of 76 BRAF and RAS wild-type classical PTCs had RET/PTC1 rearrangement. Comparison of RET/PTC1 rearrangement analysis between RT-PCR and the Nanostring showed moderate agreement with a κ value of 0.56 ( p = 0.002). The RET/PTC1 rearrangement analysis by RT-PCR using routine air-dried FNA specimen was confirmed to be technically applicable. A significant proportion (38.2%) of the BRAF and RAS wild-type PTCs harbored RET/PTC1 rearrangements.

PTC/PROP tasting: anatomy, psychophysics, and sex effects.

PubMed

Bartoshuk, L M; Duffy, V B; Miller, I J

1994-12-01

Taste worlds of humans vary because of taste blindness to phenylthiocarbamide (PTC) and its chemical relative, 6-n-propylthiouracil (PROP). We review early PTC studies and apply modern statistical analyses to show that a higher frequency of women tasted PTC crystals, and were tasters (threshold classification). In our laboratory, scaling of PROP bitterness led to the identification of a subset of tasters (supertasters) who rate PROP as intensely bitter. Supertasters also perceive stronger tastes from a variety of bitter and sweet substances, and perceive more burn from oral irritants (alcohol and capsaicin). The density of taste receptors on the anterior tongue (fungiform papillae, taste buds) correlate significantly with perceived bitterness of PROP and support the supertaster concept. Psychophysical data from studies in our laboratory also show a sex effect; women are supertasters more frequently. The anatomical data also support the sex difference; women have more fungiform papillae and more taste buds. Future investigations of PTC/PROP tasting and food behaviors should include scaling to identify supertasters and separate sex effects.

The LIM-homeodomain transcription factor LMX1B regulates expression of NF-kappa B target genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rascle, Anne; Neumann, Tanja; Raschta, Anne-Sarah

2009-01-01

LMX1B is a LIM-homeodomain transcription factor essential for development. Putative LMX1B target genes have been identified through mouse gene targeting studies, but their identity as direct LMX1B targets remains hypothetical. We describe here the first molecular characterization of LMX1B target gene regulation. Microarray analysis using a tetracycline-inducible LMX1B expression system in HeLa cells revealed that a subset of NF-{kappa}B target genes, including IL-6 and IL-8, are upregulated in LMX1B-expressing cells. Inhibition of NF-{kappa}B activity by short interfering RNA-mediated knock-down of p65 impairs, while activation of NF-{kappa}B activity by TNF-{alpha} synergizes induction of NF-{kappa}B target genes by LMX1B. Chromatin immunoprecipitation demonstratedmore » that LMX1B binds to the proximal promoter of IL-6 and IL-8 in vivo, in the vicinity of the characterized {kappa}B site, and that LMX1B recruitment correlates with increased NF-{kappa}B DNA association. IL-6 promoter-reporter assays showed that the {kappa}B site and an adjacent putative LMX1B binding motif are both involved in LMX1B-mediated transcription. Expression of NF-{kappa}B target genes is affected in the kidney of Lmx1b{sup -/-} knock-out mice, thus supporting the biological relevance of our findings. Together, these data demonstrate for the first time that LMX1B directly regulates transcription of a subset of NF-{kappa}B target genes in cooperation with nuclear p50/p65 NF-{kappa}B.« less

Opposing roles for DNA structure-specific proteins Rad1, Msh2, Msh3, and Sgs1 in yeast gene targeting.

PubMed

Langston, Lance D; Symington, Lorraine S

2005-06-15

Targeted gene replacement (TGR) in yeast and mammalian cells is initiated by the two free ends of the linear targeting molecule, which invade their respective homologous sequences in the chromosome, leading to replacement of the targeted locus with a selectable gene from the targeting DNA. To study the postinvasion steps in recombination, we examined the effects of DNA structure-specific proteins on TGR frequency and heteroduplex DNA formation. In strains deleted of RAD1, MSH2, or MSH3, we find that the frequency of TGR is reduced and the mechanism of TGR is altered while the reverse is true for deletion of SGS1, suggesting that Rad1 and Msh2:Msh3 facilitate TGR while Sgs1 opposes it. The altered mechanism of TGR in the absence of Msh2:Msh3 and Rad1 reveals a separate role for these proteins in suppressing an alternate gene replacement pathway in which incorporation of both homology regions from a single strand of targeting DNA into heteroduplex with the targeted locus creates a mismatch between the selectable gene on the targeting DNA and the targeted gene in the chromosome.

PTC MathCAD and Workgroup Manager: Implementation in a Multi-Org System

NASA Technical Reports Server (NTRS)

Jones, Corey

2015-01-01

In this presentation, the presenter will review what was done at Kennedy Space Center to deploy and implement PTC MathCAD and PTC Workgroup Manager in a multi-org system. During the presentation the presenter will explain how they configured PTC Windchill to create custom soft-types and object initialization rules for their custom numbering scheme and why they choose these methods. This presentation will also include how to modify the EPM default soft-type file in the PTC Windchill server codebase folder. The presenter will also go over the code used in a start up script to initiate PTC MathCAD and PTC Workgroup Manager in the proper order, and also set up the environment variables when running both PTC Workgroup Manager and PTC Creo. The configuration.ini file the presenter used will also be reviewed to show you how to set up the PTC Workgroup Manager and customized it to their user community. This presentation will be of interest to administrators trying to create a similar set-up in either a single org or multiple org system deployment. The big take away will be ideas and best practices learned through implementing this system, and the lessons learned what to do and not to do when setting up this configuration. Attendees will be exposed to several different sets of code used and that worked well and will hear some limitations on what the software can accomplish when configured this way.

pTC Plasmids from Sulfolobus Species in the Geothermal Area of Tengchong, China: Genomic Conservation and Naturally-Occurring Variations as a Result of Transposition by Mobile Genetic Elements

PubMed Central

Xiang, Xiaoyu; Huang, Xiaoxing; Wang, Haina; Huang, Li

2015-01-01

Plasmids occur frequently in Archaea. A novel plasmid (denoted pTC1) containing typical conjugation functions has been isolated from Sulfolobus tengchongensis RT8-4, a strain obtained from a hot spring in Tengchong, China, and characterized. The plasmid is a circular double-stranded DNA molecule of 20,417 bp. Among a total of 26 predicted pTC1 ORFs, 23 have homologues in other known Sulfolobus conjugative plasmids (CPs). pTC1 resembles other Sulfolobus CPs in genome architecture, and is most highly conserved in the genomic region encoding conjugation functions. However, attempts to demonstrate experimentally the capacity of the plasmid for conjugational transfer were unsuccessful. A survey revealed that pTC1 and its closely related plasmid variants were widespread in the geothermal area of Tengchong. Variations of the plasmids at the target sites for transposition by an insertion sequence (IS) and a miniature inverted-repeat transposable element (MITE) were readily detected. The IS was efficiently inserted into the pTC1 genome, and the inserted sequence was inactivated and degraded more frequently in an imprecise manner than in a precise manner. These results suggest that the host organism has evolved a strategy to maintain a balance between the insertion and elimination of mobile genetic elements to permit genomic plasticity while inhibiting their fast spreading. PMID:25686154

pTC Plasmids from Sulfolobus Species in the Geothermal Area of Tengchong, China: Genomic Conservation and Naturally-Occurring Variations as a Result of Transposition by Mobile Genetic Elements.

PubMed

Xiang, Xiaoyu; Huang, Xiaoxing; Wang, Haina; Huang, Li

2015-02-12

Plasmids occur frequently in Archaea. A novel plasmid (denoted pTC1) containing typical conjugation functions has been isolated from Sulfolobus tengchongensis RT8-4, a strain obtained from a hot spring in Tengchong, China, and characterized. The plasmid is a circular double-stranded DNA molecule of 20,417 bp. Among a total of 26 predicted pTC1 ORFs, 23 have homologues in other known Sulfolobus conjugative plasmids (CPs). pTC1 resembles other Sulfolobus CPs in genome architecture, and is most highly conserved in the genomic region encoding conjugation functions. However, attempts to demonstrate experimentally the capacity of the plasmid for conjugational transfer were unsuccessful. A survey revealed that pTC1 and its closely related plasmid variants were widespread in the geothermal area of Tengchong. Variations of the plasmids at the target sites for transposition by an insertion sequence (IS) and a miniature inverted-repeat transposable element (MITE) were readily detected. The IS was efficiently inserted into the pTC1 genome, and the inserted sequence was inactivated and degraded more frequently in an imprecise manner than in a precise manner. These results suggest that the host organism has evolved a strategy to maintain a balance between the insertion and elimination of mobile genetic elements to permit genomic plasticity while inhibiting their fast spreading.

49 CFR 236.1011 - PTC Implementation Plan content requirements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... technology embedded in PTC systems that does not employ all of the functionalities required by this subpart... INSTALLATION, INSPECTION, MAINTENANCE, AND REPAIR OF SIGNAL AND TRAIN CONTROL SYSTEMS, DEVICES, AND APPLIANCES Positive Train Control Systems § 236.1011 PTC Implementation Plan content requirements. (a) Contents. A...

49 CFR 236.1011 - PTC Implementation Plan content requirements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... technology embedded in PTC systems that does not employ all of the functionalities required by this subpart... INSTALLATION, INSPECTION, MAINTENANCE, AND REPAIR OF SIGNAL AND TRAIN CONTROL SYSTEMS, DEVICES, AND APPLIANCES Positive Train Control Systems § 236.1011 PTC Implementation Plan content requirements. (a) Contents. A...

49 CFR 236.1011 - PTC Implementation Plan content requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... technology embedded in PTC systems that does not employ all of the functionalities required by this subpart... INSTALLATION, INSPECTION, MAINTENANCE, AND REPAIR OF SIGNAL AND TRAIN CONTROL SYSTEMS, DEVICES, AND APPLIANCES Positive Train Control Systems § 236.1011 PTC Implementation Plan content requirements. (a) Contents. A...

Id-1 gene and gene products as therapeutic targets for treatment of breast cancer and other types of carcinoma

DOEpatents

Desprez, Pierre-Yves; Campisi, Judith

2014-08-19

A method for treatment of breast cancer and other types of cancer. The method comprises targeting and modulating Id-1 gene expression, if any, for the Id-1 gene, or gene products in breast or other epithelial cancers in a patient by delivering products that modulate Id-1 gene expression. When expressed, Id-1 gene is a prognostic indicator that cancer cells are invasive and metastatic.

The yeast Hot1 transcription factor is critical for activating a single target gene, STL1

PubMed Central

Bai, Chen; Tesker, Masha; Engelberg, David

2015-01-01

Transcription factors are commonly activated by signal transduction cascades and induce expression of many genes. They therefore play critical roles in determining the cell's fate. The yeast Hog1 MAP kinase pathway is believed to control the transcription of hundreds of genes via several transcription factors. To identify the bona fide target genes of Hog1, we inducibly expressed the spontaneously active variant Hog1D170A+F318L in cells lacking the Hog1 activator Pbs2. This system allowed monitoring the effects of Hog1 by itself. Expression of Hog1D170A+F318L in pbs2∆ cells imposed induction of just 105 and suppression of only 26 transcripts by at least twofold. We looked for the Hog1-responsive element within the promoter of the most highly induced gene, STL1 (88-fold). A novel Hog1 responsive element (HoRE) was identified and shown to be the direct target of the transcription factor Hot1. Unexpectedly, we could not find this HoRE in any other yeast promoter. In addition, the only gene whose expression was abolished in hot1∆ cells was STL1. Thus Hot1 is essential for transcription of just one gene, STL1. Hot1 may represent a class of transcription factors that are essential for transcription of a very few genes or even just one. PMID:25904326

Tumor suppressor miR-1 inhibits tumor growth and metastasis by simultaneously targeting multiple genes

PubMed Central

Liu, Cuilian; Zhang, Song; Wang, Qizhi; Zhang, Xiaobo

2017-01-01

Cancer progression depends on tumor growth and metastasis, which are activated or suppressed by multiple genes. An individual microRNA may target multiple genes, suggesting that a miRNA may suppress tumor growth and metastasis via simultaneously targeting different genes. However, thus far, this issue has not been explored. In the present study, the findings showed that miR-1 could simultaneously inhibit tumor growth and metastasis of gastric and breast cancers by targeting multiple genes. The results indicated that miR-1 was significantly downregulated in cancer tissues compared with normal tissues. The miR-1 overexpression led to cell cycle arrest in the G1 phase in gastric and breast cancer cells but not in normal cells. Furthermore, the miR-1 overexpression significantly inhibited the metastasis of gastric and breast cancer cells. An analysis of the underlying mechanism revealed that the simultaneous inhibition of tumor growth and metastasis mediated by miR-1 was due to the synchronous targeting of 6 miR-1 target genes encoding cyclin dependent kinase 4, twinfilin actin binding protein 1, calponin 3, coronin 1C, WAS protein family member 2 and thymosin beta 4, X-linked. In vivo assays demonstrated that miR-1 efficiently inhibited tumor growth and metastasis of gastric and breast cancers in nude mice. Therefore, our study contributed novel insights into the miR-1′s roles in tumorigenesis of gastric and breast cancers. PMID:28159933

Tumor suppressor miR-1 inhibits tumor growth and metastasis by simultaneously targeting multiple genes.

PubMed

Liu, Cuilian; Zhang, Song; Wang, Qizhi; Zhang, Xiaobo

2017-06-27

Cancer progression depends on tumor growth and metastasis, which are activated or suppressed by multiple genes. An individual microRNA may target multiple genes, suggesting that a miRNA may suppress tumor growth and metastasis via simultaneously targeting different genes. However, thus far, this issue has not been explored. In the present study, the findings showed that miR-1 could simultaneously inhibit tumor growth and metastasis of gastric and breast cancers by targeting multiple genes. The results indicated that miR-1 was significantly downregulated in cancer tissues compared with normal tissues. The miR-1 overexpression led to cell cycle arrest in the G1 phase in gastric and breast cancer cells but not in normal cells. Furthermore, the miR-1 overexpression significantly inhibited the metastasis of gastric and breast cancer cells. An analysis of the underlying mechanism revealed that the simultaneous inhibition of tumor growth and metastasis mediated by miR-1 was due to the synchronous targeting of 6 miR-1 target genes encoding cyclin dependent kinase 4, twinfilin actin binding protein 1, calponin 3, coronin 1C, WAS protein family member 2 and thymosin beta 4, X-linked. In vivo assays demonstrated that miR-1 efficiently inhibited tumor growth and metastasis of gastric and breast cancers in nude mice. Therefore, our study contributed novel insights into the miR-1's roles in tumorigenesis of gastric and breast cancers.

Successful Strategies for Rapidly Upgrading PTC Windchill 9.1 to Windchill 10.1 on a Light Budget

NASA Technical Reports Server (NTRS)

Shearrow, Charles A.

2013-01-01

Topics covered include: The Frugal Times Historical Upgrade Process; Planning for Possible Constraints; PTC Compatibility Matrix; In-Place Upgrade Process; Pre-Upgrade Activities; Upgrade Activities; Post Upgrade Activities; Results of the Upgrade; Tips for an Upgrade On a Shoestring Budget.

[Identification of Clonorchis sinensis metacercariae based on PCR targeting ribosomal DNA ITS regions and COX1 gene].

PubMed

Yang, Qing-Li; Shen, Ji-Qing; Jiang, Zhi-Hua; Yang, Yi-Chao; Li, Hong-Mei; Chen, Ying-Dan; Zhou, Xiao-Nong

2014-06-01

To identify Clonorchis sinensis metacercariae using PCR targeting ribosomal DNA ITS region and COX1 gene. Pseudorasbora parva were collected from Hengxian County of Guangxi at the end of May 2013. Single metacercaria of C. sinensis and other trematodes were separated from muscle tissue of P. parva by digestion method. Primers targeting ribosomal DNA ITS region and COX1 gene of C. sinensis were designed for PCR and the universal primers were used as control. The sensitivity and specificity of the PCR detection were analyzed. C. sinensis metacercariae at different stages were identified by PCR. DNA from single C. sinensis metacercaria was detected by PCR targeting ribosomal DNA ITS region and COX1 gene. The specific amplicans have sizes of 437/549, 156/249 and 195/166 bp, respectively. The ratio of the two positive numbers in PCR with universal primers and specific primers targeting C. sinensis ribosomal DNA ITS1 and ITS2 regions was 0.905 and 0.952, respectively. The target gene fragments were amplified by PCR using COX1 gene-specific primers. The PCR with specific primers did not show any non-specific amplification. However, the PCR with universal primers targeting ribosomal DNA ITS regions performed serious non-specific amplification. C. sinensis metacercariae at different stages are identified by morphological observation and PCR method. Species-specific primers targeting ribosomal DNA ITS region show higher sensitivity and specificity than the universal primers. PCR targeting COX1 gene shows similar sensitivity and specificity to PCR with specific primers targeting ribosomal DNA ITS regions.

Biological effects of RNAi targeted inhibiting Tiam1 gene expression on cholangiocarcinoma cells.

PubMed

Cheng, Wei; Liu, Yaling; Zuo, Zhi; Yin, Xinmin; Jiang, Bo; Chen, Daojin; Peng, Chuang; Yang, Jianhui

2015-01-01

To investigate the characteristics of Tiam1 gene expression in human cholangiocarcinoma tissues and benign bile duct tissues, and to analyze the correlations between Tiam1 gene expression and the degree of tumor differentiation, invasive and metastatic abilities. To explore the effect of targeted inhibiting Tiam1 gene expression on proliferation and migration activity of human cholangiocarcinoma cells. Expression of Tiam1 in 83 cases of cholangiocarcinoma tissues and 25 cases of benign bile tissues was detected using immunohistochemistry. The clinical data of patients with cholangiocarcinoma were collected. The correlations between Tiam1 gene expression and the clinicopathologic features in patients with cholangiocarcinoma were analyzed. The human cholangiocarcinoma RBE cells were divided into 3 groups. Cells in experimental group and control group were respectively transfected with Tiam1 shRNA lentiviral vectors and negative shRNA lentiviral control vectors. Cells in blank group received no treatment. Real-time PCR endogenesis was used to verify Tiam1 gene expression. Cell cycle experiments and MTT assay were used to measure cell proliferation activity. Transwell test was used to detect cell migration activity. The negative rate Tiam1 protein expression in cholangiocarcinoma tissues was significantly higher than that in benign bile tissues (P<0.001). Tiam1 protein expression in cholangiocarcinoma tissues had correlations with cholangiocarcinoma differentiation degree, TNM stage and lymph node metastasis (P<0.05), and had no significant correlations with gender, age and distant metastasis (P>0.05). Real-time PCR detection indicated that Tiam1 expression of experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that Tiam1 shRNA was effective on Tiam1 gene silencing in RBE cells. Cell cycle experiment showed that the percentage of S phase in cell cycle in experimental group was lower than that in control group

Biological effects of RNAi targeted inhibiting Tiam1 gene expression on cholangiocarcinoma cells

PubMed Central

Cheng, Wei; Liu, Yaling; Zuo, Zhi; Yin, Xinmin; Jiang, Bo; Chen, Daojin; Peng, Chuang; Yang, Jianhui

2015-01-01

Objective: To investigate the characteristics of Tiam1 gene expression in human cholangiocarcinoma tissues and benign bile duct tissues, and to analyze the correlations between Tiam1 gene expression and the degree of tumor differentiation, invasive and metastatic abilities. To explore the effect of targeted inhibiting Tiam1 gene expression on proliferation and migration activity of human cholangiocarcinoma cells. Methods: Expression of Tiam1 in 83 cases of cholangiocarcinoma tissues and 25 cases of benign bile tissues was detected using immunohistochemistry. The clinical data of patients with cholangiocarcinoma were collected. The correlations between Tiam1 gene expression and the clinicopathologic features in patients with cholangiocarcinoma were analyzed. The human cholangiocarcinoma RBE cells were divided into 3 groups. Cells in experimental group and control group were respectively transfected with Tiam1 shRNA lentiviral vectors and negative shRNA lentiviral control vectors. Cells in blank group received no treatment. Real-time PCR endogenesis was used to verify Tiam1 gene expression. Cell cycle experiments and MTT assay were used to measure cell proliferation activity. Transwell test was used to detect cell migration activity. Results: The negative rate Tiam1 protein expression in cholangiocarcinoma tissues was significantly higher than that in benign bile tissues (P<0.001). Tiam1 protein expression in cholangiocarcinoma tissues had correlations with cholangiocarcinoma differentiation degree, TNM stage and lymph node metastasis (P<0.05), and had no significant correlations with gender, age and distant metastasis (P>0.05). Real-time PCR detection indicated that Tiam1 expression of experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that Tiam1 shRNA was effective on Tiam1 gene silencing in RBE cells. Cell cycle experiment showed that the percentage of S phase in cell cycle in experimental group was lower

An experimental study of the PTC properties of polymers with carbon black fillers

NASA Astrophysics Data System (ADS)

Lin, Jianlian

The Positive Temperature Coefficient (PTC) phenomenon, first discovered by Harman in 1957, is defined as the sharp increase of the electrical resistivity of the material with temperature, especially at the Curie transition temperature. Polymeric PTC materials have been widely used since 1975 as self-regulating components, over current or over heat protectors, sensors, etc. In this project a detailed study of polymeric PTC materials has been carried out. Polymeric PTC materials consist of a non-conducting polymeric phase in which conductive particles, such as CB's, are added. Previously most of the studies of the polymer matrices of PTC materials were limited to single component semi-crystalline polymers, such as HDPE, LDPE, EVA etc. In this work, the PTC effects of carbon black filled binary polymer blends, such as LDPE/EPDM, HDPD/EPDM, HDPE/EVA, etc. are mainly studied. For the LDPE/EPDM/CB system, it is found that the PTC intensity of the blends after gamma-ray irradiation increases by almost 5 orders of magnitude compared with that of irradiated LDPE/CB compound. This increase in PTC intensity is due to the greater thermal expansion coefficient of the rubber (EPDM) phase. In addition, a comparison of E-beam and gamma-ray irradiation is made and the resulting effect on the PTC properties of LDPE/EPDM/CB blends is studied in detail. It is found that with higher dose of gamma-ray, the material undergoes significant radiation damage, while irradiation with E-beam prevents radiation damage due to shorter exposure time. The influence of using treated carbon blacks on the PTC/NTC effects of the composites is also studied. The polymer blends filled with oxidized carbon black display higher PTC intensity followed by a weaker NTC effect, which is due to stronger interactions between oxidized CB's & polymer. It is concluded that strong interactions between polymers and fillers suppress the NTC effect. Finally ESR analysis is used to study the interactions between the

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

PubMed Central

Robinson, Lois; Panayiotakis, Alexandra; Papas, Takis S.; Kola, Ismail; Seth, Arun

1997-01-01

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to

Characterization of the novel tumor-suppressor gene CCDC67 in papillary thyroid carcinoma.

PubMed

Yin, De Tao; Xu, Jianhui; Lei, Mengyuan; Li, Hongqiang; Wang, Yongfei; Liu, Zhen; Zhou, Yubing; Xing, Mingzhao

2016-02-02

Some studies showed an association of coiled-coil domain-containing (CCDC) genes with cancers. Our previous limited data specifically suggested a possible pathogenic role of CCDC67 in papillary thyroid cancer (PTC), but this has not been firmly established. The present study was to further investigate and establish this role of CCDC67 in PTC. The expression of CCDC67, both at mRNA and protein levels, was sharply down-regulated in PTC compared with normal thyroid tissues. Lower CCDC67 expression was significantly associated with aggressive tumor behaviors, such as advanced tumor stages and lymph node metastasis, as well as BRAF mutation. Introduced expression of CCDC67 in TPC-1 cells robustly inhibited cell proliferation, colony formation and migration, induced G1 phase cell cycle arrest, and increased cell apoptosis. Primary PTC tumors and matched normal thyroid tissues were obtained from 200 unselected patients at the initial surgery for detection of CCDC67 mRNA and protein by RT-PCR and Western blotting analyses, respectively. Genomic DNA sequencing was performed to detect BRAF mutation in PTC tumors. Clinicopathological data were retrospectively reviewed for correlation analyses. PTC cell line TPC-1 with stable transfection of CCDC67 was used to investigate the functions of CCDC67. This large study demonstrates down-regulation of CCDC67 in PTC, an inverse relationship between CCDC67 expression and PTC aggressiveness and BRAF mutation, and a robust inhibitory effect of CCDC67 on PTC cellular activities. These results are consistent with CCDC67 being a novel and impaired tumor suppressor gene in PTC, providing important prognostic and therapeutic implications for this cancer.

Highly Unsaturated Platinum and Palladium Carbenes PtC3 and PdC3 Isolated and Characterized in the Gas Phase

PubMed Central

Bittner, Dror M.; Zaleski, Daniel P.; Tew, David P.

2016-01-01

Abstract Carbenes of platinum and palladium, PtC3 and PdC3, were generated in the gas phase through laser vaporization of a metal target in the presence of a low concentration of a hydrocarbon precursor undergoing supersonic expansion. Rotational spectroscopy and ab initio calculations confirm that both molecules are linear. The geometry of PtC3 was accurately determined by fitting to the experimental moments of inertia of twenty‐six isotopologues. The results are consistent with the proposal of an autogenic isolobal relationship between O, Au+, and Pt atoms. PMID:26879473

Highly Unsaturated Platinum and Palladium Carbenes PtC 3 and PdC 3 Isolated and Characterized in the Gas Phase

DOE PAGES

Bittner, Dror M.; Zaleski, Daniel P.; Tew, David P.; ...

2016-02-16

Carbenes of platinum and palladium, PtC 3 and PdC 3 , were generated in the gas phase through laser vaporization of a metal target in the presence of a low concentration of a hydrocarbon precursor undergoing supersonic expansion. Rotational spectroscopy and abinitio calculations confirm that both molecules are linear. The geometry of PtC 3 was accurately determined by fitting to the experimental moments of inertia of twenty-six isotopologues. In conclusion, the results are consistent with the proposal of an autogenic isolobal relationship between O, Au + , and Ptatoms.

Genome-wide analysis of YY2 versus YY1 target genes

PubMed Central

Chen, Li; Shioda, Toshi; Coser, Kathryn R.; Lynch, Mary C.; Yang, Chuanwei; Schmidt, Emmett V.

2010-01-01

Yin Yang 1 (YY1) is a critical transcription factor controlling cell proliferation, development and DNA damage responses. Retrotranspositions have independently generated additional YY family members in multiple species. Although Drosophila YY1 [pleiohomeotic (Pho)] and its homolog [pleiohomeotic-like (Phol)] redundantly control homeotic gene expression, the regulatory contributions of YY1-homologs have not yet been examined in other species. Indeed, targets for the mammalian YY1 homolog YY2 are completely unknown. Using gene set enrichment analysis, we found that lentiviral constructs containing short hairpin loop inhibitory RNAs for human YY1 (shYY1) and its homolog YY2 (shYY2) caused significant changes in both shared and distinguishable gene sets in human cells. Ribosomal protein genes were the most significant gene set upregulated by both shYY1 and shYY2, although combined shYY1/2 knock downs were not additive. In contrast, shYY2 reversed the anti-proliferative effects of shYY1, and shYY2 particularly altered UV damage response, platelet-specific and mitochondrial function genes. We found that decreases in YY1 or YY2 caused inverse changes in UV sensitivity, and that their combined loss reversed their respective individual effects. Our studies show that human YY2 is not redundant to YY1, and YY2 is a significant regulator of genes previously identified as uniquely responding to YY1. PMID:20215434

RET/PTC Rearrangements Are Associated with Elevated Postoperative TSH Levels and Multifocal Lesions in Papillary Thyroid Cancer without Concomitant Thyroid Benign Disease

PubMed Central

Su, Xuan; He, Caiyun; Ma, Jiangjun; Tang, Tao; Zhang, Xiao; Ye, Zulu; Long, Yakang; Shao, Qiong

2016-01-01

RET/PTC rearrangements, resulting in aberrant activity of the RET protein tyrosine kinase receptor, occur exclusively in papillary thyroid cancer (PTC). In this study, we examined the association between RET/PTC rearrangements and thyroid hormone homeostasis, and explored whether concomitant diseases such as nodular goiter and Hashimoto's thyroiditis influenced this association. A total of 114 patients diagnosed with PTC were enrolled in this study. Thyroid hormone levels, clinicopathological parameters and lifestyle were obtained through medical records and surgical pathology reports. RET/PTC rearrangements were detected using TaqMan RT-PCR and validated by direct sequencing. No RET/PTC rearrangements were detected in benign thyroid tissues. RET/PTC rearrangements were detected in 23.68% (27/114) of PTC tissues. No association between thyroid function, clinicopathological parameters and lifestyle was observed either in total thyroid cancer patients or the subgroup of patients with concomitant disease. In the subgroup of PTC patients without concomitant disease, RET/PTC rearrangement was associated with multifocal cancer (P = 0.018). RET/PTC rearrangement was also correlated with higher TSH levels at one month post-surgery (P = 0.037). Based on likelihood-ratio regression analysis, the RET/PTC-positive PTC cases showed an increased risk of multifocal cancers in the thyroid gland (OR = 5.57, 95% CI, 1.39–22.33). Our findings suggest that concomitant diseases such as nodular goiter and Hashimoto's thyroiditis in PTC may be a confounding factor when examining the effects of RET/PTC rearrangements. Excluding the potential effect of this confounding factor showed that RET/PTC may confer an increased risk for the development of multifocal cancers in the thyroid gland. Aberrantly increased post-operative levels of TSH were also associated with RET/PTC rearrangement. Together, our data provides useful information for the treatment of papillary thyroid cancer. PMID

Common genetic variants related to genomic integrity and risk of papillary thyroid cancer

PubMed Central

Neta, Gila; Brenner, Alina V.; Sturgis, Erich M.; Pfeiffer, Ruth M.; Hutchinson, Amy A.; Aschebrook-Kilfoy, Briseis; Yeager, Meredith; Xu, Li; Wheeler, William; Abend, Michael; Ron, Elaine; Tucker, Margaret A.; Chanock, Stephen J.; Sigurdson, Alice J.

2011-01-01

DNA damage is an important mechanism in carcinogenesis, so genes related to maintaining genomic integrity may influence papillary thyroid cancer (PTC) risk. Candidate gene studies targeting some of these genes have identified only a few polymorphisms associated with risk of PTC. Here, we expanded the scope of previous candidate studies by increasing the number and coverage of genes related to maintenance of genomic integrity. We evaluated 5077 tag single-nucleotide polymorphisms (SNPs) from 340 candidate gene regions hypothesized to be involved in DNA repair, epigenetics, tumor suppression, apoptosis, telomere function and cell cycle control and signaling pathways in a case–control study of 344 PTC cases and 452 matched controls. We estimated odds ratios for associations of single SNPs with PTC risk and combined P values for SNPs in the same gene region or pathway to obtain gene region-specific or pathway-specific P values using adaptive rank-truncated product methods. Nine SNPs had P values <0.0005, three of which were in HDAC4 and were inversely related to PTC risk. After multiple comparisons adjustment, no SNPs remained associated with PTC risk. Seven gene regions were associated with PTC risk at P < 0.01, including HUS1, ALKBH3, HDAC4, BAK1, FAF1_CDKN2C, DACT3 and FZD6. Our results suggest a possible role of genes involved in maintenance of genomic integrity in relation to risk of PTC. PMID:21642358

Gene expression and the biological phenotype of papillary thyroid carcinomas.

PubMed

Delys, L; Detours, V; Franc, B; Thomas, G; Bogdanova, T; Tronko, M; Libert, F; Dumont, J E; Maenhaut, C

2007-12-13

The purpose of this paper is to correlate the molecular phenotype of papillary thyroid carcinoma (PTC) to their biological pathology. We hybridized 26 PTC on microarrays and showed that nearly 44% of the transcriptome was regulated in these tumors. We then combined our data set with two published PTC microarray studies to produce a platform- and study-independent list of PTC-associated genes. We further confirmed the mRNA regulation of 15 genes from this list by quantitative reverse transcription-PCR. Analysis of this list with statistical tools led to several conclusions: (1) there is a change in cell population with an increased expression of genes involved in the immune response, reflecting lymphocyte infiltration in the tumor compared to the normal tissue. (2) The c-jun N-terminal kinase pathway is activated by overexpression of its components. (3) The activation of ERKK1/2 by genetic alterations is supplemented by activation of the epidermal growth factor but not of the insulin-like growth factor signaling pathway. (4) There is a downregulation of immediate early genes. (5) We observed an overexpression of many proteases in accordance with tumor remodeling, and suggested a probable role of S100 proteins and annexin A2 in this process. (6) Numerous overexpressed genes favor the hypothesis of a collective migration mode of tumor cells.

Medulloblastomas derived from Cxcr6 mutant mice respond to treatment with a smoothened inhibitor.

PubMed

Sasai, Ken; Romer, Justyna T; Kimura, Hiromichi; Eberhart, Derek E; Rice, Dennis S; Curran, Tom

2007-04-15

The sonic hedgehog (Shh) pathway is activated in approximately 30% of human medulloblastoma resulting in increased expression of downstream target genes. In about half of these cases, this has been shown to be a consequence of mutations in regulatory genes within the pathway, including Ptc1, Smo, and Sufu. However, for some tumors, no mutations have been detected in known pathway genes. This suggests that either mutations in other genes promote tumorigenesis or that epigenetic alterations increase pathway activity in these tumors. Here, we report that 3% to 4% of mice lacking either one or both functional copies of Cxcr6 develop medulloblastoma. Although CXCR6 is not known to be involved in Shh signaling, tumors derived from Cxcr6 mutant mice expressed Shh pathway target genes including Gli1, Gli2, Ptc2, and Sfrp1, indicating elevated pathway activity. Interestingly, the level of Ptc1 expression was decreased in tumor cells although two normal copies of Ptc1 were retained. This implies that reduced CXCR6 function leads to suppression of Ptc1 thereby increasing Smoothened function and promoting tumorigenesis. We used a direct transplant model to test the sensitivity of medulloblastoma arising in Cxcr6 mutant mice to a small-molecule inhibitor of Smoothened (HhAntag). We found that transplanted tumors were dramatically inhibited in mice treated for only 4 days with HhAntag. These findings suggest that HhAntag may be effective against tumors lacking mutations in known Shh pathway genes.

Materials for damping the PTC-induced thermal fluctuations of the cold-head

NASA Astrophysics Data System (ADS)

Catarino, I.; Martins, D.; Sudiwala, R.

2015-12-01

The cold head on mechanical Pulse Tube Cryocoolers (PTCs) is subject to substantially less mechanical vibration and electromagnetic interference compared to that typically found in Gifford MacMahon coolers. However, thermal fluctuations at the PTC frequency are still present at the cold-head, typically at a level of 200 mK peak-to-peak at 1.4 Hz for a Cryomech Model PT405 cooler running at 4 K. It is highly desirable to damp out these fluctuations if PTCs are to be used successfully for running systems sensitive to such thermal fluctuations, for example, bolometeric detectors. We report here the characterization over the temperature range 2.5 K to 6 K of two materials, GOS (Gd2O2S) and GAP (GdAlO3), for use as low-pass thermal filters. These materials have antiferromagnetic transitions at around 4 K giving rise to an enhanced heat capacity and have a high thermal conductance. These are two highly desirable properties for thermal dampers in this application. Those materials were fired as ceramic discs to be tested as thermal dumpers. Thermal filter assemblies with discs of diameter 75 mm and thickness 2.5 mm and 1.6 mm (GOS and GAP, respectively) mounted in a PTC show thermal attenuation levels of x0.12 (GOS) and x0.11 (GAP) at 0.01Hz with a clean-side temperature of 4 K; the PTC induced fluctuations at 1.48 Hz are damped completely to within the noise limits (0.2 mK) of the thermometers. Experimentally determined thermal conductance and heat capacity data are reported. For this system, with a PTC cold-head (dirty-side) temperature of 3.3 K, a clean-side power dissipation of up to 30 mW is realized before its temperature rises above 4.2 K.

Vemurafenib-resistance via de novo RBM genes mutations and chromosome 5 aberrations is overcome by combined therapy with palbociclib in thyroid carcinoma with BRAFV600E.

PubMed

Antonello, Zeus A; Hsu, Nancy; Bhasin, Manoj; Roti, Giovanni; Joshi, Mukta; Van Hummelen, Paul; Ye, Emily; Lo, Agnes S; Karumanchi, S Ananth; Bryke, Christine R; Nucera, Carmelo

2017-10-17

Papillary thyroid carcinoma (PTC) is the most frequent endocrine tumor. BRAF V600E represents the PTC hallmark and is targeted with selective inhibitors (e.g. vemurafenib). Although there have been promising results in clinical trials using these inhibitors, most patients develop resistance and progress. Tumor clonal diversity is proposed as one mechanism underlying drug resistance. Here we have investigated mechanisms of primary and secondary resistance to vemurafenib in BRAF WT/V600E -positive PTC patient-derived cells with P16 -/- (CDKN2A -/- ). Following treatment with vemurafenib, we expanded a sub-population of cells with primary resistance and characterized them genetically and cytogenetically. We have used exome sequencing, metaphase chromosome analysis, FISH and oligonucleotide SNP-microarray assays to assess clonal evolution of vemurafenib-resistant cells. Furthermore, we have validated our findings by networks and pathways analyses using PTC clinical samples. Vemurafenib-resistant cells grow similarly to naïve cells but are refractory to apoptosis upon treatment with vemurafenib, and accumulate in G2-M phase. We find that vemurafenib-resistant cells show amplification of chromosome 5 and de novo mutations in the RBM (RNA-binding motifs) genes family (i.e. RBMX, RBM10). RBMX knockdown in naïve-cells contributes to tetraploidization, including expansion of clones with chromosome 5 aberrations (e.g. isochromosome 5p). RBMX elicits gene regulatory networks with chromosome 5q cancer-associated genes and pathways for G2-M and DNA damage-response checkpoint regulation in BRAF WT/V600E -PTC. Importantly, combined therapy with vemurafenib plus palbociclib (inhibitor of CDK4/6, mimicking P16 functions) synergistically induces stronger apoptosis than single agents in resistant-cells and in anaplastic thyroid tumor cells harboring the heterozygous BRAF WT/V600E mutation. Critically, our findings suggest for the first time that targeting BRAF WT/V600E and CDK4

Targeted disruption of the 3p12 gene, Dutt1/Robo1, predisposes mice to lung adenocarcinomas and lymphomas with methylation of the gene promoter.

PubMed

Xian, Jian; Aitchison, Alan; Bobrow, Linda; Corbett, Gerard; Pannell, Richard; Rabbitts, Terence; Rabbitts, Pamela

2004-09-15

The DUTT1 gene is located on human chromosome 3, band p12, within a region of nested homozygous deletions in breast and lung tumors. It is therefore a candidate tumor suppressor gene in humans and is the homologue (ROBO1) of the Drosophila axonal guidance receptor gene, Roundabout. We have shown previously that mice with a targeted homozygous deletion within the Dutt1/Robo1 gene generally die at birth due to incomplete lung development: survivors die within the first year of life with epithelial bronchial hyperplasia as a common feature. Because Dutt1/Robo1 heterozygous mice develop normally, we have determined their tumor susceptibility. Mice with a targeted deletion within one Dutt1/Robo1 allele spontaneously develop lymphomas and carcinomas in their second year of life with a 3-fold increase in incidence compared with controls: invasive lung adenocarcinomas are by far the predominant carcinoma. In addition to the mutant allele, loss of heterozygosity analysis indicates that these tumors retain the structurally normal allele but with substantial methylation of the gene's promoter. Substantial reduction of Dutt1/Robo1 protein expression in tumors is observed by Western blotting and immunohistochemistry. This suggests that Dutt1/Robo1 is a classic tumor suppressor gene requiring inactivation of both alleles to elicit tumorigenesis in these mice.

Targeting glutaminase-mediated glutamine dependence in papillary thyroid cancer.

PubMed

Yu, Yang; Yu, Xiaohui; Fan, Chenling; Wang, Hong; Wang, Renee; Feng, Chen; Guan, Haixia

2018-06-25

Papillary thyroid cancer is a prevalent endocrine malignancy. Although alterations in glutamine metabolism have been reported in several types of hematological and solid tumors, little is known about the functions of glutamine and glutaminolysis-associated proteins in papillary thyroid cancer. Here, we demonstrated the glutamine dependence of papillary thyroid cancer cells, and with the use of RT 2 -PCR arrays, we screened for the aberrant overexpression of glutaminase in human papillary thyroid cancer tissues and cells. These results were later confirmed via real-time PCR, Western blots, and immunohistochemical staining. We found that the levels of glutaminase were significantly correlated with extrathyroidal extension. Inhibition of GLS suppressed glutaminolysis and reduced mitochondrial respiration. The proliferative, viable, migratory, and invasive abilities of papillary thyroid cancer cells were impaired by both the pharmacological inhibition and the genetic knockdown of glutaminase. Additionally, the inhibition of glutaminase deactivated the mechanistic target of the rapamycin complex 1 (mTORC1) signaling pathway, promoting autophagy and apoptosis. Collectively, these findings show that glutaminase-mediated glutamine dependence may be a potential therapeutic target for papillary thyroid cancer. PTC cells are glutamine-dependent, and GLS is aberrantly overexpressed in PTC. Inhibition of GLS suppressed glutaminolysis and reduced mitochondrial respiration. Inhibition of GLS impairs the viability of PTC cells. GLS blockade causes deactivation of mTORC1 and induction of autophagy and apoptosis. GLS may be a potential therapeutic target for PTC.

STAT3 or USF2 Contributes to HIF Target Gene Specificity

PubMed Central

Pawlus, Matthew R.; Wang, Liyi; Murakami, Aya; Dai, Guanhai; Hu, Cheng-Jun

2013-01-01

The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein. PMID:23991099

The SET1 Complex Selects Actively Transcribed Target Genes via Multivalent Interaction with CpG Island Chromatin.

PubMed

Brown, David A; Di Cerbo, Vincenzo; Feldmann, Angelika; Ahn, Jaewoo; Ito, Shinsuke; Blackledge, Neil P; Nakayama, Manabu; McClellan, Michael; Dimitrova, Emilia; Turberfield, Anne H; Long, Hannah K; King, Hamish W; Kriaucionis, Skirmantas; Schermelleh, Lothar; Kutateladze, Tatiana G; Koseki, Haruhiko; Klose, Robert J

2017-09-05

Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Genetic analysis of Ikaros target genes and tumor suppressor function in BCR-ABL1+ pre–B ALL

PubMed Central

Aghajanirefah, Ali; McLaughlin, Jami; Cheng, Donghui; Geng, Huimin; Eggesbø, Linn M.; Smale, Stephen T.; Müschen, Markus

2017-01-01

Inactivation of the tumor suppressor gene encoding the transcriptional regulator Ikaros (IKZF1) is a hallmark of BCR-ABL1+ precursor B cell acute lymphoblastic leukemia (pre–B ALL). However, the mechanisms by which Ikaros functions as a tumor suppressor in pre–B ALL remain poorly understood. Here, we analyzed a mouse model of BCR-ABL1+ pre–B ALL together with a new model of inducible expression of wild-type Ikaros in IKZF1 mutant human BCR-ABL1+ pre–B ALL. We performed integrated genome-wide chromatin and expression analyses and identified Ikaros target genes in mouse and human BCR-ABL1+ pre–B ALL, revealing novel conserved gene pathways associated with Ikaros tumor suppressor function. Notably, genetic depletion of different Ikaros targets, including CTNND1 and the early hematopoietic cell surface marker CD34, resulted in reduced leukemic growth. Our results suggest that Ikaros mediates tumor suppressor function by enforcing proper developmental stage–specific expression of multiple genes through chromatin compaction at its target genes. PMID:28190001

CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice.

PubMed

Yin, Xiaojia; Biswal, Akshaya K; Dionora, Jacqueline; Perdigon, Kristel M; Balahadia, Christian P; Mazumdar, Shamik; Chater, Caspar; Lin, Hsiang-Chun; Coe, Robert A; Kretzschmar, Tobias; Gray, Julie E; Quick, Paul W; Bandyopadhyay, Anindya

2017-05-01

CRISPR-Cas9/Cpf1 system with its unique gene targeting efficiency, could be an important tool for functional study of early developmental genes through the generation of successful knockout plants. The introduction and utilization of systems biology approaches have identified several genes that are involved in early development of a plant and with such knowledge a robust tool is required for the functional validation of putative candidate genes thus obtained. The development of the CRISPR-Cas9/Cpf1 genome editing system has provided a convenient tool for creating loss of function mutants for genes of interest. The present study utilized CRISPR/Cas9 and CRISPR-Cpf1 technology to knock out an early developmental gene EPFL9 (Epidermal Patterning Factor like-9, a positive regulator of stomatal development in Arabidopsis) orthologue in rice. Germ-line mutants that were generated showed edits that were carried forward into the T2 generation when Cas9-free homozygous mutants were obtained. The homozygous mutant plants showed more than an eightfold reduction in stomatal density on the abaxial leaf surface of the edited rice plants. Potential off-target analysis showed no significant off-target effects. This study also utilized the CRISPR-LbCpf1 (Lachnospiracae bacterium Cpf1) to target the same OsEPFL9 gene to test the activity of this class-2 CRISPR system in rice and found that Cpf1 is also capable of genome editing and edits get transmitted through generations with similar phenotypic changes seen with CRISPR-Cas9. This study demonstrates the application of CRISPR-Cas9/Cpf1 to precisely target genomic locations and develop transgene-free homozygous heritable gene edits and confirms that the loss of function analysis of the candidate genes emerging from different systems biology based approaches, could be performed, and therefore, this system adds value in the validation of gene function studies.

Interleukin-10-1082 gene polymorphism is associated with papillary thyroid cancer.

PubMed

Çil, Esra; Kumral, Alkın; Kanmaz-Özer, Müge; Vural, Pervin; Doğru-Abbasoğlu, Semra; Altuntaş, Yüksel; Uysal, Müjdat

2014-05-01

The etiopathogenesis of thyroid cancer has not been clearly elucidated although the role of chronical inflammation and the imbalance between pro- and anti-inflammatory cytokines may play a role in the etiology. The aim of the present study was to investigate whether cytokine gene polymorphisms are associated with papillary thyroid cancer (PTC), and to evaluate the relationship between genotypes and clinical/laboratory manifestation of PTC. Tumor necrosis factorα (TNFα) G-308A (rs 1800629), interleukin-6 (IL-6) G-174C (rs 1800795) and IL-10 A-1082G (rs 1800896) single nucleotide polymorphisms in DNA from peripheral blood leukocytes of 190 patients with thyroid cancer and 216 healthy controls were investigated by real-time PCR combined with melting curve analysis. There was no notable risk for PTC afflicted by TNFα-308 and IL-6-174 alone. However, IL-10-1082 G allele frequency were higher among PTC patients than healthy controls (p=0.009). The patients with IL-10-1082 GG geotype have twofold increased risk of developing thyroid cancer according to AA genotype (OR 2.07, 95% CI 1.21-3.55). In addition, the concomitant presence of IL-10-1082 G allele (GG+AG genotypes) together with IL-6 -174 GG genotype has a nearly twofold increased risk for thyroid cancer (OR 1.75 with 95% CI 1.00-3.05, p=0.049). We suggest that IL-10-1082 G allele is associated with an increased risk of PTC. The polymorphism of IL-10 gene can improve our knowledge about the pathogenesis of PTC, and could provide to estimate people at the increased risk for PTC.

The BTB and CNC homology 1 (BACH1) target genes are involved in the oxidative stress response and in control of the cell cycle.

PubMed

Warnatz, Hans-Jörg; Schmidt, Dominic; Manke, Thomas; Piccini, Ilaria; Sultan, Marc; Borodina, Tatiana; Balzereit, Daniela; Wruck, Wasco; Soldatov, Alexey; Vingron, Martin; Lehrach, Hans; Yaspo, Marie-Laure

2011-07-01

The regulation of gene expression in response to environmental signals and metabolic imbalances is a key step in maintaining cellular homeostasis. BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAF recognition elements, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we combined chromatin immunoprecipitation sequencing analysis of BACH1 target genes in HEK 293 cells with knockdown of BACH1 using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. The 59 BACH1 target genes identified by chromatin immunoprecipitation sequencing were found highly enriched in genes showing expression changes after BACH1 knockdown, demonstrating the impact of BACH1 repression on transcription. In addition to known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1, and SLC48A1) and redox regulation (GCLC, GCLM, and SLC7A11), we also discovered BACH1 target genes affecting cell cycle and apoptosis pathways (ITPR2, CALM1, SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, and MAFG) as well as subcellular transport processes (CLSTN1, PSAP, MAPT, and vault RNA). The newly identified impact of BACH1 on genes involved in neurodegenerative processes and proliferation provides an interesting basis for future dissection of BACH1-mediated gene repression in neurodegeneration and virus-induced cancerogenesis.

TINAGL1 and B3GALNT1 are potential therapy target genes to suppress metastasis in non-small cell lung cancer

PubMed Central

2014-01-01

Background Non-small cell lung cancer (NSCLC) remains lethal despite the development of numerous drug therapy technologies. About 85% to 90% of lung cancers are NSCLC and the 5-year survival rate is at best still below 50%. Thus, it is important to find drugable target genes for NSCLC to develop an effective therapy for NSCLC. Results Integrated analysis of publically available gene expression and promoter methylation patterns of two highly aggressive NSCLC cell lines generated by in vivo selection was performed. We selected eleven critical genes that may mediate metastasis using recently proposed principal component analysis based unsupervised feature extraction. The eleven selected genes were significantly related to cancer diagnosis. The tertiary protein structure of the selected genes was inferred by Full Automatic Modeling System, a profile-based protein structure inference software, to determine protein functions and to specify genes that could be potential drug targets. Conclusions We identified eleven potentially critical genes that may mediate NSCLC metastasis using bioinformatic analysis of publically available data sets. These genes are potential target genes for the therapy of NSCLC. Among the eleven genes, TINAGL1 and B3GALNT1 are possible candidates for drug compounds that inhibit their gene expression. PMID:25521548

New gene targets for glucagon-like peptide-1 during embryonic development and in undifferentiated pluripotent cells.

PubMed

Sanz, Carmen; Blázquez, Enrique

2011-09-01

In humans, glucagon-like peptide (GLP-1) functions during adult life as an incretin hormone with anorexigenic and antidiabetogenic properties. Also, the therapeutic potential of GLP-1 in preventing the adipocyte hyperplasia associated with obesity and in bolstering the maintenance of human mesenchymal stem cell (hMSC) stores by promoting the proliferation and cytoprotection of hMSC seems to be relevant. Since these observations suggest a role for GLP-1 during developmental processes, the aim of the present work was to characterize GLP-1 in early development as well as its gene targets in mouse embryonic stem (mES) cells. Mouse embryos E6, E8, and E10.5 and pluripotent mES were used for the inmunodetection of GLP-1 and GLP-1 receptor. Quantitative real-time PCR was used to determine the expression levels of GLP-1R in several tissues from E12.5 mouse embryos. Additionally, GLP-1 gene targets were studied in mES by multiple gene expression analyses. GLP-1 and its receptors were identified in mES and during embryonic development. In pluripotent mES, GLP-1 modified the expression of endodermal, ectodermal, and mesodermal gene markers as well as sonic hedgehog, noggin, members of the fibroblast and hepatic growth factor families, and others involved in pancreatic development. Additionally, GLP-1 promoted the expression of the antiapoptotic gene bcl2 and at the same time reduced proapoptotic caspase genes. Our results indicate that apart from the effects and therapeutic benefits of GLP-1 in adulthood, it may have additional gene targets in mES cells during embryonic life. Furthermore, the pathophysiological implications of GLP-1 imbalance in adulthood may have a counterpart during development.

E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

PubMed

Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

2017-01-01

Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

Evolving phage vectors for cell targeted gene delivery.

PubMed

Larocca, David; Burg, Michael A; Jensen-Pergakes, Kristen; Ravey, Edward Prenn; Gonzalez, Ana Maria; Baird, Andrew

2002-03-01

We adapted filamentous phage vectors for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (GFP) into the vector backbone and fusing the pIII coat protein to a cell targeting ligand (i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage gene delivery is concentration, time, and ligand dependent. Importantly, targeted phage particles are specific for the appropriate target cell surface receptor. Phage have distinct advantages over existing gene therapy vectors because they are simple, economical to produce at high titer, have no intrinsic tropism for mammalian cells, and are relatively simple to genetically modify and evolve. Initially transduction by targeted phage particles was low resulting in foreign gene expression in 1-2% of transfected cells. We increased transduction efficiency by modifying both the transfection protocol and vector design. For example, we stabilized the display of the targeting ligand to create multivalent phagemid-based vectors with transduction efficiencies of up to 45% in certain cell lines when combined with genotoxic treatment. Taken together, these studies establish that the efficiency of phage-mediated gene transfer can be significantly improved through genetic modification. We are currently evolving phage vectors with enhanced cell targeting, increased stability, reduced immunogenicity and other properties suitable for gene therapy.

[A mini-review of targeting gene-virotherapy of cancer].

PubMed

Liu, Xin-Yuan; Gu, Jin-Fa

2006-10-01

New progress has been made on the project "targeting gene-virotherapy of cancer" proposed by us, which is "targeting dual gene-virotherapy of cancer". By the use of two genes, all the xenograft tumors in nude mice could be completely eliminated. The researches have been published in international journals, such as Hepatology and Cancer Research (a highlight paper). In this study, a further superior strategy--"double targeting virus-dual gene therapy" was introduced. This strategy was specialized by the use of tumor specific promoter to control the tumor specific suppressor gene, such as alpha-fetoprotein (AFP), which controls hepatoma specific suppressor gene LFIRE or HCCS1. In addition, a second tumor specific promoter, such as hTERT or survivin was used to control E1A or E1B in the construct, as hTERT-E1A-AFP-E1B-HCCS1 or LFIRE, a double tumor specific promoter controlling hepatoma specific LFIRE or HCCS1 gene. By the combined use of this construct with a very strong antitumor construct, such as hTERT-E1A-AFP-E1B-IL-24, a strategy with both excellent tumor killing effect and excellent safety with very little damage to normal cells was obtained. Therefore, double targeting virus-dual gene therapy might be one of the most potential strategies for cancer treatment. Furthermore, a new type of interferon was also introduced, which might be an ideal antitumor drug.

PTC test bed upgrades to provide ACSES testing support capabilities at transportation technology center.

DOT National Transportation Integrated Search

2015-06-01

FRA Task Order 314 upgraded the Positive Train Control (PTC) Test Bed at the Transportation Technology Center to support : testing of PTC systems, components, and related equipment associated with the Advanced Civil Speed Enforcement System : (ACSES)...

Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

PubMed Central

Jacobs, Frank M. J.; van der Linden, Annemarie J. A.; Wang, Yuhui; von Oerthel, Lars; Sul, Hei Sook; Burbach, J. Peter H.; Smidt, Marten P.

2009-01-01

The orphan nuclear receptor Nurr1 is essential for the development of meso-diencephalic dopamine (mdDA) neurons and is required, together with the homeobox transcription factor Pitx3, for the expression of genes involved in dopamine metabolism. In order to elucidate the molecular mechanisms that underlie the neuronal deficits in Nurr1-/- mice, we performed combined gene expression microarrays and ChIP-on-chip analysis and thereby identified Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in vivo. In line with the previously described cooperativity between Nurr1 and Pitx3, we show that the expression of Ptpru and Klhl1 in mdDA neurons is also dependent on Pitx3. Furthermore, we demonstrate that Nurr1 interacts with the Ptpru promoter directly and requires Pitx3 for full expression of Ptpru in mdDA neurons. By contrast, the expression of Dlk1 is maintained in Pitx3-/- embryos and is even expanded into the rostral part of the mdDA area, suggesting a unique position of Dlk1 in the Nurr1 and Pitx3 transcriptional cascades. Expression analysis in Dlk1-/- embryos reveals that Dlk1 is required to prevent premature expression of Dat in mdDA neuronal precursors as part of the multifaceted process of mdDA neuronal differentiation driven by Nurr1 and Pitx3. Taken together, the involvement of Nurr1 and Pitx3 in the expression of novel target genes involved in important neuronal processes such as neuronal patterning, axon outgrowth and terminal differentiation, opens up new avenues to study the properties of mdDA neurons during development and in neuronal pathology as observed in Parkinson's disease. PMID:19515692

Association between the KRAS Gene Polymorphisms and Papillary Thyroid Carcinoma in a Chinese Han Population.

PubMed

Ning, Lifeng; Rao, Wenwang; Yu, Yaqin; Liu, Xiaoli; Pan, Yuchen; Ma, Yuan; Liu, Rui; Zhang, Shangchao; Sun, Hui; Yu, Qiong

2016-01-01

Several studies have reported the association between MAPK signaling pathway gene polymorphisms and papillary thyroid carcinoma (PTC). KRAS gene, an oncogene from the mammalian RAS gene family plays an important role in the MAPK pathway. This study aimed to identify the potential association of KRAS gene polymorphisms with susceptibility to PTC in a Han Chinese population. A total of 861 patients with PTC, 562 disease controls with nodular goiter and 897 healthy controls were recruited. Four tagSNP polymorphisms (rs12427141, rs712, rs7315339 and rs7960917) of KRAS gene were genotyped by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) . Statistical analyses and haplotype estimations were conducted using Haploview and Unphased softwares. Only significant differences were observed in genotypic frequencies of the rs7315339 polymorphism (χ 2 =7.234, df=2, p=0.027) between PTC and disease controls. Statistically significant differences in both allelic and genotypic genotypes frequencies for rs712 (Genotype, χ 2 =8.258, p=0.016) and rs12427141 (Allele, χ 2 =3.992, p=0.046; Genotype, χ 2 =8.140, p=0.017) were observed between PTC patients and controls. Haplotype analyses revealed higher frequencies of GA and TA haplotypes (p=0.039 and p=0.003, respectively) from rs712- rs12427141 (two-SNP) or TGA and TTG haplotype containing the alleles from rs7960917, rs712 and rs12427141, as well as the GAT haplotype containing the alleles from rs712, rs12427141 and rs7315339 in PTC patients than in healthy controls (p=0.042, p=0.037, p=0.027, respectively). Inversely, the haplotype TTA from rs7960917, rs712 and rs12427141 or the haplotype TAC from rs712, rs12427141 and rs7315339 was significantly less frequent in the PTC patients than in normal control (p=0.003, p=0.003, respectively). These findings suggest the role of these KRAS gene variants in susceptibility to PTC. Moreover, significant differences of the KRAS gene polymorphisms may

49 CFR 236.1027 - PTC system exclusions.

Code of Federal Regulations, 2010 CFR

2010-10-01

... distributed computer-based system that directly or indirectly controls the active movement of trains in a rail... 49 Transportation 4 2010-10-01 2010-10-01 false PTC system exclusions. 236.1027 Section 236.1027..., MAINTENANCE, AND REPAIR OF SIGNAL AND TRAIN CONTROL SYSTEMS, DEVICES, AND APPLIANCES Positive Train Control...

49 CFR 236.1027 - PTC system exclusions.

Code of Federal Regulations, 2013 CFR

2013-10-01

... distributed computer-based system that directly or indirectly controls the active movement of trains in a rail... 49 Transportation 4 2013-10-01 2013-10-01 false PTC system exclusions. 236.1027 Section 236.1027..., MAINTENANCE, AND REPAIR OF SIGNAL AND TRAIN CONTROL SYSTEMS, DEVICES, AND APPLIANCES Positive Train Control...

49 CFR 236.1027 - PTC system exclusions.

Code of Federal Regulations, 2011 CFR

2011-10-01

... distributed computer-based system that directly or indirectly controls the active movement of trains in a rail... 49 Transportation 4 2011-10-01 2011-10-01 false PTC system exclusions. 236.1027 Section 236.1027..., MAINTENANCE, AND REPAIR OF SIGNAL AND TRAIN CONTROL SYSTEMS, DEVICES, AND APPLIANCES Positive Train Control...

49 CFR 236.1027 - PTC system exclusions.

Code of Federal Regulations, 2014 CFR

2014-10-01

... distributed computer-based system that directly or indirectly controls the active movement of trains in a rail... 49 Transportation 4 2014-10-01 2014-10-01 false PTC system exclusions. 236.1027 Section 236.1027..., MAINTENANCE, AND REPAIR OF SIGNAL AND TRAIN CONTROL SYSTEMS, DEVICES, AND APPLIANCES Positive Train Control...

49 CFR 236.1027 - PTC system exclusions.

Code of Federal Regulations, 2012 CFR

2012-10-01

... distributed computer-based system that directly or indirectly controls the active movement of trains in a rail... 49 Transportation 4 2012-10-01 2012-10-01 false PTC system exclusions. 236.1027 Section 236.1027..., MAINTENANCE, AND REPAIR OF SIGNAL AND TRAIN CONTROL SYSTEMS, DEVICES, AND APPLIANCES Positive Train Control...

Targeted Mutagenesis of Duplicated Genes in Soybean with Zinc-Finger Nucleases1[W][OA

PubMed Central

Curtin, Shaun J.; Zhang, Feng; Sander, Jeffry D.; Haun, William J.; Starker, Colby; Baltes, Nicholas J.; Reyon, Deepak; Dahlborg, Elizabeth J.; Goodwin, Mathew J.; Coffman, Andrew P.; Dobbs, Drena; Joung, J. Keith; Voytas, Daniel F.; Stupar, Robert M.

2011-01-01

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform—a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting DICER-LIKE (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome. PMID:21464476

The preparation and characterization of novel Pt/C electrocatalysts with controlled porosity and cluster size

DOE PAGES

Coker, Eric N.; Steen, William A.; Miller, Jeffrey T.; ...

2007-05-23

Small platinum clusters have been prepared in zeolite hosts through ion exchange and controlled calcination/reduction processes. In order to enable electrochemical application, the pores of the Pt-zeolite were filled with electrically conductive carbon via infiltration with carbon precursors, polymerization, and pyrolysis. The zeolite host was then removed by acid washing, to leave a Pt/C electrocatalyst possessing quasi-zeolitic porosity and Pt clusters of well-controlled size. The electrocatalysts were characterized by TEM, XRD, EXAFS, nitrogen adsorption and electrochemical techniques. Depending on the synthesis conditions, average Pt cluster sizes in the Pt/C catalysts ranged from 1.3 to 2.0 nm. The presence of orderedmore » porosity/structure in the catalysts was evident in TEM images as lattice fringes, and in XRD as a low-angle diffraction peak with d-spacing similar to the parent zeolite. The catalysts possess micro- and meso-porosity, with pore size distributions that depend upon synthesis variables. Finally, electroactive surface areas as high as 112 m 2 g Pt -1 have been achieved in Pt/C electrocatalysts which show oxygen reduction performance comparable to standard industrial catalysts.« less

Specific c-Jun target genes in malignant melanoma.

PubMed

Schummer, Patrick; Kuphal, Silke; Vardimon, Lily; Bosserhoff, Anja K; Kappelmann, Melanie

2016-05-03

A fundamental event in the development and progression of malignant melanoma is the de-regulation of cancer-relevant transcription factors. We recently showed that c-Jun is a main regulator of melanoma progression and, thus, is the most important member of the AP-1 transcription factor family in this disease. Surprisingly, no cancer-related specific c-Jun target genes in melanoma were described in the literature, so far. Therefore, we focused on pre-existing ChIP-Seq data (Encyclopedia of DNA Elements) of 3 different non-melanoma cell lines to screen direct c-Jun target genes. Here, a specific c-Jun antibody to immunoprecipitate the associated promoter DNA was used. Consequently, we identified 44 direct c-Jun targets and a detailed analysis of 6 selected genes confirmed their deregulation in malignant melanoma. The identified genes were differentially regulated comparing 4 melanoma cell lines and normal human melanocytes and we confirmed their c-Jun dependency. Direct interaction between c-Jun and the promoter/enhancer regions of the identified genes was confirmed by us via ChIP experiments. Interestingly, we revealed that the direct regulation of target gene expression via c-Jun can be independent of the existence of the classical AP-1 (5´-TGA(C/G)TCA-3´) consensus sequence allowing for the subsequent down- or up-regulation of the expression of these cancer-relevant genes. In summary, the results of this study indicate that c-Jun plays a crucial role in the development and progression of malignant melanoma via direct regulation of cancer-relevant target genes and that inhibition of direct c-Jun targets through inhibition of c-Jun is a potential novel therapeutic option for treatment of malignant melanoma.

Hypoxia regulates alternative splicing of HIF and non-HIF target genes.

PubMed

Sena, Johnny A; Wang, Liyi; Heasley, Lynn E; Hu, Cheng-Jun

2014-09-01

Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma cells and characterized the role of HIF in regulating AS of HIF-induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia-reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets, including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing. This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. ©2014 American Association for Cancer Research.

About miRNAs, miRNA seeds, target genes and target pathways.

PubMed

Kehl, Tim; Backes, Christina; Kern, Fabian; Fehlmann, Tobias; Ludwig, Nicole; Meese, Eckart; Lenhof, Hans-Peter; Keller, Andreas

2017-12-05

miRNAs are typically repressing gene expression by binding to the 3' UTR, leading to degradation of the mRNA. This process is dominated by the eight-base seed region of the miRNA. Further, miRNAs are known not only to target genes but also to target significant parts of pathways. A logical line of thoughts is: miRNAs with similar (seed) sequence target similar sets of genes and thus similar sets of pathways. By calculating similarity scores for all 3.25 million pairs of 2,550 human miRNAs, we found that this pattern frequently holds, while we also observed exceptions. Respective results were obtained for both, predicted target genes as well as experimentally validated targets. We note that miRNAs target gene set similarity follows a bimodal distribution, pointing at a set of 282 miRNAs that seems to target genes with very high specificity. Further, we discuss miRNAs with different (seed) sequences that nonetheless regulate similar gene sets or pathways. Most intriguingly, we found miRNA pairs that regulate different gene sets but similar pathways such as miR-6886-5p and miR-3529-5p. These are jointly targeting different parts of the MAPK signaling cascade. The main goal of this study is to provide a general overview on the results, to highlight a selection of relevant results on miRNAs, miRNA seeds, target genes and target pathways and to raise awareness for artifacts in respective comparisons. The full set of information that allows to infer detailed results on each miRNA has been included in miRPathDB, the miRNA target pathway database (https://mpd.bioinf.uni-sb.de).

Mitochondrial Metabolism as a Treatment Target in Anaplastic Thyroid Cancer

PubMed Central

Johnson, Jennifer M; Lai, Stephen Y.; Cotzia, Paolo; Cognetti, David; Luginbuhl, Adam; Pribitkin, Edmund A.; Zhan, Tingting; Mollaee, Mehri; Domingo-Vidal, Marina; Chen, Yunyun; Campling, Barbara; Bar-Ad, Voichita; Birbe, Ruth; Tuluc, Madalina; Outschoorn, Ubaldo Martinez; Curry, Joseph

2015-01-01

Aims Anaplastic thyroid cancer (ATC) is one of the most aggressive human cancers. Key signal transduction pathways that regulate mitochondrial metabolism are frequently altered in ATC. Our goal was to determine the mitochondrial metabolic phenotype of ATC by studying markers of mitochondrial metabolism, specifically Monocarboxylate Transporter 1 (MCT1) and Translocase of the Outer Mitochondrial Membrane Member 20 (TOMM20). Methods Staining patterns of MCT1 and TOMM20 in 35 human thyroid samples (15 ATC, 12 papillary thyroid cancer (PTC), and 8 non-cancerous thyroid) and 9 ATC mouse orthotopic xenografts were assessed by visual and Aperio digital scoring. Staining patterns of areas involved with cancer versus areas with no evidence of cancer were evaluated independently where available. Results MCT1 is highly expressed in human anaplastic thyroid cancer when compared to both non-cancerous thyroid tissues and papillary thyroid cancers (p<0.001 for both). TOMM20 is also highly expressed in both ATC and PTC compared to non-cancerous thyroid tissue (p<0.01 for both). High MCT1 and TOMM20 expression is also found in ATC mouse xenograft tumors compared to non-cancerous thyroid tissue (p<0.001). These xenograft tumors have high 13C- pyruvate uptake. Conclusions Anaplastic thyroid cancer has metabolic features that distinguish it from PTC and non-cancerous thyroid tissue, including high expression of MCT1 and TOMM20. PTC has low expression of MCT1 and non-cancerous thyroid tissue has low expression of both MCT1 and TOMM20. This work suggests that MCT1 blockade may specifically target ATC cells presenting an opportunity for a new drug target. PMID:26615136

Mitochondrial Metabolism as a Treatment Target in Anaplastic Thyroid Cancer.

PubMed

Johnson, Jennifer M; Lai, Stephen Y; Cotzia, Paolo; Cognetti, David; Luginbuhl, Adam; Pribitkin, Edmund A; Zhan, Tingting; Mollaee, Mehri; Domingo-Vidal, Marina; Chen, Yunyun; Campling, Barbara; Bar-Ad, Voichita; Birbe, Ruth; Tuluc, Madalina; Martinez Outschoorn, Ubaldo; Curry, Joseph

2015-12-01

Anaplastic thyroid cancer (ATC) is one of the most aggressive human cancers. Key signal transduction pathways that regulate mitochondrial metabolism are frequently altered in ATC. Our goal was to determine the mitochondrial metabolic phenotype of ATC by studying markers of mitochondrial metabolism, specifically monocarboxylate transporter 1 (MCT1) and translocase of the outer mitochondrial membrane member 20 (TOMM20). Staining patterns of MCT1 and TOMM20 in 35 human thyroid samples (15 ATC, 12 papillary thyroid cancer [PTC], and eight non-cancerous thyroid) and nine ATC mouse orthotopic xenografts were assessed by visual and Aperio digital scoring. Staining patterns of areas involved with cancer versus areas with no evidence of cancer were evaluated independently where available. MCT1 is highly expressed in human anaplastic thyroid cancer when compared to both non-cancerous thyroid tissues and papillary thyroid cancers (P<.001 for both). TOMM20 is also highly expressed in both ATC and PTC compared to non-cancerous thyroid tissue (P<.01 for both). High MCT1 and TOMM20 expression is also found in ATC mouse xenograft tumors compared to non-cancerous thyroid tissue (P<.001). These xenograft tumors have high (13)C- pyruvate uptake. ATC has metabolic features that distinguish it from PTC and non-cancerous thyroid tissue, including high expression of MCT1 and TOMM20. PTC has low expression of MCT1 and non-cancerous thyroid tissue has low expression of both MCT1 and TOMM20. This work suggests that MCT1 blockade may specifically target ATC cells presenting an opportunity for a new drug target. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

A comparison of Agrobacterium-mediated transformation and protoplast-mediated transformation with CRISPR-Cas9 and bipartite gene targeting substrates, as effective gene targeting tools for Aspergillus carbonarius.

PubMed

Weyda, István; Yang, Lei; Vang, Jesper; Ahring, Birgitte K; Lübeck, Mette; Lübeck, Peter S

2017-04-01

In recent years, versatile genetic tools have been developed and applied to a number of filamentous fungi of industrial importance. However, the existing techniques have limitations when it comes to achieve the desired genetic modifications, especially for efficient gene targeting. In this study, we used Aspergillus carbonarius as a host strain due to its potential as a cell factory, and compared three gene targeting techniques by disrupting the ayg1 gene involved in the biosynthesis of conidial pigment in A. carbonarius. The absence of the ayg1 gene leads to phenotypic change in conidia color, which facilitated the analysis on the gene targeting frequency. The examined transformation techniques included Agrobacterium-mediated transformation (AMT) and protoplast-mediated transformation (PMT). Furthermore, the PMT for the disruption of the ayg1 gene was carried out with bipartite gene targeting fragments and the recently adapted CRISPR-Cas9 system. All three techniques were successful in generating Δayg1 mutants, but showed different efficiencies. The most efficient method for gene targeting was AMT, but further it was shown to be dependent on the choice of Agrobacterium strain. However, there are different advantages and disadvantages of all three gene targeting methods which are discussed, in order to facilitate future approaches for fungal strain improvements. Copyright © 2017 Elsevier B.V. All rights reserved.

49 CFR 236.1031 - Previously approved PTC systems.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 4 2010-10-01 2010-10-01 false Previously approved PTC systems. 236.1031 Section 236.1031 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD....1005 and 236.1007 and otherwise conform to this subpart. (d) Previous approval or recognition of a...

Screening for microsatellite instability target genes in colorectal cancers

PubMed Central

Vilkki, S; Launonen, V; Karhu, A; Sistonen, P; Vastrik, I; Aaltonen, L

2002-01-01

Background: Defects in the DNA repair system lead to genetic instability because replication errors are not corrected. This type of genetic instability is a key event in the malignant progression of HNPCC and a subset of sporadic colon cancers and mutation rates are particularly high at short repetitive sequences. Somatic deletions of coding mononucleotide repeats have been detected, for example, in the TGFßRII and BAX genes, and recently many novel target genes for microsatellite instability (MSI) have been proposed. Novel target genes are likely to be discovered in the future. More data should be created on background mutation rates in MSI tumours to evaluate mutation rates observed in the candidate target genes. Methods: Mutation rates in 14 neutral intronic repeats were evaluated in MSI tumours. Bioinformatic searches combined with keywords related to cancer and tumour suppressor or CRC related gene homology were used to find new candidate MSI target genes. By comparison of mutation frequencies observed in intronic mononucleotide repeats versus exonic coding repeats of potential MSI target genes, the significance of the exonic mutations was estimated. Results: As expected, the length of an intronic mononucleotide repeat correlated positively with the number of slippages for both G/C and A/T repeats (p=0.0020 and p=0.0012, respectively). BRCA1, CtBP1, and Rb1 associated CtIP and other candidates were found in a bioinformatic search combined with keywords related to cancer. Sequencing showed a significantly increased mutation rate in the exonic A9 repeat of CtIP (25/109=22.9%) as compared with similar intronic repeats (p≤0.001). Conclusions: We propose a new candidate MSI target gene CtIP to be evaluated in further studies. PMID:12414815

Progress in gene targeting and gene therapy for retinitis pigmentosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Farrar, G.J.; Humphries, M.M.; Erven, A.

1994-09-01

Previously, we localized disease genes involved in retinitis pigmentosa (RP), an inherited retinal degeneration, close to the rhodopsin and peripherin genes on 3q and 6p. Subsequently, we and others identified mutations in these genes in RP patients. Currently animal models for human retinopathies are being generated using gene targeting by homologous recombination in embryonic stem (ES) cells. Genomic clones for retinal genes including rhodopsin and peripherin have been obtained from a phage library carrying mouse DNA isogenic with the ES cell line (CC1.2). The peripherin clone has been sequenced to establish the genomic structure of the mouse gene. Targeting vectorsmore » for rhodopsin and peripherin including a neomycin cassette for positive selection and thymidine kinase genes enabling selection against random intergrants are under construction. Progress in vector construction will be presented. Simultaneously we are developing systems for delivery of gene therapies to retinal tissues utilizing replication-deficient adenovirus (Ad5). Efficacy of infection subsequent to various methods of intraocular injection and with varying viral titers is being assayed using an adenovirus construct containing a CMV promoter LacZ fusion as reporter and the range of tissues infected and the level of duration of LacZ expression monitored. Viral constructs with the LacZ reporter gene under the control of retinal specific promoters such as rhodopsin and IRBP cloned into pXCJL.1 are under construction. An update on developments in photoreceptor cell-directed expression of virally delivered genes will be presented.« less

Ultraviolet brightness of celestial targets for Apollo 17

NASA Technical Reports Server (NTRS)

Fastie, W. G.

1972-01-01

An evaluation of the ultraviolet flux from the stars expected in the various inertial-hold pointing directions and PTC scans during the Apollo 17 mission is presented. These directions and PTC scan poles for the nominal mission are listed. The methodology used in evaluating the flux, and the individual targets themselves is explained.

BLISTER Regulates Polycomb-Target Genes, Represses Stress-Regulated Genes and Promotes Stress Responses in Arabidopsis thaliana.

PubMed

Kleinmanns, Julia A; Schatlowski, Nicole; Heckmann, David; Schubert, Daniel

2017-01-01

HIGHLIGHTS The PRC2 interacting protein BLISTER likely acts downstream of PRC2 to silence Polycomb target genes and is a key regulator of specific stress responses in Arabidopsis . Polycomb group (PcG) proteins are key epigenetic regulators of development. The highly conserved Polycomb repressive complex 2 (PRC2) represses thousands of target genes by trimethylating H3K27 (H3K27me3). Plant specific PcG components and functions are largely unknown, however, we previously identified the plant-specific protein BLISTER (BLI) as a PRC2 interactor. BLI regulates PcG target genes and promotes cold stress resistance. To further understand the function of BLI , we analyzed the transcriptional profile of bli-1 mutants. Approximately 40% of the up-regulated genes in bli are PcG target genes, however, bli-1 mutants did not show changes in H3K27me3 levels at all tested genes, indicating that BLI regulates PcG target genes downstream of or in parallel to PRC2. Interestingly, a significant number of BLI regulated H3K27me3 target genes is regulated by the stress hormone absciscic acid (ABA). We further reveal an overrepresentation of genes responding to abiotic stresses such as drought, high salinity, or heat stress among the up-regulated genes in bli mutants. Consistently, bli mutants showed reduced desiccation stress tolerance. We conclude that the PRC2 associated protein BLI is a key regulator of stress-responsive genes in Arabidopsis : it represses ABA-responsive PcG target genes, likely downstream of PRC2, and promotes resistance to several stresses such as cold and drought.

Targeted gene therapy and cell reprogramming in Fanconi anemia

PubMed Central

Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

2014-01-01

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. PMID:24859981

AP1 binding site is another target of FGF2 regulation of bone sialoprotein gene transcription.

PubMed

Takai, Hideki; Araki, Shouta; Mezawa, Masaru; Kim, Dong-Soon; Li, Xinyue; Yang, Li; Li, Zhengyang; Wang, Zhitao; Nakayama, Youhei; Ogata, Yorimasa

2008-02-29

Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. We previously reported that fibroblast growth factor 2 (FGF2) regulates BSP gene transcription via FGF2 response element (FRE) in the proximal promoter of rat BSP gene. We here report that activator protein 1 (AP1) binding site overlapping with glucocorticoid response element (GRE) AP1/GRE in the rat BSP gene promoter is another target of FGF2. Using the osteoblastic cell line ROS17/2.8, we determined that BSP mRNA levels increased by 10 ng/ml FGF2 at 6 and 12 h. Runx2 protein levels increased by FGF2 (10 ng/ml) at 3 h. Treatment of ROS17/2.8 cells with FGF2 (10 ng/ml, 12 h) increased luciferase activities of constructs including -116 to +60 and -938 to +60 of the rat BSP gene promoter. Effects of FGF2 abrogated in constructs included 2 bp mutations in the FRE and AP1/GRE elements. Luciferase activities induced by FGF2 were blocked by tyrosine kinase inhibitor herbimycin A, src-tyrosine kinase inhibitor PP1 and MAP kinase kinase inhibitor U0126. Gel shift analyses showed that FGF2 increased binding of FRE and AP1/GRE elements. Notably, the AP1/GRE-protein complexes were supershifted by Smad1 and c-Fos antibodies, c-Jun and Dlx5 antibodies disrupted the complexes formation, on the other hand AP1/GRE-protein complexes did not change by Runx2 antibody. These studies demonstrate that FGF2 stimulates BSP gene transcription by targeting the FRE and AP1/GRE elements in the rat BSP gene promoter.

Expression of Spindle and Kinetochore-Associated Protein 1 Is Associated with Poor Prognosis in Papillary Thyroid Carcinoma

PubMed Central

Dong, Chao; Wang, Xiao-li; Ma, Bin-lin

2015-01-01

Aim. Spindle and kinetochore-associated protein 1 (SKA1) is one subtype of SKA, whose protein can make spindle microtubules attach steadily to the kinetochore in the middle of mitosis. At present, there are fewer researches on the relationship between SKA1 expression and tumor development. Methods. In this study, immunohistochemical analysis was used to determine the expression of SKA1 in papillary thyroid carcinoma (PTC) and adjacent tissues. We used quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis to further verify the results. Results. We found that SKA1 expression was significantly higher in PTC tissues than normal adjacent tissues (P < 0.05). There existed a significant correlation among a higher SKA1 expression, including lymphoid node (P = 0.005), clinical stage (P = 0.015), and extrathyroid invasion (P = 0.004). Survival analysis showed high SKA1 expression in PTC patients more likely to relapse after surgery. Conclusion. High SKA1 expression is predictive of poor prognosis of PTC, implying that SKA1 may be a promising new target for targeted therapies for PTC. PMID:26063960

The Receptor Tyrosine Kinase EphA2 Is a Direct Target Gene of Hypermethylated in Cancer 1 (HIC1)*

PubMed Central

Foveau, Bénédicte; Boulay, Gaylor; Pinte, Sébastien; Van Rechem, Capucine; Rood, Brian R.; Leprince, Dominique

2012-01-01

The tumor suppressor gene hypermethylated in cancer 1 (HIC1), which encodes a transcriptional repressor, is epigenetically silenced in many human tumors. Here, we show that ectopic expression of HIC1 in the highly malignant MDA-MB-231 breast cancer cell line severely impairs cell proliferation, migration, and invasion in vitro. In parallel, infection of breast cancer cell lines with a retrovirus expressing HIC1 also induces decreased mRNA and protein expression of the tyrosine kinase receptor EphA2. Moreover, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments demonstrate that endogenous HIC1 proteins are bound, together with the MTA1 corepressor, to the EphA2 promoter in WI38 cells. Taken together, our results identify EphA2 as a new direct target gene of HIC1. Finally, we observe that inactivation of endogenous HIC1 through RNA interference in normal breast epithelial cells results in the up-regulation of EphA2 and is correlated with increased cellular migration. To conclude, our results involve the tumor suppressor HIC1 in the transcriptional regulation of the tyrosine kinase receptor EphA2, whose ligand ephrin-A1 is also a HIC1 target gene. Thus, loss of the regulation of this Eph pathway through HIC1 epigenetic silencing could be an important mechanism in the pathogenesis of epithelial cancers. PMID:22184117

Endothelial sirtuin 1 inactivation enhances capillary rarefaction and fibrosis following kidney injury through Notch activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kida, Yujiro; Zullo, Joseph A.; Renal Research Institute, Department of Physiology, New York Medical College, Valhalla, NY

Peritubular capillary (PTC) rarefaction along with tissue fibrosis is a hallmark of chronic kidney disease (CKD). However, molecular mechanisms of PTC loss have been poorly understood. Previous studies have demonstrated that functional loss of endothelial sirtuin 1 (SIRT1) impairs angiogenesis during development and tissue damage. Here, we found that endothelial SIRT1 dysfunction causes activation of endothelial Notch1 signaling, which leads to PTC rarefaction and fibrosis following kidney injury. In mice lacking functional SIRT1 in the endothelium (Sirt1 mutant), kidney injury enhanced apoptosis and senescence of PTC endothelial cells with impaired endothelial proliferation and expanded myofibroblast population and collagen deposition. Comparedmore » to wild-type kidneys, Sirt1 mutant kidneys up-regulated expression of Delta-like 4 (DLL4, a potent Notch1 ligand), Hey1 and Hes1 (Notch target genes), and Notch intracellular domain-1 (NICD1, active form of Notch1) in microvascular endothelial cells (MVECs) post-injury. Sirt1 mutant primary kidney MVECs reduced motility and vascular assembly and enhanced senescence compared to wild-type kidney MVECs. This difference in the phenotype was negated with Notch inhibition. Concurrent stimulation of DLL4 and transforming growth factor (TGF)-β1 increased trans-differentiation of primary kidney pericytes into myofibroblast more than TGF-β1 treatment alone. Collectively, these results indicate that endothelial SIRT1 counteracts PTC rarefaction by repression of Notch1 signaling and antagonizes fibrosis via suppression of endothelial DLL4 expression. - Highlights: • SIRT1 represses Notch1 signaling in capillary endothelial cells in the kidney. • Endothelial SIRT1 is depleted in the kidney following injury. • Activation of endothelial Notch impairs angiogenesis in the kidney. • Increased expression of endothelial DLL4 enhances renal fibrosis.« less

COX-2 expression in papillary thyroid carcinoma (PTC) in cytological material obtained by fine needle aspiration biopsy (FNAB)

PubMed Central

2011-01-01

Background COX-2 is an enzyme isoform that catalyses the formation of prostanoids from arachidonic acid. An increased COX-2 gene expression is believed to participate in carcinogenesis. Recent studies have shown that COX-2 up-regulation is associated with the development of numerous neoplasms, including skin, colorectal, breast, lung, stomach, pancreas and liver cancers. COX-2 products stimulate endothelial cell proliferation and their overexpression has been demonstrated to be involved in the mechanism of decreased resistance to apoptosis. Suppressed angiogenesis was found in experimental animal studies as a consequence of null mutation of COX-2 gene in mice. Despite the role of COX-2 expression remains a subject of numerous studies, its participation in carcinogenesis or the thyroid cancer progression remains unclear. Methods Twenty three (23) patients with cytological diagnosis of PTC were evaluated. After FNAB examination, the needle was washed out with a lysis buffer and the obtained material was used for COX-2 expression estimation. Total RNA was isolated (RNeasy Micro Kit), and RT reactions were performed. β-actin was used as endogenous control. Relative COX-2 expression was assessed in real-time PCR reactions by an ABI PRISM 7500 Sequence Detection System, using the ΔΔCT method. Results COX-2 gene expression was higher in patients with PTC, when compared to specimens from patients with non-toxic nodular goitre (NTG). Conclusions The preliminary results may indicate COX-2 role in thyroid cancer pathogenesis, however the observed variability in results among particular subjects requires additional clinical data and tumor progression analysis. PMID:21214962

COX-2 expression in papillary thyroid carcinoma (PTC) in cytological material obtained by fine needle aspiration biopsy (FNAB).

PubMed

Krawczyk-Rusiecka, Kinga; Wojciechowska-Durczyńska, Katarzyna; Cyniak-Magierska, Anna; Adamczewski, Zbigniew; Gałecka, Elżbieta; Lewiński, Andrzej

2011-01-10

COX-2 is an enzyme isoform that catalyses the formation of prostanoids from arachidonic acid. An increased COX-2 gene expression is believed to participate in carcinogenesis. Recent studies have shown that COX-2 up-regulation is associated with the development of numerous neoplasms, including skin, colorectal, breast, lung, stomach, pancreas and liver cancers. COX-2 products stimulate endothelial cell proliferation and their overexpression has been demonstrated to be involved in the mechanism of decreased resistance to apoptosis. Suppressed angiogenesis was found in experimental animal studies as a consequence of null mutation of COX-2 gene in mice. Despite the role of COX-2 expression remains a subject of numerous studies, its participation in carcinogenesis or the thyroid cancer progression remains unclear. Twenty three (23) patients with cytological diagnosis of PTC were evaluated. After FNAB examination, the needle was washed out with a lysis buffer and the obtained material was used for COX-2 expression estimation. Total RNA was isolated (RNeasy Micro Kit), and RT reactions were performed. β-actin was used as endogenous control. Relative COX-2 expression was assessed in real-time PCR reactions by an ABI PRISM 7500 Sequence Detection System, using the ΔΔCT method. COX-2 gene expression was higher in patients with PTC, when compared to specimens from patients with non-toxic nodular goitre (NTG). The preliminary results may indicate COX-2 role in thyroid cancer pathogenesis, however the observed variability in results among particular subjects requires additional clinical data and tumor progression analysis.

Targeted gene therapy and cell reprogramming in Fanconi anemia.

PubMed

Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

2014-06-01

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Duplicate Maize Wrinkled1 Transcription Factors Activate Target Genes Involved in Seed Oil Biosynthesis1[C][W

PubMed Central

Pouvreau, Benjamin; Baud, Sébastien; Vernoud, Vanessa; Morin, Valérie; Py, Cyrille; Gendrot, Ghislaine; Pichon, Jean-Philippe; Rouster, Jacques; Paul, Wyatt; Rogowsky, Peter M.

2011-01-01

WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs. PMID:21474435

Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

PubMed

Osato, Naoki

2018-01-19

Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional

Long noncoding RNA PVT1 enhances the viability and invasion of papillary thyroid carcinoma cells by functioning as ceRNA of microRNA-30a through mediating expression of insulin like growth factor 1 receptor.

PubMed

Feng, Kun; Liu, Yu; Xu, Li-Juan; Zhao, Ling-Fei; Jia, Chao-Wen; Xu, Ming-Yan

2018-08-01

Invasion and metastasis of papillary thyroid carcinoma (PTC) significantly affects prognosis and quality of life of patients. Herein, we explored the binding relationship of long noncoding RNA PVT1 as ceRNA to microRNA-30a (miR-30a), and their effect on the development of PTC through regulating insulin like growth factor 1 receptor (IGF1R). PTC and adjacent normal tissues were collected, where the qRT-PCR and western blot assay were employed to evaluate the expression levels of PVT1, miR-30a and IGF1R. The correlation between PVT1 expression and clinicopathological characteristics of PTC patients was observed. PTC cell lines with the most/least significant difference from normal thyroid cells were selected and treated with siRNA PVT1 or overexpression PVT1 plasmids, miR-30a mimics or miR-30a inhibitors. Nucleus and cytoplasm segmentation was used to identify subcellular fractionation of PVT1. The binding relationship of PVT1 to miR-30a and the targeting relationship of miR-30a to IGF1R were confirmed by using bioinformatic prediction program, dual-luciferase reporter gene assay and RNA-pull down. Cell viability, cell cycle and apoptosis, invasion and migration capacities were assessed by MTT, flow cytometry, Transwell assay and scratch test, respectively. Western blot assay was employed to examine protein expression of IGF1R, apoptosis-related factors (caspase-3, cleaved capase-3) and epithelial-mesenchymal transition (EMT)-related factors (E-cadherin, Vimentin). In the PTC tissues and cells, PVT1 and IGF1R were highly expressed and miR-30a was poorly expressed. PVT1 exerted its effects on PTC mainly in the cytoplasm. The PVT1 expression was correlated with TNM staging, LNM and tumor infiltration of PTC. The competitive binding of PVT1 to miR-30a enhanced expression of IGF1R. In the in vitro experiments, BCPAP and TPC-1 cells were selected. When subjected to siRNA PVT1 or miR-30a mimics, BCPAP and TPC-1 cells exhibited inhibited proliferation, cell cycle

Targeted gene insertion for molecular medicine.

PubMed

Voigt, Katrin; Izsvák, Zsuzsanna; Ivics, Zoltán

2008-11-01

Genomic insertion of a functional gene together with suitable transcriptional regulatory elements is often required for long-term therapeutical benefit in gene therapy for several genetic diseases. A variety of integrating vectors for gene delivery exist. Some of them exhibit random genomic integration, whereas others have integration preferences based on attributes of the targeted site, such as primary DNA sequence and physical structure of the DNA, or through tethering to certain DNA sequences by host-encoded cellular factors. Uncontrolled genomic insertion bears the risk of the transgene being silenced due to chromosomal position effects, and can lead to genotoxic effects due to mutagenesis of cellular genes. None of the vector systems currently used in either preclinical experiments or clinical trials displays sufficient preferences for target DNA sequences that would ensure appropriate and reliable expression of the transgene and simultaneously prevent hazardous side effects. We review in this paper the advantages and disadvantages of both viral and non-viral gene delivery technologies, discuss mechanisms of target site selection of integrating genetic elements (viruses and transposons), and suggest distinct molecular strategies for targeted gene delivery.

Cyclin D1 in well differentiated thyroid tumour of uncertain malignant potential.

PubMed

Lamba Saini, Monika; Weynand, Birgit; Rahier, Jacques; Mourad, Michel; Hamoir, Marc; Marbaix, Etienne

2015-04-18

Encapsulated follicular tumours with equivocal papillary thyroid carcinoma (PTC) type nuclear features continue to remain a challenge despite the recent attempts to classify these borderline lesions. The term 'well differentiated tumour of uncertain malignant potential (WDT-UMP)' was introduced to classify these tumours. The present study aimed to evaluate the role of a cell cycle regulator like cyclin D1 in these tumours along with assessment of other well established PTC markers like galectin-3, HBME-1, CK19. Thirteen cases of metastatic PTC, papillary microcarcinoma and follicular variant of PTC (FVPTC) were identified from a histological review of 510 cases. In addition, 13 cases of a subset of follicular adenomatoid nodules with focal areas showing nuclear features characteristic of PTC, identified as WDT-UMP, were also analyzed. Immunohistochemical analysis of galectin-3, HBME-1, CK19 and the proliferation markers Ki67 and cyclin D1 was performed. Lesions were analyzed for cyclin D1 gene amplification by fluorescent in-situ hybridization. All WDT-UMP lesions showed immunolabelling of cyclin D1, Ki67; 11/ 13 cases showed immunolabelling of CK19; 10/13 cases showed immunolabelling of HBME-1 and 4/13 cases showed immunolabelling of galectin-3. Surrounding benign adenomatoid areas showed no to faint focal staining in all thirteen cases of cyclin D1, HBME-1 and galectin-3. A low rate of cyclin D1 gene amplification was identified in a significant proportion of cells in the WDT-UMP lesions as compared to surrounding benign adenomatoid areas. Increased expression of cyclin D1 and amplification of its gene along with immunolabelling of HBME-1 in WDT-UMP lesions showing cytological features of papillary thyroid carcinoma within follicular adenomatoid nodules suggest that these areas could correspond to a precursor lesion of follicular variant of PTC. Overexpression of cyclin D1, associated with the amplification of the gene suggests that these WDT-UMP lesions are an

49 CFR 236.1006 - Equipping locomotives operating in PTC territory.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 31, 2015, a train controlled by a locomotive with an onboard PTC apparatus that has failed en route... III railroad, including a tourist or excursion railroad, and controlled by a locomotive not equipped...

Endoplasmic Reticulum Stress in Mice Increases Hepatic Expression of Genes Carrying a Premature Termination Codon via a Nutritional Status-Independent GRP78-Dependent Mechanism.

PubMed

Harada, Nagakatsu; Okuyama, Maiko; Yoshikatsu, Aya; Yamamoto, Hironori; Ishiwata, Saori; Hamada, Chikako; Hirose, Tomoyo; Shono, Masayuki; Kuroda, Masashi; Tsutsumi, Rie; Takeo, Jiro; Taketani, Yutaka; Nakaya, Yutaka; Sakaue, Hiroshi

2017-11-01

Nonsense-mediated mRNA decay (NMD) degrades mRNAs carrying a premature termination codon (PTC) in eukaryotes. Cellular stresses, including endoplasmic reticulum (ER) stress, inhibit NMD, and up-regulate PTC-containing mRNA (PTC-mRNA) levels in several cell lines. However, whether similar effects exist under in vivo conditions that involve systemic nutritional status is unclear. Here, we compared the effects of pharmacological induction of ER stress with those of nutritional interventions on hepatic PTC-mRNA levels in mice. In mouse livers, the ER stress inducer tunicamycin increased PTC-mRNA levels of endogenous marker genes. Tunicamycin decreased body weight and perturbed nutrient metabolism in mice. Food restriction or deprivation mimicked the effect of tunicamycin on weight loss and metabolism, but did not increase PTC-mRNA levels. Hyperphagia-induced obesity also had little effect on hepatic PTC-mRNA levels. Meanwhile, in mouse liver phosphorylation of eIF2α, a factor that regulates NMD, was increased by both tunicamycin and nutritional interventions. Hepatic expression of GRP78, a central chaperone in ER stress responses, was increased by tunicamycin but not by the nutritional interventions. In cultured liver cells (Hepa), exogenous overexpression of a phosphomimetic eIF2α failed to increase PTC-mRNA levels. However, GRP78 overexpression in Hepa cells increased PTC-mRNA and PTC-mRNA-derived protein levels. ER stress promoted localization of GRP78 to mitochondria, and exogenous expression of a GRP78 fusion protein targeted to mitochondria mimicked the effect of wild type GRP78. These results indicate that GRP78, but not nutritional status, is a potent up-regulator of hepatic PTC-mRNA levels during induction of ER stress in vivo. J. Cell. Biochem. 118: 3810-3824, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Towards β-globin gene-targeting with integrase-defective lentiviral vectors.

PubMed

Inanlou, Davoud Nouri; Yakhchali, Bagher; Khanahmad, Hossein; Gardaneh, Mossa; Movassagh, Hesam; Cohan, Reza Ahangari; Ardestani, Mehdi Shafiee; Mahdian, Reza; Zeinali, Sirous

2010-11-01

We have developed an integrase-defective lentiviral (LV) vector in combination with a gene-targeting approach for gene therapy of β-thalassemia. The β-globin gene-targeting construct has two homologous stems including sequence upstream and downstream of the β-globin gene, a β-globin gene positioned between hygromycin and neomycin resistant genes and a herpes simplex virus type 1 thymidine kinase (HSVtk) suicide gene. Utilization of integrase-defective LV as a vector for the β-globin gene increased the number of selected clones relative to non-viral methods. This method represents an important step toward the ultimate goal of a clinical gene therapy for β-thalassemia.

Genomic identification of direct target genes of LEAFY

PubMed Central

William, Dilusha A.; Su, Yanhui; Smith, Michael R.; Lu, Meina; Baldwin, Don A.; Wagner, Doris

2004-01-01

The switch from vegetative to reproductive development in plants necessitates a switch in the developmental program of the descendents of the stem cells in the shoot apical meristem. Genetic and molecular investigations have demonstrated that the plant-specific transcription factor and meristem identity regulator LEAFY (LFY) controls this developmental transition by inducing expression of a second transcription factor, APETALA1, and by regulating the expression of additional, as yet unknown, genes. Here we show that the additional LFY targets include the APETALA1-related factor, CAULI-FLOWER, as well as three transcription factors and two putative signal transduction pathway components. These genes are up-regulated by LFY even when protein synthesis is inhibited and, hence, appear to be direct targets of LFY. Supporting this conclusion, cis-regulatory regions upstream of these genes are bound by LFY in vivo. The newly identified LFY targets likely initiate the transcriptional changes that are required for the switch from vegetative to reproductive development in Arabidopsis. PMID:14736918

[Expression of miR-21 and Its Acat1, Armcx1, and Pten Target Genes in Liver of Female Rats Treated with DDT and Benzo[a]pyrene].

PubMed

Chanyshev, M D; Ushakov, D S; Gulyaeva, L F

2017-01-01

MiR-21 is the most studied cancer-promoting oncomiR, which target numerous tumor suppressor genes associated with proliferation, apoptosis, and invasion. Here we have studied the synthesis of miR-21 and quantified the mRNA and protein levels for miR-21 potential target genes, i.e., Acat1, Armcx1, and Pten, in the livers of female Wistar rats after their treatment with either 1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) or benzo[a]pyrene (BP). The most important finding appears to be the significant decrease in the miR-21 level the day after treatment with DDT with subsequent rebound. These changes are accompanied by an increase and subsequent drop in the levels of mRNAs and proteins of the Acat1, Armcx1, and Pten genes. These observations indicate the involvement of miR-21 in the posttranscriptional regulation of the Acat1, Armcx1, and Pten genes in response to xenobiotics. We hypothesize that the toxic effects of xenobiotics may be indirect and may manifest by inducing epigenetic changes, particularly through the regulation of miRNAs and their target genes.

49 CFR 236.1011 - PTC Implementation Plan content requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... the following risk factors by track segment: (i) Segment traffic characteristics such as typical...; (4) How, to the extent practical, the PTC system will be implemented to address areas of greater risk to the public and railroad employees before areas of lesser risk; (5) The sequence and schedule in...

ChIP-Chip Identifies SEC23A, CFDP1, and NSD1 as TFII-I Target Genes in Human Neural Crest Progenitor Cells.

PubMed

Makeyev, Aleksandr V; Bayarsaihan, Dashzeveg

2013-05-01

Objectives :  GTF2I and GTF2IRD1 genes located in Williams-Beuren syndrome (WBS) critical region encode TFII-I family transcription factors. The aim of this study was to map genomic sites bound by these proteins across promoter regions of developmental regulators associated with craniofacial development. Design :  Chromatin was isolated from human neural crest progenitor cells and the DNA-binding profile was generated using the human RefSeq tiling promoter ChIP-chip arrays. Results :  TFII-I transcription factors are recruited to the promoters of SEC23A, CFDP1, and NSD1 previously defined as TFII-I target genes. Moreover, our analysis revealed additional binding elements that contain E-boxes and initiator-like motifs. Conclusions :  Genome-wide promoter binding studies revealed SEC23A, CFDP1, and NSD1 linked to craniofacial or dental development as direct TFII-I targets. Developmental regulation of these genes by TFII-I factors could contribute to the WBS-specific facial dysmorphism.

Targeting gene therapy to cancer: a review.

PubMed

Dachs, G U; Dougherty, G J; Stratford, I J; Chaplin, D J

1997-01-01

In recent years the idea of using gene therapy as a modality in the treatment of diseases other than genetically inherited, monogenic disorders has taken root. This is particularly obvious in the field of oncology where currently more than 100 clinical trials have been approved worldwide. This report will summarize some of the exciting progress that has recently been made with respect to both targeting the delivery of potentially therapeutic genes to tumor sites and regulating their expression within the tumor microenvironment. In order to specifically target malignant cells while at the same time sparing normal tissue, cancer gene therapy will need to combine highly selective gene delivery with highly specific gene expression, specific gene product activity, and, possibly, specific drug activation. Although the efficient delivery of DNA to tumor sites remains a formidable task, progress has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods. In this report emphasis will be placed on targeted rather than high-efficiency delivery, although those would need to be combined in the future for effective therapy. To date delivery has been targeted to tumor-specific and tissue-specific antigens, such as epithelial growth factor receptor, c-kit receptor, and folate receptor, and these will be described in some detail. To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue. Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements. Another novel possibility that will be

Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

PubMed Central

Tan, Siyuan; Guschin, Dmitry; Davalos, Albert; Lee, Ya-Li; Snowden, Andrew W.; Jouvenot, Yann; Zhang, H. Steven; Howes, Katherine; McNamara, Andrew R.; Lai, Albert; Ullman, Chris; Reynolds, Lindsey; Moore, Michael; Isalan, Mark; Berg, Lutz-Peter; Campos, Bradley; Qi, Hong; Spratt, S. Kaye; Case, Casey C.; Pabo, Carl O.; Campisi, Judith; Gregory, Philip D.

2003-01-01

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics. PMID:14514889

Differential Effects of HNF-1α Mutations Associated with Familial Young-Onset Diabetes on Target Gene Regulation

PubMed Central

Galán, Maria; García-Herrero, Carmen-Maria; Azriel, Sharona; Gargallo, Manuel; Durán, Maria; Gorgojo, Juan-Jose; Andía, Victor-Manuel; Navas, Maria-Angeles

2011-01-01

Hepatocyte nuclear factor 1-α (HNF-1α) is a homeodomain transcription factor expressed in a variety of tissues (including liver and pancreas) that regulates a wide range of genes. Heterozygous mutations in the gene encoding HNF-1α (HNF1A) cause familial young-onset diabetes, also known as maturity-onset diabetes of the young, type 3 (MODY3). The variability of the MODY3 clinical phenotype can be due to environmental and genetic factors as well as to the type and position of mutations. Thus, functional characterization of HNF1A mutations might provide insight into the molecular defects explaining the variability of the MODY3 phenotype. We have functionally characterized six HNF1A mutations identified in diabetic patients: two novel ones, p.Glu235Gly and c-57-64delCACGCGGT;c-55G>C; and four previously described, p.Val133Met, p.Thr196Ala, p.Arg271Trp and p.Pro379Arg. The effects of mutations on transcriptional activity have been measured by reporter assays on a subset of HNF-1α target promoters in Cos7 and Min6 cells. Target DNA binding affinities have been quantified by electrophoretic mobility shift assay using bacterially expressed glutathione-S-transferase (GST)-HNF-1α fusion proteins and nuclear extracts of transfected Cos7 cells. Our functional studies revealed that mutation c-57-64delCACGCGGT;c-55G>C reduces HNF1A promoter activity in Min6 cells and that missense mutations have variable effects. Mutation p.Arg271Trp impairs HNF-1α activity in all conditions tested, whereas mutations p.Val133Met, p.Glu235Gly and p.Pro379Arg exert differential effects depending on the target promoter. In contrast, substitution p.Thr196Ala does not appear to alter HNF-1α function. Our results suggest that HNF1A mutations may have differential effects on the regulation of specific target genes, which could contribute to the variability of the MODY3 clinical phenotype. PMID:21170474

Dynamics of 1α,25-dihydroxyvitamin D3-dependent chromatin accessibility of early vitamin D receptor target genes.

PubMed

Seuter, Sabine; Pehkonen, Petri; Heikkinen, Sami; Carlberg, Carsten

2013-12-01

The signaling cascade of the transcription factor vitamin D receptor (VDR) is triggered by its specific ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study we demonstrate that in THP-1 human monocytic leukemia cells 87.4% of the 1034 most prominent genome-wide VDR binding sites co-localize with loci of open chromatin. At 165 of them 1α,25(OH)2D3 strongly increases chromatin accessibility and has at further 217 sites weaker effects. Interestingly, VDR binding sites in 1α,25(OH)2D3-responsive chromatin regions are far more often composed of direct repeats with 3 intervening nucleotides (DR3s) than those in ligand insensitive regions. DR3-containing VDR sites are enriched in the neighborhood of genes that are involved in controling cellular growth, while non-DR3 VDR binding is often found close to genes related to immunity. At the example of six early VDR target genes we show that the slope of their 1α,25(OH)2D3-induced transcription correlates with the basal chromatin accessibility of their major VDR binding regions. However, the chromatin loci controlling these genes are indistinguishable in their VDR association kinetics. Taken together, ligand responsive chromatin loci represent dynamically regulated contact points of VDR with the genome, from where it controls early 1α,25(OH)2D3 target genes. © 2013.

Artificial Neural Network Modeling of Pt/C Cathode Degradation in PEM Fuel Cells

NASA Astrophysics Data System (ADS)

Maleki, Erfan; Maleki, Nasim

2016-08-01

Use of computational modeling with a few experiments is considered useful to obtain the best possible result for a final product, without performing expensive and time-consuming experiments. Proton exchange membrane fuel cells (PEMFCs) can produce clean electricity, but still require further study. An oxygen reduction reaction (ORR) takes place at the cathode, and carbon-supported platinum (Pt/C) is commonly used as an electrocatalyst. The harsh conditions during PEMFC operation result in Pt/C degradation. Observation of changes in the Pt/C layer under operating conditions provides a tool to study the lifetime of PEMFCs and overcome durability issues. Recently, artificial neural networks (ANNs) have been used to solve, predict, and optimize a wide range of scientific problems. In this study, several rates of change at the cathode were modeled using ANNs. The backpropagation (BP) algorithm was used to train the network, and experimental data were employed for network training and testing. Two different models are constructed in the present study. First, the potential cycles, temperature, and humidity are used as inputs to predict the resulting Pt dissolution rate of the Pt/C at the cathode as the output parameter of the network. Thereafter, the Pt dissolution rate and Pt ion diffusivity are regarded as inputs to obtain values of the Pt particle radius change rate, Pt mass loss rate, and surface area loss rate as outputs. The networks are finely tuned, and the modeling results agree well with experimental data. The modeled responses of the ANNs are acceptable for this application.

Targeted polymeric nanoparticles for cancer gene therapy

PubMed Central

Kim, Jayoung; Wilson, David R.; Zamboni, Camila G.; Green, Jordan J.

2015-01-01

In this article, advances in designing polymeric nanoparticles for targeted cancer gene therapy are reviewed. Characterization and evaluation of biomaterials, targeting ligands, and transcriptional elements are each discussed. Advances in biomaterials have driven improvements to nanoparticle stability and tissue targeting, conjugation of ligands to the surface of polymeric nanoparticles enable binding to specific cancer cells, and the design of transcriptional elements has enabled selective DNA expression specific to the cancer cells. Together, these features have improved the performance of polymeric nanoparticles as targeted non-viral gene delivery vectors to treat cancer. As polymeric nanoparticles can be designed to be biodegradable, non-toxic, and to have reduced immunogenicity and tumorigenicity compared to viral platforms, they have significant potential for clinical use. Results of polymeric gene therapy in clinical trials and future directions for the engineering of nanoparticle systems for targeted cancer gene therapy are also presented. PMID:26061296

In papillary thyroid carcinoma, expression by immunohistochemistry of BRAF V600E, PD-L1, and PD-1 is closely related.

PubMed

Bai, Yanhua; Guo, Ting; Huang, Xiaozheng; Wu, Qi; Niu, Dongfeng; Ji, Xinqiang; Feng, Qin; Li, Zhongwu; Kakudo, Kennichi

2018-05-01

Immune checkpoint inhibitor therapies targeting PD-L1/PD-1 have been shown to be effective in treating several types of human cancer. In papillary thyroid carcinoma (PTC), little is known about the expression of PD-L1/PD-1 in the tumor microenvironment or its potential correlation with BRAF V600E mutation status. In this study, we examined the expression of PD-L1, PD-1, and BRAF V600E in PTC by immunohistochemistry and investigated the clinical significance of expression status. We studied the expression of PD-L1, PD-1, and BRAF V600E by immunohistochemical staining in 110 cases of PTC with a diameter > 1 cm. Cases with a background of chronic lymphocytic thyroiditis (CLT) were excluded, as differentiating lymphocytes in the context of CLT from tumor-infiltrating lymphocytes (TILs) is difficult. We classified PD-L1+/PD-1+ expression as type 1 (41%), PD-L1-/PD-1- as type 2 (17%), PD-L1+/PD-1- as type 3 (5%), and PD-L1-/PD-1+ as type 4 (37%). Significant correlations were found between expression of BRAF V600E and that of PD-L1 and PD-1. The positive correlation observed between expression of BRAF V600E and PD-L1/PD-1 suggests that immunotherapies targeting PD-L1/PD-1 might be effective for PTC patients with the BRAF V600E mutation, which are refractory to radioiodine therapy.

Gene Therapy by Targeted Adenovirus-mediated Knockdown of Pulmonary Endothelial Tph1 Attenuates Hypoxia-induced Pulmonary Hypertension

PubMed Central

Morecroft, Ian; White, Katie; Caruso, Paola; Nilsen, Margaret; Loughlin, Lynn; Alba, Raul; Reynolds, Paul N; Danilov, Sergei M; Baker, Andrew H; MacLean, Margaret R

2012-01-01

Serotonin is produced by pulmonary arterial endothelial cells (PAEC) via tryptophan hydroxylase-1 (Tph1). Pathologically, serotonin acts on underlying pulmonary arterial cells, contributing to vascular remodeling associated with pulmonary arterial hypertension (PAH). The effects of hypoxia on PAEC-Tph1 activity are unknown. We investigated the potential of a gene therapy approach to PAH using selective inhibition of PAEC-Tph1 in vivo in a hypoxic model of PAH. We exposed cultured bovine pulmonary arterial smooth muscle cells (bPASMCs) to conditioned media from human PAECs (hPAECs) before and after hypoxic exposure. Serotonin levels were increased in hypoxic PAEC media. Conditioned media evoked bPASMC proliferation, which was greater with hypoxic PAEC media, via a serotonin-dependent mechanism. In vivo, adenoviral vectors targeted to PAECs (utilizing bispecific antibody to angiotensin-converting enzyme (ACE) as the selective targeting system) were used to deliver small hairpin Tph1 RNA sequences in rats. Hypoxic rats developed PAH and increased lung Tph1. PAEC-Tph1 expression and development of PAH were attenuated by our PAEC-Tph1 gene knockdown strategy. These results demonstrate that hypoxia induces Tph1 activity and selective knockdown of PAEC-Tph1 attenuates hypoxia-induced PAH in rats. Further investigation of pulmonary endothelial-specific Tph1 inhibition via gene interventions is warranted. PMID:22525513

Ultrasound assisted PTC catalyzed saponification of vegetable oils using aqueous alkali.

PubMed

Bhatkhande, B S; Samant, S D

1998-03-01

A few vegetable oils were saponified using aqueous KOH and different PTCs at room temperature in the presence of ultrasound. The extent of saponification was studied using the saponification value as a reference. Optimizations of various parameters such as time, selection of PTC, quantity of PTC, quantity of KOH and quantity of water were carried out using soyabean oil as a sample oil under sonication with stirring. To study the effect of ultrasound, the saponification was also carried out at 35 +/- 2 degrees C under different conditions, namely stirring, sonication, stirring and sonication, and heating at 100 degrees C. It was found that the heterogeneous liquid-liquid phase saponification of different vegetable oils using aq. KOH/CTAB was remarkably accelerated at 35 +/- 2 degrees C in the presence of ultrasound along with stirring.

Gene targeting in mosquito cells: a demonstration of 'knockout' technology in extrachromosomal gene arrays

PubMed Central

Eggleston, Paul; Zhao, Yuguang

2001-01-01

Background Gene targeting would offer a number of advantages over current transposon-based strategies for insect transformation. These include freedom from both position effects associated with quasi-random integration and concerns over transgene instability mediated by endogenous transposases, independence from phylogenetic restrictions on transposon mobility and the ability to generate gene knockouts. Results We describe here our initial investigations of gene targeting in the mosquito. The target site was a hygromycin resistance gene, stably maintained as part of an extrachromosomal array. Using a promoter-trap strategy to enrich for targeted events, a neomycin resistance gene was integrated into the target site. This resulted in knockout of hygromycin resistance concurrent with the expression of high levels of neomycin resistance from the resident promoter. PCR amplification of the targeted site generated a product that was specific to the targeted cell line and consistent with precise integration of the neomycin resistance gene into the 5' end of the hygromycin resistance gene. Sequencing of the PCR product and Southern analysis of cellular DNA subsequently confirmed this molecular structure. Conclusions These experiments provide the first demonstration of gene targeting in mosquito tissue and show that mosquito cells possess the necessary machinery to bring about precise integration of exogenous sequences through homologous recombination. Further development of these procedures and their extension to chromosomally located targets hold much promise for the exploitation of gene targeting in a wide range of medically and economically important insect species. PMID:11513755

Swift heavy ion irradiation effects in Pt/C and Ni/C multilayers

NASA Astrophysics Data System (ADS)

Gupta, Ajay; Pandita, Suneel; Avasthi, D. K.; Lodha, G. S.; Nandedkar, R. V.

1998-12-01

Irradiation effects of 100 MeV Ag ion irradiation on Ni/C and Pt/C multilayers have been studied using X-ray reflectivity measurements. Modifications are observed in both the multilayers at (dE/dx)e values much below the threshold values for Ni and Pt. This effect is attributed to the discontinuous nature of the metal layers. In both the multilayers interfacial roughness increases with irradiation dose. While Ni/C multilayers exhibit large ion-beam induced intermixing, no observable intermixing is observed in the case of Pt/C multilayer. This difference in the behavior of the two systems suggests a significant role for chemically guided defect motion in the mixing process associated with swift heavy ion irradiation.

A targeted resequencing gene panel for focal epilepsy.

PubMed

Hildebrand, Michael S; Myers, Candace T; Carvill, Gemma L; Regan, Brigid M; Damiano, John A; Mullen, Saul A; Newton, Mark R; Nair, Umesh; Gazina, Elena V; Milligan, Carol J; Reid, Christopher A; Petrou, Steven; Scheffer, Ingrid E; Berkovic, Samuel F; Mefford, Heather C

2016-04-26

We report development of a targeted resequencing gene panel for focal epilepsy, the most prevalent phenotypic group of the epilepsies. The targeted resequencing gene panel was designed using molecular inversion probe (MIP) capture technology and sequenced using massively parallel Illumina sequencing. We demonstrated proof of principle that mutations can be detected in 4 previously genotyped focal epilepsy cases. We searched for both germline and somatic mutations in 251 patients with unsolved sporadic or familial focal epilepsy and identified 11 novel or very rare missense variants in 5 different genes: CHRNA4, GRIN2B, KCNT1, PCDH19, and SCN1A. Of these, 2 were predicted to be pathogenic or likely pathogenic, explaining ∼0.8% of the cohort, and 8 were of uncertain significance based on available data. We have developed and validated a targeted resequencing panel for focal epilepsies, the most important clinical class of epilepsies, accounting for about 60% of all cases. Our application of MIP technology is an innovative approach that will be advantageous in the clinical setting because it is highly sensitive, efficient, and cost-effective for screening large patient cohorts. Our findings indicate that mutations in known genes likely explain only a small proportion of focal epilepsy cases. This is not surprising given the established clinical and genetic heterogeneity of these disorders and underscores the importance of further gene discovery studies in this complex syndrome. © 2016 American Academy of Neurology.

A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast

DTIC Science & Technology

2004-05-01

AD Award Number: DAMD17-03-1-0232 TITLE: A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast PRINCIPAL INVESTIGATOR...Approach to Identify Novel Breast DAMD17-03-1-0232 Cancer Gene Targets in Yeast 6. A UTHOR(S) Craig Bennett, Ph.D. 7. PERFORMING ORGANIZA TION NAME(S...Unlimited 13. ABSTRACT (Maximum 200 Words) We are using the yeast Saccharomyces cerevisiae to identify new cancer gene targets that interact with the

The Mechanism of Gene Targeting in Human Somatic Cells

PubMed Central

Kan, Yinan; Ruis, Brian; Lin, Sherry; Hendrickson, Eric A.

2014-01-01

Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB) repair known as homologous recombination (HR). The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells. PMID:24699519

Simple Monitoring of Gene Targeting Efficiency in Human Somatic Cell Lines Using the PIGA Gene

PubMed Central

Karnan, Sivasundaram; Konishi, Yuko; Ota, Akinobu; Takahashi, Miyuki; Damdindorj, Lkhagvasuren; Hosokawa, Yoshitaka; Konishi, Hiroyuki

2012-01-01

Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines. PMID:23056640

Identification of STAT target genes in adipocytes

PubMed Central

Zhao, Peng; Stephens, Jacqueline M.

2013-01-01

Adipocytes play important roles in lipid storage, energy homeostasis and whole body insulin sensitivity. Studies in the last two decades have identified the hormones and cytokines that activate specific STATs in adipocytes in vitro and in vivo. Five of the seven STAT family members are expressed in adipocyte (STATs 1, 3, 5A, 5B and 6). Many transcription factors, including STATs, have been shown to play an important role in adipose tissue development and function. This review will summarize the importance of adipocytes, indicate the cytokines and hormones that utilize the JAK-STAT signaling pathway in fat cells and focus on the identification of STAT target genes in mature adipocytes. To date, specific target genes have been identified for STATs, 1, 5A and 5B, but not for STATs 3 and 6. PMID:24058802

Combined serial analysis of gene expression and transcription factor binding site prediction identifies novel-candidate-target genes of Nr2e1 in neocortex development.

PubMed

Schmouth, Jean-François; Arenillas, David; Corso-Díaz, Ximena; Xie, Yuan-Yun; Bohacec, Slavita; Banks, Kathleen G; Bonaguro, Russell J; Wong, Siaw H; Jones, Steven J M; Marra, Marco A; Simpson, Elizabeth M; Wasserman, Wyeth W

2015-07-24

Nr2e1 (nuclear receptor subfamily 2, group e, member 1) encodes a transcription factor important in neocortex development. Previous work has shown that nuclear receptors can have hundreds of target genes, and bind more than 300 co-interacting proteins. However, recognition of the critical role of Nr2e1 in neural stem cells and neocortex development is relatively recent, thus the molecular mechanisms involved for this nuclear receptor are only beginning to be understood. Serial analysis of gene expression (SAGE), has given researchers both qualitative and quantitative information pertaining to biological processes. Thus, in this work, six LongSAGE mouse libraries were generated from laser microdissected tissue samples of dorsal VZ/SVZ (ventricular zone and subventricular zone) from the telencephalon of wild-type (Wt) and Nr2e1-null embryos at the critical development ages E13.5, E15.5, and E17.5. We then used a novel approach, implementing multiple computational methods followed by biological validation to further our understanding of Nr2e1 in neocortex development. In this work, we have generated a list of 1279 genes that are differentially expressed in response to altered Nr2e1 expression during in vivo neocortex development. We have refined this list to 64 candidate direct-targets of NR2E1. Our data suggested distinct roles for Nr2e1 during different neocortex developmental stages. Most importantly, our results suggest a possible novel pathway by which Nr2e1 regulates neurogenesis, which includes Lhx2 as one of the candidate direct-target genes, and SOX9 as a co-interactor. In conclusion, we have provided new candidate interacting partners and numerous well-developed testable hypotheses for understanding the pathways by which Nr2e1 functions to regulate neocortex development.

EBF factors drive expression of multiple classes of target genes governing neuronal development.

PubMed

Green, Yangsook S; Vetter, Monica L

2011-04-30

Early B cell factor (EBF) family members are transcription factors known to have important roles in several aspects of vertebrate neurogenesis, including commitment, migration and differentiation. Knowledge of how EBF family members contribute to neurogenesis is limited by a lack of detailed understanding of genes that are transcriptionally regulated by these factors. We performed a microarray screen in Xenopus animal caps to search for targets of EBF transcriptional activity, and identified candidate targets with multiple roles, including transcription factors of several classes. We determined that, among the most upregulated candidate genes with expected neuronal functions, most require EBF activity for some or all of their expression, and most have overlapping expression with ebf genes. We also found that the candidate target genes that had the most strongly overlapping expression patterns with ebf genes were predicted to be direct transcriptional targets of EBF transcriptional activity. The identification of candidate targets that are transcription factor genes, including nscl-1, emx1 and aml1, improves our understanding of how EBF proteins participate in the hierarchy of transcription control during neuronal development, and suggests novel mechanisms by which EBF activity promotes migration and differentiation. Other candidate targets, including pcdh8 and kcnk5, expand our knowledge of the types of terminal differentiated neuronal functions that EBF proteins regulate.

Gene-carried hepatoma targeting complex induced high gene transfection efficiency with low toxicity and significant antitumor activity.

PubMed

Zhao, Qing-Qing; Hu, Yu-Lan; Zhou, Yang; Li, Ni; Han, Min; Tang, Gu-Ping; Qiu, Feng; Tabata, Yasuhiko; Gao, Jian-Qing

2012-01-01

The success of gene transfection is largely dependent on the development of a vehicle or vector that can efficiently deliver a gene to cells with minimal toxicity. A liver cancer-targeted specific peptide (FQHPSF sequence) was successfully synthesized and linked with chitosan-linked polyethylenimine (CP) to form a new targeted gene delivery vector called CPT (CP/peptide). The structure of CPT was confirmed by (1)H nuclear magnetic resonance spectroscopy and ultraviolet spectrophotometry. The particle size of CPT/ DNA complexes was measured using laser diffraction spectrometry and the cytotoxicity of the copolymer was evaluated by methylthiazol tetrazolium method. The transfection efficiency evaluation of the CP copolymer was performed using luciferase activity assay. Cellular internalization of the CP/DNA complex was observed under confocal laser scanning microscopy. The targeting specificity of the polymer coupled to peptide was measured by competitive inhibition transfection study. The liver targeting specificity of the CPT copolymer in vivo was demonstrated by combining the copolymer with a therapeutic gene, interleukin-12, and assessed by its abilities in suppressing the growth of ascites tumor in mouse model. The results showed that the liver cancer-targeted specific peptide was successfully synthesized and linked with CP to form a new targeted gene delivery vector called CPT. The composition of CPT was confirmed and the vector showed low cytotoxicity and strong targeting specificity to liver tumors in vitro. The in vivo study results showed that interleukin-12 delivered by the new gene vector CPT/DNA significantly enhanced the antitumor effect on ascites tumor-bearing imprinting control region mice as compared with polyethylenimine (25 kDa), CP, and other controls, which further demonstrate the targeting specificity of the new synthesized polymer. The synthesized CPT copolymer was proven to be an effective liver cancer-targeted vector for therapeutic gene

Expression profiling identifies novel Hh/Gli regulated genes in developing zebrafish embryos.

PubMed Central

Bergeron, Sadie A.; Milla, Luis A.; Villegas, Rosario; Shen, Meng-Chieh; Burgess, Shawn M.; Allende, Miguel L.; Karlstrom, Rolf O.; Palma, Verónica

2008-01-01

The Hedgehog (Hh) signaling pathway plays critical instructional roles during embryonic development. Mis-regulation of Hh/Gli signaling is a major causative factor in human congenital disorders and in a variety of cancers. The zebrafish is a powerful genetic model for the study of Hh signaling during embryogenesis, as a large number of mutants have been identified affecting different components of the Hh/Gli signaling system. By performing global profiling of gene expression in different Hh/Gli gain- and loss-of-function scenarios we identified several known (e.g. ptc1 and nkx2.2a) as well as a large number of novel Hh regulated genes that are differentially expressed in embryos with altered Hh/Gli signaling function. By uncovering changes in tissue specific gene expression, we revealed new embryological processes that are influenced by Hh signaling. We thus provide a comprehensive survey of Hh/Gli regulated genes during embryogenesis and we identify new Hh-regulated genes that may be targets of mis-regulation during tumorogenesis. PMID:18055165

Study on the PTC/NTC effect of carbon black-filled polymer composites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Hao; Chen, Xinfang; Luo, Yunxia

1995-12-01

In this work, the effect of processing condition and radiation-crosslinking on the electrical and dynamic behaviors of carbon black filled low density polyethylene (LDPE) composites were investigated. Compared with the solution counterpart, the mechanical composites have a strong PTC effect and a great dynamic elastic mold, which results from the strong interaction between carbon black and LDPE. The experiment result shows that the NTC effect is caused by the decrease of elastic mold of LDPE at high temperature, and it can be declined significantly by radiation-crosslinking. We conclude that the strong interaction between polymer and carbon black is essentially importantmore » for composites to have a great PTC intensity good electrical reproducibility and high dynamic elastic sold.« less

Targeted gene flow for conservation.

PubMed

Kelly, Ella; Phillips, Ben L

2016-04-01

Anthropogenic threats often impose strong selection on affected populations, causing rapid evolutionary responses. Unfortunately, these adaptive responses are rarely harnessed for conservation. We suggest that conservation managers pay close attention to adaptive processes and geographic variation, with an eye to using them for conservation goals. Translocating pre-adapted individuals into recipient populations is currently considered a potentially important management tool in the face of climate change. Targeted gene flow, which involves moving individuals with favorable traits to areas where these traits would have a conservation benefit, could have a much broader application in conservation. Across a species' range there may be long-standing geographic variation in traits or variation may have rapidly developed in response to a threatening process. Targeted gene flow could be used to promote natural resistance to threats to increase species resilience. We suggest that targeted gene flow is a currently underappreciated strategy in conservation that has applications ranging from the management of invasive species and their impacts to controlling the impact and virulence of pathogens. © 2015 Society for Conservation Biology.

Bacteriophage-Derived Vectors for Targeted Cancer Gene Therapy

PubMed Central

Pranjol, Md Zahidul Islam; Hajitou, Amin

2015-01-01

Cancer gene therapy expanded and reached its pinnacle in research in the last decade. Both viral and non-viral vectors have entered clinical trials, and significant successes have been achieved. However, a systemic administration of a vector, illustrating safe, efficient, and targeted gene delivery to solid tumors has proven to be a major challenge. In this review, we summarize the current progress and challenges in the targeted gene therapy of cancer. Moreover, we highlight the recent developments of bacteriophage-derived vectors and their contributions in targeting cancer with therapeutic genes following systemic administration. PMID:25606974

Bacteriophage-derived vectors for targeted cancer gene therapy.

PubMed

Pranjol, Md Zahidul Islam; Hajitou, Amin

2015-01-19

Cancer gene therapy expanded and reached its pinnacle in research in the last decade. Both viral and non-viral vectors have entered clinical trials, and significant successes have been achieved. However, a systemic administration of a vector, illustrating safe, efficient, and targeted gene delivery to solid tumors has proven to be a major challenge. In this review, we summarize the current progress and challenges in the targeted gene therapy of cancer. Moreover, we highlight the recent developments of bacteriophage-derived vectors and their contributions in targeting cancer with therapeutic genes following systemic administration.

Control of aflatoxin production of Aspergillus flavus and Aspergillus parasiticus using RNA silencing technology by targeting aflD (nor-1) gene.

PubMed

Abdel-Hadi, Ahmed M; Caley, Daniel P; Carter, David R F; Magan, Naresh

2011-06-01

Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B(1 )(AFB(1)), and aflatoxin G(1) (AFG(1)) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB(1) production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB(1 )production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB(1) production by A. flavus EGP9 and AFG(1 )production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG(1) production by A. parasiticus SSWT 2999. Changes in AFB(1) production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.

Gene Therapy and Targeted Toxins for Glioma

PubMed Central

Castro, Maria G.; Candolfi, Marianela; Kroeger, Kurt; King, Gwendalyn D.; Curtin, James F.; Yagiz, Kader; Mineharu, Yohei; Assi, Hikmat; Wibowo, Mia; Muhammad, AKM Ghulam; Foulad, David; Puntel, Mariana; Lowenstein, Pedro R.

2011-01-01

The most common primary brain tumor in adults is glioblastoma. These tumors are highly invasive and aggressive with a mean survival time of nine to twelve months from diagnosis to death. Current treatment modalities are unable to significantly prolong survival in patients diagnosed with glioblastoma. As such, glioma is an attractive target for developing novel therapeutic approaches utilizing gene therapy. This review will examine the available preclinical models for glioma including xenographs, syngeneic and genetic models. Several promising therapeutic targets are currently being pursued in pre-clinical investigations. These targets will be reviewed by mechanism of action, i.e., conditional cytotoxic, targeted toxins, oncolytic viruses, tumor suppressors/oncogenes, and immune stimulatory approaches. Preclinical gene therapy paradigms aim to determine which strategies will provide rapid tumor regression and long-term protection from recurrence. While a wide range of potential targets are being investigated preclinically, only the most efficacious are further transitioned into clinical trial paradigms. Clinical trials reported to date are summarized including results from conditionally cytotoxic, targeted toxins, oncolytic viruses and oncogene targeting approaches. Clinical trial results have not been as robust as preclinical models predicted; this could be due to the limitations of the GBM models employed. Once this is addressed, and we develop effective gene therapies in models that better replicate the clinical scenario, gene therapy will provide a powerful approach to treat and manage brain tumors. PMID:21453286

Utilization of DR1 as true RARE in regulating the Ssm, a novel retinoic acid-target gene in the mouse testis.

PubMed

Han, Kyuyong; Song, Haengseok; Moon, Irene; Augustin, Robert; Moley, Kelle; Rogers, Melissa; Lim, Hyunjung

2007-03-01

Various nuclear receptors form dimers to activate target genes via specific response elements located within promoters or enhancers. Retinoid X receptor (RXR) serves as a dimerization partner for many nuclear receptors including retinoic acid receptor (RAR) and peroxisome proliferator-activated receptor (PPAR). Dimers show differential preference towards directly repeated response elements with 1-5 nucleotide spacing, and direct repeat 1 (DR1) is a promiscuous element which recruits RAR/RXR, RXR/RXR, and PPAR/RXR in vitro. In the present investigation, we report identification of a novel RAR/RXR target gene which is regulated by DR1s in the promoter region. This gene, namely spermatocyte-specific marker (Ssm), recruits all the three combinations of nuclear receptors in vitro, but in vivo regulation is observed by trans-retinoic acid-activated RAR/RXR dimer. Indeed, chromatin immunoprecipitation experiment demonstrates binding of RARbeta and RXRalpha in the promoter region of the Ssm. Interestingly, expression of Ssm is almost exclusively observed in spermatocytes in the adult mouse testis, where RA signaling is known to regulate developmental program of male germ cells. The results show that Ssm is a RAR/RXR target gene uniquely using DR1 and exhibits stage-specific expression in the mouse testis with potential function in later stages of spermatogenesis. This finding exemplifies usage of DR1s as retinoic acid response element (RARE) under a specific in vivo context.

Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.

PubMed

Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

2016-02-08

Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription-activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

Generation of novel resistance genes using mutation and targeted gene editing.

PubMed

Gal-On, Amit; Fuchs, Marc; Gray, Stewart

2017-10-01

Classical breeding for virus resistance is a lengthy process and is restricted by the availability of resistance genes. Precise genome editing is a 'dream technology' to improve plants for virus resistance and these tools have opened new and very promising ways to generate virus resistant plants by disrupting host susceptibility genes, or by increasing the expression of viral resistance genes. However, precise targets must be identified and their roles understood to minimize potential negative effects on the plant. Nonetheless, the opportunities for genome editing are expanding, as are the technologies to generate effective and broad-spectrum resistance against plant viruses. Here we provide insights into recent progress related to gene targets and gene editing technologies. Published by Elsevier B.V.

Behavioral and genetic investigations of low exploratory behavior in Il18r1−/− mice: We can’t always blame it on the targeted gene

PubMed Central

Eisener-Dorman, Amy F.; Lawrence, David A.; Bolivar, Valerie J.

2010-01-01

The development of gene targeting technologies has enabled research with immune system-related knockout mouse strains to advance our understanding of how cytokines and their receptors interact and influence a number of body systems, including the central nervous system. A critical issue when we are interpreting phenotypic data from these knockout strains is the potential role of genes other than the targeted one. Although many of the knockout strains have been made congenic on a C57BL/6 (B6) genetic background, there remains a certain amount of genetic material from the129 substrain that was used in the development of these strains. This genetic material could result in phenotypes incorrectly attributed to the targeted gene. We recently reported low activity behavior in Il10−/− mice that was linked to this genetic material rather than the targeted gene itself. In the current study we confirm the generalizability of those earlier findings, by assessing behavior in Il18−/− and Il18r1−/− knockout mice. We identified low activity and high anxiety-like behaviors in Il18r1−/− mice, whereas Il18−/− mice displayed little anxiety-like behavior. Although Il18r1−/− mice are considered a congenic strain, we have identified substantial regions of 129P2-derived genetic material not only flanking the ablated Il18r1 on Chromosome 1, but also on Chromosomes 4, 5, 8, 10, and 14. Our studies suggest that residual 129-derived gene(s), rather than the targeted Il18r1 gene, is/are responsible for the low level of activity seen in the Il18r1−/− mice. Mapping studies are necessary to identify the gene or genes contributing to the low activity phenotype. PMID:20580925

Activation of Ftz-F1-Responsive Genes through Ftz/Ftz-F1 Dependent Enhancers

PubMed Central

Field, Amanda; Xiang, Jie; Anderson, W. Ray; Graham, Patricia; Pick, Leslie

2016-01-01

The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either ftz-f1 or ftz mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of ftz itself and engrailed by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success. To discern rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and ftz-f1 mutant embryos. Ftz-F1-responsive genes most highly regulated included engrailed and nine additional genes expressed in patterns dependent on both ftz and ftz-f1. Candidate enhancers for these genes were identified by combining BDTNP Ftz ChIP-chip data with a computational search for Ftz-F1 binding sites. Of eight enhancer reporter genes tested in transgenic embryos, six generated expression patterns similar to the corresponding endogenous gene and expression was lost in ftz mutants. These studies identified a new set of Ftz-F1 targets, all of which are co-regulated by Ftz. Comparative analysis of enhancers containing Ftz/Ftz-F1 binding sites that were or were not bona fide targets in vivo suggested that GAF negatively regulates enhancers that contain Ftz/Ftz-F1 binding sites but are not actually utilized. These targets include other regulatory factors as well as genes involved directly in morphogenesis, providing insight into how pair-rule genes establish the body pattern. PMID:27723822

Core Promoter Functions in the Regulation of Gene Expression of Drosophila Dorsal Target Genes*

PubMed Central

Zehavi, Yonathan; Kuznetsov, Olga; Ovadia-Shochat, Avital; Juven-Gershon, Tamar

2014-01-01

Developmental processes are highly dependent on transcriptional regulation by RNA polymerase II. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters consist of core promoter motifs, e.g. the initiator, TATA box, and the downstream core promoter element (DPE), which confer specific properties to the core promoter. Here, we explored the importance of core promoter functions in the dorsal-ventral developmental gene regulatory network. This network includes multiple genes that are activated by different nuclear concentrations of Dorsal, an NFκB homolog transcription factor, along the dorsal-ventral axis. We show that over two-thirds of Dorsal target genes contain DPE sequence motifs, which is significantly higher than the proportion of DPE-containing promoters in Drosophila genes. We demonstrate that multiple Dorsal target genes are evolutionarily conserved and functionally dependent on the DPE. Furthermore, we have analyzed the activation of key Dorsal target genes by Dorsal, as well as by another Rel family transcription factor, Relish, and the dependence of their activation on the DPE motif. Using hybrid enhancer-promoter constructs in Drosophila cells and embryo extracts, we have demonstrated that the core promoter composition is an important determinant of transcriptional activity of Dorsal target genes. Taken together, our results provide evidence for the importance of core promoter composition in the regulation of Dorsal target genes. PMID:24634215

Development of an operationally efficient PTC braking enforcement algorithm for freight trains.

DOT National Transportation Integrated Search

2013-08-01

Software algorithms used in positive train control (PTC) systems designed to predict freight train stopping distance and enforce a penalty brake application have been shown to be overly conservative, which can lead to operational inefficiencies by in...

In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation.

PubMed

Moreno, Ana M; Fu, Xin; Zhu, Jie; Katrekar, Dhruva; Shih, Yu-Ru V; Marlett, John; Cabotaje, Jessica; Tat, Jasmine; Naughton, John; Lisowski, Leszek; Varghese, Shyni; Zhang, Kang; Mali, Prashant

2018-04-25

Development of efficacious in vivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Variants in the ATM-CHEK2-BRCA1 axis determine genetic predisposition and clinical presentation of papillary thyroid carcinoma.

PubMed

Wójcicka, Anna; Czetwertyńska, Małgorzata; Świerniak, Michał; Długosińska, Joanna; Maciąg, Monika; Czajka, Agnieszka; Dymecka, Kinga; Kubiak, Anna; Kot, Adam; Płoski, Rafał; de la Chapelle, Albert; Jażdżewski, Krystian

2014-06-01

The risk of developing papillary thyroid carcinoma (PTC), the most frequent form of thyroid malignancy, is elevated up to 8.6-fold in first-degree relatives of PTC patients. The familial risk could be explained by high-penetrance mutations in yet unidentified genes, or polygenic action of low-penetrance alleles. Since the DNA-damaging exposure to ionizing radiation is a known risk factor for thyroid cancer, polymorphisms in DNA repair genes are likely to affect this risk. In a search for low-penetrance susceptibility alleles we employed Sequenom technology to genotype deleterious polymorphisms in ATM, CHEK2, and BRCA1 in 1,781 PTC patients and 2,081 healthy controls. As a result of the study, we identified CHEK2 rs17879961 (OR = 2.2, P = 2.37e-10) and BRCA1 rs16941 (odds ratio [OR] = 1.16, P = 0.005) as risk alleles for PTC. The ATM rs1801516 variant modifies the risk associated with the BRCA1 variant by 0.78 (P = 0.02). Both the ATM and BRCA1 variants modify the impact of male gender on clinical variables: T status (P = 0.007), N status (P = 0.05), and stage (P = 0.035). Our findings implicate an important role of variants in the ATM- CHEK2- BRCA1 axis in modification of the genetic predisposition to PTC and its clinical manifestations. Copyright © 2014 Wiley Periodicals, Inc.

Identification of apoptosis-related PLZF target genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernardo, Maria Victoria; Yelo, Estefania; Gimeno, Lourdes

2007-07-27

The PLZF gene encodes a BTB/POZ-zinc finger-type transcription factor, involved in physiological development, proliferation, differentiation, and apoptosis. In this paper, we investigate proliferation, survival, and gene expression regulation in stable clones from the human haematopoietic K562, DG75, and Jurkat cell lines with inducible expression of PLZF. In Jurkat cells, but not in K562 and DG75 cells, PLZF induced growth suppression and apoptosis in a cell density-dependent manner. Deletion of the BTB/POZ domain of PLZF abrogated growth suppression and apoptosis. PLZF was expressed with a nuclear speckled pattern distinctively in the full-length PLZF-expressing Jurkat clones, suggesting that the nuclear speckled localizationmore » is required for PLZF-induced apoptosis. By microarray analysis, we identified that the apoptosis-inducer TP53INP1, ID1, and ID3 genes were upregulated, and the apoptosis-inhibitor TERT gene was downregulated. The identification of apoptosis-related PLZF target genes may have biological and clinical relevance in cancer typified by altered PLZF expression.« less

Problem-Solving Test: Targeted Gene Disruption

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2008-01-01

Mutational inactivation of a specific gene is the most powerful technique to analyze the biological function of the gene. This approach has been used for a long time in viruses, bacteria, yeast, and fruit fly, but looked quite hopeless in more complex organisms. Targeted inactivation of specific genes (also known as knock-out mutation) in mice is…

49 CFR Appendix F to Part 236 - Minimum Requirements of FRA Directed Independent Third-Party Assessment of PTC System Safety...

Code of Federal Regulations, 2012 CFR

2012-10-01

... Third-Party Assessment of PTC System Safety Verification and Validation F Appendix F to Part 236... Safety Verification and Validation (a) This appendix provides minimum requirements for mandatory independent third-party assessment of PTC system safety verification and validation pursuant to subpart H or I...

49 CFR Appendix F to Part 236 - Minimum Requirements of FRA Directed Independent Third-Party Assessment of PTC System Safety...

Code of Federal Regulations, 2014 CFR

2014-10-01

... Third-Party Assessment of PTC System Safety Verification and Validation F Appendix F to Part 236... Safety Verification and Validation (a) This appendix provides minimum requirements for mandatory independent third-party assessment of PTC system safety verification and validation pursuant to subpart H or I...

49 CFR Appendix F to Part 236 - Minimum Requirements of FRA Directed Independent Third-Party Assessment of PTC System Safety...

Code of Federal Regulations, 2011 CFR

2011-10-01

... Third-Party Assessment of PTC System Safety Verification and Validation F Appendix F to Part 236... Safety Verification and Validation (a) This appendix provides minimum requirements for mandatory independent third-party assessment of PTC system safety verification and validation pursuant to subpart H or I...

49 CFR Appendix F to Part 236 - Minimum Requirements of FRA Directed Independent Third-Party Assessment of PTC System Safety...

Code of Federal Regulations, 2013 CFR

2013-10-01

... Third-Party Assessment of PTC System Safety Verification and Validation F Appendix F to Part 236... Safety Verification and Validation (a) This appendix provides minimum requirements for mandatory independent third-party assessment of PTC system safety verification and validation pursuant to subpart H or I...

Next-generation sequencing reveals the mutational landscape of clinically diagnosed Usher syndrome: copy number variations, phenocopies, a predominant target for translational read-through, and PEX26 mutated in Heimler syndrome.

PubMed

Neuhaus, Christine; Eisenberger, Tobias; Decker, Christian; Nagl, Sandra; Blank, Cornelia; Pfister, Markus; Kennerknecht, Ingo; Müller-Hofstede, Cornelie; Charbel Issa, Peter; Heller, Raoul; Beck, Bodo; Rüther, Klaus; Mitter, Diana; Rohrschneider, Klaus; Steinhauer, Ute; Korbmacher, Heike M; Huhle, Dagmar; Elsayed, Solaf M; Taha, Hesham M; Baig, Shahid M; Stöhr, Heidi; Preising, Markus; Markus, Susanne; Moeller, Fabian; Lorenz, Birgit; Nagel-Wolfrum, Kerstin; Khan, Arif O; Bolz, Hanno J

2017-09-01

Combined retinal degeneration and sensorineural hearing impairment is mostly due to autosomal recessive Usher syndrome (USH1: congenital deafness, early retinitis pigmentosa (RP); USH2: progressive hearing impairment, RP). Sanger sequencing and NGS of 112 genes (Usher syndrome, nonsyndromic deafness, overlapping conditions), MLPA, and array-CGH were conducted in 138 patients clinically diagnosed with Usher syndrome. A molecular diagnosis was achieved in 97% of both USH1 and USH2 patients, with biallelic mutations in 97% (USH1) and 90% (USH2), respectively. Quantitative readout reliably detected CNVs (confirmed by MLPA or array-CGH), qualifying targeted NGS as one tool for detecting point mutations and CNVs. CNVs accounted for 10% of identified USH2A alleles, often in trans to seemingly monoallelic point mutations. We demonstrate PTC124-induced read-through of the common p.Trp3955* nonsense mutation (13% of detected USH2A alleles), a potential therapy target. Usher gene mutations were found in most patients with atypical Usher syndrome, but the diagnosis was adjusted in case of double homozygosity for mutations in OTOA and NR2E3 , genes implicated in isolated deafness and RP. Two patients with additional enamel dysplasia had biallelic PEX26 mutations, for the first time linking this gene to Heimler syndrome. Targeted NGS not restricted to Usher genes proved beneficial in uncovering conditions mimicking Usher syndrome.

Ultrasound-Mediated Vascular Gene Transfection by Cavitation of Endothelial-Targeted Cationic Microbubbles

PubMed Central

Xie, Aris; Belcik, Todd; Qi, Yue; Morgan, Terry K.; Champaneri, Shivam A.; Taylor, Sarah; Davidson, Brian P.; Zhao, Yan; Klibanov, Alexander L.; Kuliszewski, Michael A.; Leong-Poi, Howard; Ammi, Azzdine; Lindner, Jonathan R.

2013-01-01

OBJECTIVES Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. BACKGROUND Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. METHODS Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)–stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. RESULTS Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm2). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1–targeted microbubbles and by ultrasound molecular imaging of P-selectin–targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin–targeted microbubbles but was associated with

Microarray‑based bioinformatics analysis of the prospective target gene network of key miRNAs influenced by long non‑coding RNA PVT1 in HCC.

PubMed

Zhang, Yu; Mo, Wei-Jia; Wang, Xiao; Zhang, Tong-Tong; Qin, Yuan; Wang, Han-Lin; Chen, Gang; Wei, Dan-Ming; Dang, Yi-Wu

2018-05-02

The long non‑coding RNA (lncRNA) PVT1 plays vital roles in the tumorigenesis and development of various types of cancer. However, the potential expression profiling, functions and pathways of PVT1 in HCC remain unknown. PVT1 was knocked down in SMMC‑7721 cells, and a miRNA microarray analysis was performed to detect the differentially expressed miRNAs. Twelve target prediction algorithms were used to predict the underlying targets of these differentially expressed miRNAs. Bioinformatics analysis was performed to explore the underlying functions, pathways and networks of the targeted genes. Furthermore, the relationship between PVT1 and the clinical parameters in HCC was confirmed based on the original data in the TCGA database. Among the differentially expressed miRNAs, the top two upregulated and downregulated miRNAs were selected for further analysis based on the false discovery rate (FDR), fold‑change (FC) and P‑values. Based on the TCGA database, PVT1 was obviously highly expressed in HCC, and a statistically higher PVT1 expression was found for sex (male), ethnicity (Asian) and pathological grade (G3+G4) compared to the control groups (P<0.05). Furthermore, Gene Ontology (GO) analysis revealed that the target genes were involved in complex cellular pathways, such as the macromolecule biosynthetic process, compound metabolic process, and transcription. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the MAPK and Wnt signaling pathways may be correlated with the regulation of the four candidate miRNAs. The results therefore provide significant information on the differentially expressed miRNAs associated with PVT1 in HCC, and we hypothesized that PVT1 may play vital roles in HCC by regulating different miRNAs or target gene expression (particularly MAPK8) via the MAPK or Wnt signaling pathways. Thus, further investigation of the molecular mechanism of PVT1 in HCC is needed.

Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression

PubMed Central

Abruzzi, Katharine Compton; Rodriguez, Joseph; Menet, Jerome S.; Desrochers, Jennifer; Zadina, Abigail; Luo, Weifei; Tkachev, Sasha; Rosbash, Michael

2011-01-01

CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation (ChIP) tiling array assays (ChIP–chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night. The CLK partner protein CYCLE (CYC) is on most of these sites. The CLK/CYC heterodimer is joined 4–6 h later by the transcriptional repressor PERIOD (PER), indicating that the majority of CLK targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. CLK has specific targets in different tissues, implying that important CLK partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation. PMID:22085964

TARGET Researchers Identify Mutations in SIX1/2 and microRNA Processing Genes in Favorable Histology Wilms Tumor | Office of Cancer Genomics

Cancer.gov

TARGET researchers molecularly characterized favorable histology Wilms tumor (FHWT), a pediatric renal cancer. Comprehensive genome and transcript analyses revealed single-nucleotide substitution/deletion mutations in microRNA processing genes (15% of FHWT patients) and Sine Oculis Homeobox Homolog 1/2 (SIX1/2) genes (7% of FHWT patients). SIX1/2 genes play a critical role in renal development and were not previously associated with FHWT, thus presenting a novel role for SIX1/2 pathway aberrations in this disease.

Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9.

PubMed

Li, Jun; Meng, Xiangbing; Zong, Yuan; Chen, Kunling; Zhang, Huawei; Liu, Jinxing; Li, Jiayang; Gao, Caixia

2016-09-12

Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants(1), but replacing a fragment and even obtaining gene insertions at specific loci in plant genomes remain a serious challenge. Here, we report efficient intron-mediated site-specific gene replacement and insertion approaches that generate mutations using the non-homologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%. We also obtained targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant. Furthermore, the site-specific gene replacements and insertions were faithfully transmitted to the next generation. These newly developed approaches can be generally used to replace targeted gene fragments and to insert exogenous DNA sequences into specific genomic sites in rice and other plants.

PAI-1, a target gene of miR-143, regulates invasion and metastasis by upregulating MMP-13 expression of human osteosarcoma.

PubMed

Hirahata, Mio; Osaki, Mitsuhiko; Kanda, Yusuke; Sugimoto, Yui; Yoshioka, Yusuke; Kosaka, Nobuyoshi; Takeshita, Fumitaka; Fujiwara, Tomohiro; Kawai, Akira; Ito, Hisao; Ochiya, Takahiro; Okada, Futoshi

2016-05-01

Despite recent improvements in the therapy for osteosarcoma, 30-40% of osteosarcoma patients die of this disease, mainly due to its lung metastasis. We have previously reported that intravenous injection of miR-143 significantly suppresses lung metastasis of human osteosarcoma cells (143B) in a mouse model. In this study, we examined the biological role and mechanism of miR-143 in the metastasis of human osteosarcoma cells. We identified plasminogen activator inhibitor-1 (PAI-1) as a direct target gene of miR-143. To determine the role of PAI-1 in human osteosarcoma cells, siRNA was transfected into 143B cells for knockdown of PAI-1 expression. An in vitro study showed that downregulation of PAI-1 suppressed cell invasion activity, but not proliferation. Moreover, injection of PAI-1 siRNA into a primary lesion in the osteosarcoma mouse model inhibited lung metastasis compared to control siRNA-injected mice, without influencing the proliferative activity of the tumor cells. Subsequent examination using 143B cells revealed that knockdown of PAI-1 expression resulted in downregulation of the expression and secretion of matrix metalloproteinase-13 (MMP-13), which is also a target gene of miR-143 and a proteolytic enzyme that regulates tumor-induced osteolysis. Immunohistochemical analysis using clinical samples showed that higher miR-143 expressing cases showed poor expression of PAI-1 in the primary tumor cells. All such cases belonged to the lung metastasis-negative group. Moreover, the frequency of lung metastasis-positive cases was significantly higher in PAI-1 and MMP-13 double-positive cases than in PAI-1 or MMP-13 single-positive or double-negative cases (P < 0.05). These results indicated that PAI-1, a target gene of miR-143, regulates invasion and lung metastasis via enhancement of MMP-13 expression and secretion in human osteosarcoma cells, suggesting that these molecules could be potential therapeutic target genes for preventing lung metastasis in

Cellular and molecular mechanisms of HIV-1 integration targeting.

PubMed

Engelman, Alan N; Singh, Parmit K

2018-07-01

Integration is central to HIV-1 replication and helps mold the reservoir of cells that persists in AIDS patients. HIV-1 interacts with specific cellular factors to target integration to interior regions of transcriptionally active genes within gene-dense regions of chromatin. The viral capsid interacts with several proteins that are additionally implicated in virus nuclear import, including cleavage and polyadenylation specificity factor 6, to suppress integration into heterochromatin. The viral integrase protein interacts with transcriptional co-activator lens epithelium-derived growth factor p75 to principally position integration within gene bodies. The integrase additionally senses target DNA distortion and nucleotide sequence to help fine-tune the specific phosphodiester bonds that are cleaved at integration sites. Research into virus-host interactions that underlie HIV-1 integration targeting has aided the development of a novel class of integrase inhibitors and may help to improve the safety of viral-based gene therapy vectors.

Target genes discovery through copy number alteration analysis in human hepatocellular carcinoma.

PubMed

Gu, De-Leung; Chen, Yen-Hsieh; Shih, Jou-Ho; Lin, Chi-Hung; Jou, Yuh-Shan; Chen, Chian-Feng

2013-12-21

High-throughput short-read sequencing of exomes and whole cancer genomes in multiple human hepatocellular carcinoma (HCC) cohorts confirmed previously identified frequently mutated somatic genes, such as TP53, CTNNB1 and AXIN1, and identified several novel genes with moderate mutation frequencies, including ARID1A, ARID2, MLL, MLL2, MLL3, MLL4, IRF2, ATM, CDKN2A, FGF19, PIK3CA, RPS6KA3, JAK1, KEAP1, NFE2L2, C16orf62, LEPR, RAC2, and IL6ST. Functional classification of these mutated genes suggested that alterations in pathways participating in chromatin remodeling, Wnt/β-catenin signaling, JAK/STAT signaling, and oxidative stress play critical roles in HCC tumorigenesis. Nevertheless, because there are few druggable genes used in HCC therapy, the identification of new therapeutic targets through integrated genomic approaches remains an important task. Because a large amount of HCC genomic data genotyped by high density single nucleotide polymorphism arrays is deposited in the public domain, copy number alteration (CNA) analyses of these arrays is a cost-effective way to reveal target genes through profiling of recurrent and overlapping amplicons, homozygous deletions and potentially unbalanced chromosomal translocations accumulated during HCC progression. Moreover, integration of CNAs with other high-throughput genomic data, such as aberrantly coding transcriptomes and non-coding gene expression in human HCC tissues and rodent HCC models, provides lines of evidence that can be used to facilitate the identification of novel HCC target genes with the potential of improving the survival of HCC patients.

Methylation of WNT target genes AXIN2 and DKK1 as robust biomarkers for recurrence prediction in stage II colon cancer

PubMed Central

Kandimalla, R; Linnekamp, J F; van Hooff, S; Castells, A; Llor, X; Andreu, M; Jover, R; Goel, A; Medema, J P

2017-01-01

Stage II colon cancer (CC) still remains a clinical challenge with patient stratification for adjuvant therapy (AT) largely relying on clinical parameters. Prognostic biomarkers are urgently needed for better stratification. Previously, we have shown that WNT target genes AXIN2, DKK1, APCDD1, ASCL2 and LGR5 are silenced by DNA methylation and could serve as prognostic markers in stage II CC patients using methylation-specific PCR. Here, we have extended our discovery cohort AMC90-AJCC-II (N=65) and methylation was analyzed by quantitative pyrosequencing. Subsequently, we validated the results in an independent EPICOLON1 CC cohort (N=79). Methylation of WNT target genes is negatively correlated to mRNA expression. A combination of AXIN2 and DKK1 methylation significantly predicted recurrences in univariate (area under the curve (AUC)=0.83, confidence interval (CI): 0.72–0.94, P<0.0001) analysis in stage II microsatellite stable (MSS) CC patients. This two marker combination showed an AUC of 0.80 (CI: 0.68–0.91, P<0.0001) in the EPICOLON1 validation cohort. Multivariate analysis in the Academic Medical Center (AMC) cohort revealed that both WNT target gene methylation and consensus molecular subtype 4 (CMS4) are significantly associated with poor recurrence-free survival (hazard ratio (HR)methylation: 3.84, 95% CI: 1.14–12.43; HRCMS4: 3.73, 95% CI: 1.22–11.48). CMS4 subtype tumors with WNT target methylation showed worse prognosis. Combining WNT target gene methylation and CMS4 subtype lead to an AUC of 0.89 (0.791–0.982, P<0.0001) for recurrence prediction. Notably, we observed that methylation of DKK1 is high in BRAF mutant and CIMP (CpG island methylator phenotype)-positive cancers, whereas AXIN2 methylation appears to be associated with CMS4. Methylation of AXIN2 and DKK1 were found to be robust markers for recurrence prediction in stage II MSS CC patients. Further validation of these findings in a randomized and prospective manner could pave a way to

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

PubMed Central

Ochiai, Hiroshi; Sakamoto, Naoaki; Fujita, Kazumasa; Nishikawa, Masatoshi; Suzuki, Ken-ichi; Matsuura, Shinya; Miyamoto, Tatsuo; Sakuma, Tetsushi; Shibata, Tatsuo; Yamamoto, Takashi

2012-01-01

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B–GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos. PMID:22711830

Deletion of a target gene in Indica rice via CRISPR/Cas9.

PubMed

Wang, Ying; Geng, Lizhao; Yuan, Menglong; Wei, Juan; Jin, Chen; Li, Min; Yu, Kun; Zhang, Ya; Jin, Huaibing; Wang, Eric; Chai, Zhijian; Fu, Xiangdong; Li, Xianggan

2017-08-01

Using CRISPR/Cas9, we successfully deleted large fragments of the yield-related gene DENSE AND ERECT PANICLE1 in Indica rice at relatively high frequency and generated gain-of-function dep1 mutants. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a rapidly developing technology used to produce gene-specific modifications in both mammalian and plant systems. Most CRISPR-induced modifications in plants reported to date have been small insertions or deletions. Few large target gene deletions have thus far been reported, especially for Indica rice. In this study, we designed multiple CRISPR sgRNAs and successfully deleted DNA fragments in the gene DENSE AND ERECT PANICLE1 (DEP1) in the elite Indica rice line IR58025B. We achieved deletion frequencies of up to 21% for a 430 bp target and 9% for a 10 kb target among T0 events. Constructs with four sgRNAs did not generate higher full-length deletion frequencies than constructs with two sgRNAs. The multiple mutagenesis frequency reached 93% for four targets, and the homozygous mutation frequency reached 21% at the T0 stage. Important yield-related trait characteristics, such as dense and erect panicles and reduced plant height, were observed in dep1 homozygous T0 mutant plants produced by CRISPR/Cas9. Therefore, we successfully obtained deletions in DEP1 in the Indica background using the CRISPR/Cas9 editing tool at relatively high frequency.

Multi-targeted priming for genome-wide gene expression assays.

PubMed

Adomas, Aleksandra B; Lopez-Giraldez, Francesc; Clark, Travis A; Wang, Zheng; Townsend, Jeffrey P

2010-08-17

Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays. We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA. Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression. Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and precise assay of the transcribed sequences within the genome.

Fe₃O₄ Nanoparticles in Targeted Drug/Gene Delivery Systems.

PubMed

Shen, Lazhen; Li, Bei; Qiao, Yongsheng

2018-02-23

Fe₃O₄ nanoparticles (NPs), the most traditional magnetic nanoparticles, have received a great deal of attention in the biomedical field, especially for targeted drug/gene delivery systems, due to their outstanding magnetism, biocompatibility, lower toxicity, biodegradability, and other features. Naked Fe₃O₄ NPs are easy to aggregate and oxidize, and thus are often made with various coatings to realize superior properties for targeted drug/gene delivery. In this review, we first list the three commonly utilized synthesis methods of Fe₃O₄ NPs, and their advantages and disadvantages. In the second part, we describe coating materials that exhibit noticeable features that allow functionalization of Fe₃O₄ NPs and summarize their methods of drug targeting/gene delivery. Then our efforts will be devoted to the research status and progress of several different functionalized Fe₃O₄ NP delivery systems loaded with chemotherapeutic agents, and we present targeted gene transitive carriers in detail. In the following section, we illuminate the most effective treatment systems of the combined drug and gene therapy. Finally, we propose opportunities and challenges of the clinical transformation of Fe₃O₄ NPs targeting drug/gene delivery systems.

Pediatric, Adolescent, and Young Adult Thyroid Carcinoma Harbors Frequent and Diverse Targetable Genomic Alterations, Including Kinase Fusions

PubMed Central

Schrock, Alexa B.; Anderson, Peter M.; Morris, John C.; Heilmann, Andreas M.; Holmes, Oliver; Wang, Kai; Johnson, Adrienne; Waguespack, Steven G.; Ou, Sai‐Hong Ignatius; Khan, Saad; Fung, Kar‐Ming; Stephens, Philip J.; Erlich, Rachel L.; Miller, Vincent A.; Ross, Jeffrey S.; Ali, Siraj M.

2017-01-01

Background. Thyroid carcinoma, which is rare in pediatric patients (age 0–18 years) but more common in adolescent and young adult (AYA) patients (age 15–39 years), carries the potential for morbidity and mortality. Methods. Hybrid‐capture‐based comprehensive genomic profiling (CGP) was performed prospectively on 512 consecutively submitted thyroid carcinomas, including 58 from pediatric and AYA (PAYA) patients, to identify genomic alterations (GAs), including base substitutions, insertions/deletions, copy number alterations, and rearrangements. This PAYA data series includes 41 patients with papillary thyroid carcinoma (PTC), 3 with anaplastic thyroid carcinoma (ATC), and 14 with medullary thyroid carcinoma (MTC). Results. GAs were detected in 93% (54/58) of PAYA cases, with a mean of 1.4 GAs per case. In addition to BRAF V600E mutations, detected in 46% (19/41) of PAYA PTC cases and in 1 of 3 AYA ATC cases, oncogenic fusions involving RET, NTRK1, NTRK3, and ALK were detected in 37% (15/41) of PAYA PTC and 33% (1/3) of AYA ATC cases. Ninety‐three percent (13/14) of MTC patients harbored RET alterations, including 3 novel insertions/deletions in exons 6 and 11. Two of these MTC patients with novel alterations in RET experienced clinical benefit from vandetanib treatment. Conclusion. CGP identified diverse clinically relevant GAs in PAYA patients with thyroid carcinoma, including 83% (34/41) of PTC cases harboring activating kinase mutations or activating kinase rearrangements. These genomic observations and index cases exhibiting clinical benefit from targeted therapy suggest that young patients with advanced thyroid carcinoma can benefit from CGP and rationally matched targeted therapy. Implications for Practice. The detection of diverse clinically relevant genomic alterations in the majority of pediatric, adolescent, and young adult patients with thyroid carcinoma in this study suggests that comprehensive genomic profiling may be beneficial for young

Selective elimination of long INterspersed element-1 expressing tumour cells by targeted expression of the HSV-TK suicide gene

PubMed Central

Chendeb, Mariam; Schneider, Robert; Davidson, Irwin; Fadloun, Anas

2017-01-01

In gene therapy, effective and selective suicide gene expression is crucial. We exploited the endogenous Long INterspersed Element-1 (L1) machinery often reactivated in human cancers to integrate the Herpes Simplex Virus Thymidine Kinase (HSV-TK) suicide gene selectively into the genome of cancer cells. We developed a plasmid-based system directing HSV-TK expression only when reverse transcribed and integrated in the host genome via the endogenous L1 ORF1/2 proteins and an Alu element. Delivery of these new constructs into cells followed by Ganciclovir (GCV) treatment selectively induced mortality of L1 ORF1/2 protein expressing cancer cells, but had no effect on primary cells that do not express L1 ORF1/2. This novel strategy for selective targeting of tumour cells provides high tolerability as the HSV-TK gene cannot be expressed without reverse transcription and integration, and high selectivity as these processes take place only in cancer cells expressing high levels of functional L1 ORF1/2. PMID:28415677

The cytoplasmic expression of MUC1 in papillary thyroid carcinoma of different histological variants and its correlation with cyclin D1 overexpression.

PubMed

Abrosimov, Alexander; Saenko, Vladimir; Meirmanov, Serik; Nakashima, Masahiro; Rogounovitch, Tatiana; Shkurko, Olesya; Lushnikov, Eugeny; Mitsutake, Norisato; Namba, Hiroyuki; Yamashita, Shunichi

2007-01-01

This study addressed the immunohistochemical expression of MUC1 in papillary thyroid carcinoma (PTC) of different histotypes, sizes, and morphological features of aggressiveness, and its correlation with the overexpression of cyclin D1, a target molecule of the Wnt pathway. MUC1 expression was examined in a total of 209 PTCs. Cytoplasmic MUC1 expression was elevated in the tall, columnar cell and oncocytic variants (100%), Warthin-like (78%), and conventional PTCs (61%), and in papillary microcarcinoma (PMC) with the conventional growth pattern (52%). On the contrary, it was low in the follicular variant (27%) of PTC and PMCs with follicular architecture (13%). Cytoplasmic MUC1 accumulation did not associate with any clinicopathological features except peritumoral lymphoid infiltration in PTCs and in PMCs with the conventional growth pattern. MUC1 staining correlated with cyclin D1 overexpression in conventional PTCs and PMCs and PMCs with follicular architecture. The results demonstrate that MUC1 expression varies broadly in different histological variants of PTC, being the lowest in tumors with follicular structure. In general, it does not prove to be a prognosticator of PTC aggressiveness. A high correlation between MUC1 and cyclin D1 implies MUC1 involvement in the Wnt cascade functioning in a large subset of human PTCs and PMCs.

Targeted Gene Knock Out Using Nuclease-Assisted Vector Integration: Hemi- and Homozygous Deletion of JAG1.

PubMed

Gapinske, Michael; Tague, Nathan; Winter, Jackson; Underhill, Gregory H; Perez-Pinera, Pablo

2018-01-01

Gene editing technologies are revolutionizing fields such as biomedicine and biotechnology by providing a simple means to manipulate the genetic makeup of essentially any organism. Gene editing tools function by introducing double-stranded breaks at targeted sites within the genome, which the host cells repair preferentially by Non-Homologous End Joining. While the technologies to introduce double-stranded breaks have been extensively optimized, this progress has not been matched by the development of methods to integrate heterologous DNA at the target sites or techniques to detect and isolate cells that harbor the desired modification. We present here a technique for rapid introduction of vectors at target sites in the genome that enables efficient isolation of successfully edited cells.

Large scale RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as prime target.

PubMed

Ulrich, Julia; Dao, Van Anh; Majumdar, Upalparna; Schmitt-Engel, Christian; Schwirz, Jonas; Schultheis, Dorothea; Ströhlein, Nadi; Troelenberg, Nicole; Grossmann, Daniela; Richter, Tobias; Dönitz, Jürgen; Gerischer, Lizzy; Leboulle, Gérard; Vilcinskas, Andreas; Stanke, Mario; Bucher, Gregor

2015-09-03

Insect pest control is challenged by insecticide resistance and negative impact on ecology and health. One promising pest specific alternative is the generation of transgenic plants, which express double stranded RNAs targeting essential genes of a pest species. Upon feeding, the dsRNA induces gene silencing in the pest resulting in its death. However, the identification of efficient RNAi target genes remains a major challenge as genomic tools and breeding capacity is limited in most pest insects impeding whole-animal-high-throughput-screening. We use the red flour beetle Tribolium castaneum as a screening platform in order to identify the most efficient RNAi target genes. From about 5,000 randomly screened genes of the iBeetle RNAi screen we identify 11 novel and highly efficient RNAi targets. Our data allowed us to determine GO term combinations that are predictive for efficient RNAi target genes with proteasomal genes being most predictive. Finally, we show that RNAi target genes do not appear to act synergistically and that protein sequence conservation does not correlate with the number of potential off target sites. Our results will aid the identification of RNAi target genes in many pest species by providing a manageable number of excellent candidate genes to be tested and the proteasome as prime target. Further, the identified GO term combinations will help to identify efficient target genes from organ specific transcriptomes. Our off target analysis is relevant for the sequence selection used in transgenic plants.

Ultrasound-mediated vascular gene transfection by cavitation of endothelial-targeted cationic microbubbles.

PubMed

Xie, Aris; Belcik, Todd; Qi, Yue; Morgan, Terry K; Champaneri, Shivam A; Taylor, Sarah; Davidson, Brian P; Zhao, Yan; Klibanov, Alexander L; Kuliszewski, Michael A; Leong-Poi, Howard; Ammi, Azzdine; Lindner, Jonathan R

2012-12-01

Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)-stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm(2)). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1-targeted microbubbles and by ultrasound molecular imaging of P-selectin-targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin-targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa

Genotoxic chemical carcinogens target inducible genes in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamilton, J.W.; McCaffrey, J.; Caron, R.M.

1994-12-31

Our laboratory is interested in whether carcinogen-induced DNA damage is distributed nonrandomly in the genome - that is, {open_quotes}targeted{close_quotes} to specific genes or gene regions in vivo. As an indirect measure of whether targeting occurs at the gene level, we have examined whether carcinogens differentially alter the expression of individual genes. We have compared the effects of model genotoxic carcinogens that principally induce either strand breaks, simple alkylations, bulky lesions, or DNA cross-links on the expression of several constitutive and inducible genes in a simple in vivo system, the chick embryo. Each agent was examined for its effects on genemore » expression over a 24 hour period corresponding to the period of maximal DNA damage and repair induced by each compound. The doses used in these studies represented the maximum doses that caused no overt toxicity over a 96 hour period but that induced significant levels of DNA damage. Our results demonstrate that inducible genes are targeted by chemical carcinogens. We hypothesize that such effects may be a result of DNA damage specifically altering DNA-protein interactions within the promoters of inducible genes.« less

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens.

PubMed

Kamisugi, Yasuko; Schaefer, Didier G; Kozak, Jaroslav; Charlot, Florence; Vrielynck, Nathalie; Holá, Marcela; Angelis, Karel J; Cuming, Andrew C; Nogué, Fabien

2012-04-01

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development.

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens

PubMed Central

Kamisugi, Yasuko; Schaefer, Didier G.; Kozak, Jaroslav; Charlot, Florence; Vrielynck, Nathalie; Holá, Marcela; Angelis, Karel J.; Cuming, Andrew C.; Nogué, Fabien

2012-01-01

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development. PMID:22210882

Ndrg2 is a PGC-1α/ERRα target gene that controls protein synthesis and expression of contractile-type genes in C2C12 myotubes.

PubMed

Foletta, Victoria C; Brown, Erin L; Cho, Yoshitake; Snow, Rod J; Kralli, Anastasia; Russell, Aaron P

2013-12-01

The stress-responsive, tumor suppressor N-myc downstream-regulated gene 2 (Ndrg2) is highly expressed in striated muscle. In response to anabolic and catabolic signals, Ndrg2 is suppressed and induced, respectively, in mouse C2C12 myotubes. However, little is known about the mechanisms regulating Ndrg2 expression in muscle, as well as the biological role for Ndrg2 in differentiated myotubes. Here, we show that Ndrg2 is a target of a peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and estrogen-related receptor alpha (ERRα) transcriptional program and is induced in response to endurance exercise, a physiological stress known also to increase PGC-1α/ERRα activity. Analyses of global gene and protein expression profiles in C2C12 myotubes with reduced levels of NDRG2, suggest that NDRG2 affects muscle growth, contractile properties, MAPK signaling, ion and vesicle transport and oxidative phosphorylation. Indeed, suppression of NDRG2 in myotubes increased protein synthesis and the expression of fast glycolytic myosin heavy chain isoforms, while reducing the expression of embryonic myosin Myh3, other contractile-associated genes and the MAPK p90 RSK1. Conversely, enhanced expression of NDRG2 reduced protein synthesis, and furthermore, partially blocked the increased protein synthesis rates elicited by a constitutively active form of ERRα. In contrast, suppressing or increasing levels of NDRG2 did not affect mRNA expression of genes involved in mitochondrial biogenesis that are regulated by PGC-1α or ERRα. This study shows that in C2C12 myotubes Ndrg2 is a novel PGC-1α/ERRα transcriptional target, which influences protein turnover and the regulation of genes involved in muscle contraction and function. © 2013 Elsevier B.V. All rights reserved.

The targeting expression of the vascular endothelial growth factor gene in endothelial cells regulated by HRE.ppET-1.

PubMed

Zheng, Xiangrong; Zhang, Shangshang; Yang, Yujia; Wang, Xia; Zhong, Le; Yu, Xiaohe

2008-11-01

The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 mumol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The expression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regulated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (P<0.05). The MTT test showed that the proliferation rate of HUVEC transfected with HPVA under hypoxia exceeded that of the control group. We conclude that the HRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation of

Aberrant status and clinicopathologic characteristic associations of 11 target genes in 1,321 Chinese patients with lung adenocarcinoma.

PubMed

Zhao, Mengnan; Zhan, Cheng; Li, Ming; Yang, Xiaodong; Yang, Xinyu; Zhang, Yong; Lin, Miao; Xia, Yifeng; Feng, Mingxiang; Wang, Qun

2018-01-01

The aberrant status of target genes and their associations with clinicopathologic characteristics are still unclear in primary lung adenocarcinoma. The common mutations and translocations of nine target genes were evaluated in 1,247 specimens of surgically-resected primary lung adenocarcinoma. Immunohistochemistry was used to analyze the expressions of programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) in 731 specimens. The frequency of the aberrations and their associations with clinicopathologic characteristics were analyzed. Overall, 952 (76.3%) of 1,247 patients harbored at least one target mutation or translocation: epidermal growth factor receptor ( EGFR ) (729, 58.5%), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog ( KRAS ) (83, 6.7%), human epidermal growth factor receptor 2 ( HER2 ) (82, 6.6%), anaplastic lymphoma kinase ( ALK) (23, 1.8%), phosphoinositide-3-kinase catalytic alpha polypeptide ( PIK3CA ) (20, 1.6%), Ret proto-oncogene RET (15, 1.2%), ROS proto-oncogene 1 receptor tyrosine kinase ( ROS1 ) (12, 1.0%), B-raf proto-oncogene ( BRAF ) (9, 0.7%), neuroblastoma RAS viral (v-ras) oncogene homolog ( NRAS ) (3, 0.2%). Fourteen (1.9%) of 731 patients were PD-1 positive and 95 (13.0%) were PD-L1 positive in tumor cells. In men and smokers, there were more frequent KRAS mutations (both P<0.001) and PD-L1 positive tumors (P<0.001, P=0.005, respectively), and less frequent EGFR mutations (P=0.049, <0.001, respectively). In ground-glass opacity (GGO) or ground-glass nodules (GGN), there were more HER2 (P=0.033) but less EGFR (P=0.025) and PIK3CA mutations (P=0.012), and ALK translocations (P=0.014). EGFR (P<0.001), KRAS mutations (P=0.004) and PD-L1 positive tumors (P=0.046) were more frequent in older patients, while HER2 (P<0.001), ALK (P=0.005) and ROS1 aberrations (P=0.044) were less frequent. Invasive mucinous adenocarcinoma was significantly associated with KRAS and ALK aberrations (both P<0.001), while solid predominant adenocarcinoma

New TFII-I family target genes involved in embryonic development.

PubMed

Makeyev, Aleksandr V; Bayarsaihan, Dashzeveg

2009-09-04

Two members of the TFII-I family transcription factor genes, GTF2I and GTF2IRD1, are the prime candidates responsible for the craniofacial and cognitive abnormalities of Williams syndrome patients. We have previously generated mouse lines with targeted disruption of Gtf2i and Gtf2ird1. Microarray analysis revealed significant changes in the expression profile of mutant embryos. Here we described three unknown genes that were dramatically down-regulated in mutants. The 2410018M08Rik/Scand3 gene encodes a protein of unknown function with CHCH and hATC domains. Scand3 is down-regulated during mouse embryonic stem cell (ES) differentiation. 4933436H12Rik is a testis-specific gene, which encodes a protein with no known domains. It is expressed in mouse ES cells. 1110008P08Rik/Kbtbd7 encodes an adapter protein with BTB/POZ, BACK, and Kelch motifs, previously shown to recruit substrates to the enzymatic complexes of the histone modifying or E3 ubiquitin ligase activities. Based on its expression pattern Kbtbd7 may have a specific role in brain development and function. All three genes possess well-conserved TFII-I-binding consensus sites within proximal promoters. Therefore our analysis suggests that these genes can be direct targets of TFII-I proteins and their impaired expression, as a result of the GTF2I and GTF2IRD1 haploinsufficiency, could contribute to the etiology of Williams syndrome.

Prediction of target genes for miR-140-5p in pulmonary arterial hypertension using bioinformatics methods.

PubMed

Li, Fangwei; Shi, Wenhua; Wan, Yixin; Wang, Qingting; Feng, Wei; Yan, Xin; Wang, Jian; Chai, Limin; Zhang, Qianqian; Li, Manxiang

2017-12-01

The expression of microRNA (miR)-140-5p is known to be reduced in both pulmonary arterial hypertension (PAH) patients and monocrotaline-induced PAH models in rat. Identification of target genes for miR-140-5p with bioinformatics analysis may reveal new pathways and connections in PAH. This study aimed to explore downstream target genes and relevant signaling pathways regulated by miR-140-5p to provide theoretical evidences for further researches on role of miR-140-5p in PAH. Multiple downstream target genes and upstream transcription factors (TFs) of miR-140-5p were predicted in the analysis. Gene ontology (GO) enrichment analysis indicated that downstream target genes of miR-140-5p were enriched in many biological processes, such as biological regulation, signal transduction, response to chemical stimulus, stem cell proliferation, cell surface receptor signaling pathways. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis found that downstream target genes were mainly located in Notch, TGF-beta, PI3K/Akt, and Hippo signaling pathway. According to TF-miRNA-mRNA network, the important downstream target genes of miR-140-5p were PPI, TGF-betaR1, smad4, JAG1, ADAM10, FGF9, PDGFRA, VEGFA, LAMC1, TLR4, and CREB. After thoroughly reviewing published literature, we found that 23 target genes and seven signaling pathways were truly inhibited by miR-140-5p in various tissues or cells; most of these verified targets were in accordance with our present prediction. Other predicted targets still need further verification in vivo and in vitro .

[Discovery of the target genes inhibited by formic acid in Candida shehatae].

PubMed

Cai, Peng; Xiong, Xujie; Xu, Yong; Yong, Qiang; Zhu, Junjun; Shiyuan, Yu

2014-01-04

At transcriptional level, the inhibitory effects of formic acid was investigated on Candida shehatae, a model yeast strain capable of fermenting xylose to ethanol. Thereby, the target genes were regulated by formic acid and the transcript profiles were discovered. On the basis of the transcriptome data of C. shehatae metabolizing glucose and xylose, the genes responsible for ethanol fermentation were chosen as candidates by the combined method of yeast metabolic pathway analysis and manual gene BLAST search. These candidates were then quantitatively detected by RQ-PCR technique to find the regulating genes under gradient doses of formic acid. By quantitative analysis of 42 candidate genes, we finally identified 10 and 5 genes as markedly down-regulated and up-regulated targets by formic acid, respectively. With regard to gene transcripts regulated by formic acid in C. shehatae, the markedly down-regulated genes ranking declines as follows: xylitol dehydrogenase (XYL2), acetyl-CoA synthetase (ACS), ribose-5-phosphate isomerase (RKI), transaldolase (TAL), phosphogluconate dehydrogenase (GND1), transketolase (TKL), glucose-6-phosphate dehydrogenase (ZWF1), xylose reductase (XYL1), pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC); and a declining rank for up-regulated gens as follows: fructose-bisphosphate aldolase (ALD), glucokinase (GLK), malate dehydrogenase (MDH), 6-phosphofructokinase (PFK) and alcohol dehydrogenase (ADH).

CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation.

PubMed

Miki, Daisuke; Zhang, Wenxin; Zeng, Wenjie; Feng, Zhengyan; Zhu, Jian-Kang

2018-05-17

Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as Drosophila and mouse. However, gene targeting in higher plants, including the most widely used model plant Arabidopsis thaliana, remains challenging. Here we report a sequential transformation method for gene targeting in Arabidopsis. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific DD45 gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the Arabidopsis genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the Arabidopsis genome.

Enrichment of putative PAX8 target genes at serous epithelial ovarian cancer susceptibility loci.

PubMed

Kar, Siddhartha P; Adler, Emily; Tyrer, Jonathan; Hazelett, Dennis; Anton-Culver, Hoda; Bandera, Elisa V; Beckmann, Matthias W; Berchuck, Andrew; Bogdanova, Natalia; Brinton, Louise; Butzow, Ralf; Campbell, Ian; Carty, Karen; Chang-Claude, Jenny; Cook, Linda S; Cramer, Daniel W; Cunningham, Julie M; Dansonka-Mieszkowska, Agnieszka; Doherty, Jennifer Anne; Dörk, Thilo; Dürst, Matthias; Eccles, Diana; Fasching, Peter A; Flanagan, James; Gentry-Maharaj, Aleksandra; Glasspool, Rosalind; Goode, Ellen L; Goodman, Marc T; Gronwald, Jacek; Heitz, Florian; Hildebrandt, Michelle A T; Høgdall, Estrid; Høgdall, Claus K; Huntsman, David G; Jensen, Allan; Karlan, Beth Y; Kelemen, Linda E; Kiemeney, Lambertus A; Kjaer, Susanne K; Kupryjanczyk, Jolanta; Lambrechts, Diether; Levine, Douglas A; Li, Qiyuan; Lissowska, Jolanta; Lu, Karen H; Lubiński, Jan; Massuger, Leon F A G; McGuire, Valerie; McNeish, Iain; Menon, Usha; Modugno, Francesmary; Monteiro, Alvaro N; Moysich, Kirsten B; Ness, Roberta B; Nevanlinna, Heli; Paul, James; Pearce, Celeste L; Pejovic, Tanja; Permuth, Jennifer B; Phelan, Catherine; Pike, Malcolm C; Poole, Elizabeth M; Ramus, Susan J; Risch, Harvey A; Rossing, Mary Anne; Salvesen, Helga B; Schildkraut, Joellen M; Sellers, Thomas A; Sherman, Mark; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa; Terry, Kathryn L; Tworoger, Shelley S; Walsh, Christine; Wentzensen, Nicolas; Whittemore, Alice S; Wu, Anna H; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Freedman, Matthew L; Gayther, Simon A; Pharoah, Paul D P; Lawrenson, Kate

2017-02-14

Genome-wide association studies (GWAS) have identified 18 loci associated with serous ovarian cancer (SOC) susceptibility but the biological mechanisms driving these findings remain poorly characterised. Germline cancer risk loci may be enriched for target genes of transcription factors (TFs) critical to somatic tumorigenesis. All 615 TF-target sets from the Molecular Signatures Database were evaluated using gene set enrichment analysis (GSEA) and three GWAS for SOC risk: discovery (2196 cases/4396 controls), replication (7035 cases/21 693 controls; independent from discovery), and combined (9627 cases/30 845 controls; including additional individuals). The PAX8-target gene set was ranked 1/615 in the discovery (P GSEA <0.001; FDR=0.21), 7/615 in the replication (P GSEA =0.004; FDR=0.37), and 1/615 in the combined (P GSEA <0.001; FDR=0.21) studies. Adding other genes reported to interact with PAX8 in the literature to the PAX8-target set and applying an alternative to GSEA, interval enrichment, further confirmed this association (P=0.006). Fifteen of the 157 genes from this expanded PAX8 pathway were near eight loci associated with SOC risk at P<10 -5 (including six with P<5 × 10 -8 ). The pathway was also associated with differential gene expression after shRNA-mediated silencing of PAX8 in HeyA8 (P GSEA =0.025) and IGROV1 (P GSEA =0.004) SOC cells and several PAX8 targets near SOC risk loci demonstrated in vitro transcriptomic perturbation. Putative PAX8 target genes are enriched for common SOC risk variants. This finding from our agnostic evaluation is of particular interest given that PAX8 is well-established as a specific marker for the cell of origin of SOC.

Enrichment of putative PAX8 target genes at serous epithelial ovarian cancer susceptibility loci

PubMed Central

Kar, Siddhartha P; Adler, Emily; Tyrer, Jonathan; Hazelett, Dennis; Anton-Culver, Hoda; Bandera, Elisa V; Beckmann, Matthias W; Berchuck, Andrew; Bogdanova, Natalia; Brinton, Louise; Butzow, Ralf; Campbell, Ian; Carty, Karen; Chang-Claude, Jenny; Cook, Linda S; Cramer, Daniel W; Cunningham, Julie M; Dansonka-Mieszkowska, Agnieszka; Doherty, Jennifer Anne; Dörk, Thilo; Dürst, Matthias; Eccles, Diana; Fasching, Peter A; Flanagan, James; Gentry-Maharaj, Aleksandra; Glasspool, Rosalind; Goode, Ellen L; Goodman, Marc T; Gronwald, Jacek; Heitz, Florian; Hildebrandt, Michelle A T; Høgdall, Estrid; Høgdall, Claus K; Huntsman, David G; Jensen, Allan; Karlan, Beth Y; Kelemen, Linda E; Kiemeney, Lambertus A; Kjaer, Susanne K; Kupryjanczyk, Jolanta; Lambrechts, Diether; Levine, Douglas A; Li, Qiyuan; Lissowska, Jolanta; Lu, Karen H; Lubiński, Jan; Massuger, Leon F A G; McGuire, Valerie; McNeish, Iain; Menon, Usha; Modugno, Francesmary; Monteiro, Alvaro N; Moysich, Kirsten B; Ness, Roberta B; Nevanlinna, Heli; Paul, James; Pearce, Celeste L; Pejovic, Tanja; Permuth, Jennifer B; Phelan, Catherine; Pike, Malcolm C; Poole, Elizabeth M; Ramus, Susan J; Risch, Harvey A; Rossing, Mary Anne; Salvesen, Helga B; Schildkraut, Joellen M; Sellers, Thomas A; Sherman, Mark; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa; Terry, Kathryn L; Tworoger, Shelley S; Walsh, Christine; Wentzensen, Nicolas; Whittemore, Alice S; Wu, Anna H; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Freedman, Matthew L; Gayther, Simon A; Pharoah, Paul D P; Lawrenson, Kate

2017-01-01

Background: Genome-wide association studies (GWAS) have identified 18 loci associated with serous ovarian cancer (SOC) susceptibility but the biological mechanisms driving these findings remain poorly characterised. Germline cancer risk loci may be enriched for target genes of transcription factors (TFs) critical to somatic tumorigenesis. Methods: All 615 TF-target sets from the Molecular Signatures Database were evaluated using gene set enrichment analysis (GSEA) and three GWAS for SOC risk: discovery (2196 cases/4396 controls), replication (7035 cases/21 693 controls; independent from discovery), and combined (9627 cases/30 845 controls; including additional individuals). Results: The PAX8-target gene set was ranked 1/615 in the discovery (PGSEA<0.001; FDR=0.21), 7/615 in the replication (PGSEA=0.004; FDR=0.37), and 1/615 in the combined (PGSEA<0.001; FDR=0.21) studies. Adding other genes reported to interact with PAX8 in the literature to the PAX8-target set and applying an alternative to GSEA, interval enrichment, further confirmed this association (P=0.006). Fifteen of the 157 genes from this expanded PAX8 pathway were near eight loci associated with SOC risk at P<10−5 (including six with P<5 × 10−8). The pathway was also associated with differential gene expression after shRNA-mediated silencing of PAX8 in HeyA8 (PGSEA=0.025) and IGROV1 (PGSEA=0.004) SOC cells and several PAX8 targets near SOC risk loci demonstrated in vitro transcriptomic perturbation. Conclusions: Putative PAX8 target genes are enriched for common SOC risk variants. This finding from our agnostic evaluation is of particular interest given that PAX8 is well-established as a specific marker for the cell of origin of SOC. PMID:28103614

Identification of neuronal target genes for CCAAT/Enhancer Binding Proteins

PubMed Central

Kfoury, N.; Kapatos, G.

2009-01-01

CCAAT/Enhancer Binding Proteins (C/EBPs) play pivotal roles in development and plasticity of the nervous system. Identification of the physiological targets of C/EBPs (C/EBP target genes) should therefore provide insight into the underlying biology of these processes. We used unbiased genome-wide mapping to identify 115 C/EBPβ target genes in PC12 cells that include transcription factors, neurotransmitter receptors, ion channels, protein kinases and synaptic vesicle proteins. C/EBPβ binding sites were located primarily within introns, suggesting novel regulatory functions, and were associated with binding sites for other developmentally important transcription factors. Experiments using dominant negatives showed C/EBPβ to repress transcription of a subset of target genes. Target genes in rat brain were subsequently found to preferentially bind C/EBPα, β and δ. Analysis of the hippocampal transcriptome of C/EBPβ knockout mice revealed dysregulation of a high percentage of transcripts identified as C/EBP target genes. These results support the hypothesis that C/EBPs play non-redundant roles in the brain. PMID:19103292

The Kto-Skd complex can regulate ptc expression by interacting with Cubitus interruptus (Ci) in the Hedgehog signaling pathway.

PubMed

Mao, Feifei; Yang, Xiaofeng; Fu, Lin; Lv, Xiangdong; Zhang, Zhao; Wu, Wenqing; Yang, Siqi; Zhou, Zhaocai; Zhang, Lei; Zhao, Yun

2014-08-08

The hedgehog (Hh) signaling pathway plays a very important role in metazoan development by controlling pattern formation. Drosophila imaginal discs are subdivided into anterior and posterior compartments that derive from adjacent cell populations. The anterior/posterior (A/P) boundaries, which are critical to maintaining the position of organizers, are established by a complex mechanism involving Hh signaling. Here, we uncover the regulation of ptc in the Hh signaling pathway by two subunits of mediator complex, Kto and Skd, which can also regulate boundary location. Collectively, we provide further evidence that Kto-Skd affects the A/P-axial development of the whole wing disc. Kto can interact with Cubitus interruptus (Ci), bind to the Ci-binding region on ptc promoter, which are both regulated by Hh signals to down-regulate ptc expression. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Targeted Antiangiogenesis Gene Therapy Using Targeted Cationic Microbubbles Conjugated with CD105 Antibody Compared with Untargeted Cationic and Neutral Microbubbles

PubMed Central

Zhou, Yu; Gu, Haitao; Xu, Yan; Li, Fan; Kuang, Shaojing; Wang, Zhigang; Zhou, Xiyuan; Ma, Huafeng; Li, Pan; Zheng, Yuanyi; Ran, Haitao; Jian, Jia; Zhao, Yajing; Song, Weixiang; Wang, Qiushi; Wang, Dong

2015-01-01

Objective This study aimed to develop targeted cationic microbubbles conjugated with a CD105 antibody (CMB105) for use in targeted vascular endothelial cell gene therapy and ultrasound imaging. We compared the results with untargeted cationic microbubbles (CMB) and neutral microbubbles (NMB). Methods CMB105 were prepared and compared with untargeted CMB and NMB. First, the microbubbles were characterized in terms of size, zeta-potential, antibody binding ability and plasmid DNA loading capacity. A tumor model of subcutaneous breast cancer in nude mice was used for our experiments. The ability of different types of microbubbles to target HUVECs in vitro and tumor neovascularization in vivo was measured. The endostatin gene was selected for its outstanding antiangiogenesis effect. For in vitro experiments, the transfection efficiency and cell cycle were analyzed using flow cytometry, and the transcription and expression of endostatin were measured by qPCR and Western blotting, respectively. Vascular tube cavity formation and tumor cell invasion were used to evaluate the antiangiogenesis gene therapy efficiency in vitro. Tumors were exposed to ultrasound irradiation with different types of microbubbles, and the gene therapy effects were investigated by detecting apoptosis induction and changes in tumor volume. Results CMB105 and CMB differed significantly from NMB in terms of zeta-potential, and the DNA loading capacities were 16.76±1.75 μg, 18.21±1.22 μg, and 0.48±0.04 μg per 5×108 microbubbles, respectively. The charge coupling of plasmid DNA to CMB105 was not affected by the presence of the CD105 antibody. Both CMB105 and CMB could target to HUVECs in vitro, whereas only CMB105 could target to tumor neovascularization in vivo. In in vitro experiments, the transfection efficiency of CMB105 was 24.7-fold higher than the transfection efficiency of NMB and 1.47-fold higher than the transfection efficiency of CMB (P<0.05). With ultrasound-targeted microbubble

Targeted antiangiogenesis gene therapy using targeted cationic microbubbles conjugated with CD105 antibody compared with untargeted cationic and neutral microbubbles.

PubMed

Zhou, Yu; Gu, Haitao; Xu, Yan; Li, Fan; Kuang, Shaojing; Wang, Zhigang; Zhou, Xiyuan; Ma, Huafeng; Li, Pan; Zheng, Yuanyi; Ran, Haitao; Jian, Jia; Zhao, Yajing; Song, Weixiang; Wang, Qiushi; Wang, Dong

2015-01-01

This study aimed to develop targeted cationic microbubbles conjugated with a CD105 antibody (CMB105) for use in targeted vascular endothelial cell gene therapy and ultrasound imaging. We compared the results with untargeted cationic microbubbles (CMB) and neutral microbubbles (NMB). CMB105 were prepared and compared with untargeted CMB and NMB. First, the microbubbles were characterized in terms of size, zeta-potential, antibody binding ability and plasmid DNA loading capacity. A tumor model of subcutaneous breast cancer in nude mice was used for our experiments. The ability of different types of microbubbles to target HUVECs in vitro and tumor neovascularization in vivo was measured. The endostatin gene was selected for its outstanding antiangiogenesis effect. For in vitro experiments, the transfection efficiency and cell cycle were analyzed using flow cytometry, and the transcription and expression of endostatin were measured by qPCR and Western blotting, respectively. Vascular tube cavity formation and tumor cell invasion were used to evaluate the antiangiogenesis gene therapy efficiency in vitro. Tumors were exposed to ultrasound irradiation with different types of microbubbles, and the gene therapy effects were investigated by detecting apoptosis induction and changes in tumor volume. CMB105 and CMB differed significantly from NMB in terms of zeta-potential, and the DNA loading capacities were 16.76±1.75 μg, 18.21±1.22 μg, and 0.48±0.04 μg per 5×10(8) microbubbles, respectively. The charge coupling of plasmid DNA to CMB105 was not affected by the presence of the CD105 antibody. Both CMB105 and CMB could target to HUVECs in vitro, whereas only CMB105 could target to tumor neovascularization in vivo. In in vitro experiments, the transfection efficiency of CMB105 was 24.7-fold higher than the transfection efficiency of NMB and 1.47-fold higher than the transfection efficiency of CMB (P<0.05). With ultrasound-targeted microbubble destruction (UTMD

SO2-tolerant and H2O-promoting Pt/C catalysts for efficient NO removal via fixed-bed H2-SCR.

PubMed

Tu, Baosheng; Shi, Nian; Sun, Wei; Cao, Limei; Yang, Ji

2017-01-01

In this paper, Pt supports on carbon black powder (Vulcan XC-72) were synthesized via a hydrothermal method for selective catalytic reduction (SCR) of NO with H 2 in the presence of 2 vol% O 2 over a wide temperature of 20-300 °C. The results showed that the 3 and 5 wt% Pt/C catalysts resulted in high NO conversion (>90 %) over a temperature range of 120 to 300 °C, and the maximum NO conversion of 98.6 % was achieved over 5 wt% Pt/C at 120 °C. Meanwhile, the influence of SO 2 and H 2 O on the catalyst performance of 3 wt% Pt/C was investigated. The catalysts exhibited good SO 2 poisoning resistance when the SO 2 concentration was lower than 260 ppm. Moreover, a positive effect on NO conversion was detected with the addition of 3 and 5 vol% H 2 O in the feed gas stream. Graphical abstract TEM image and good NO conversion performance of the Pt/C catalysts.

Carbon Black - Polyethylene Composites for PTC (Positive Temperature Coefficient) Thermistor Applications

DTIC Science & Technology

1987-12-01

triphasic composite. Addition of the third filler yielded two effects. First, the filler provided sufficient mechanical stabilization of the composite...2000C. The stabilization was accomplished without diminishing the magnitude of the PTC effect. The triphasic composites also displayed equivalent or...differences in conductor and insulator --.-+icle size lead to the formation of quasi-compo- -s, resulting in a 3(0-3)-0 connectivity. Triphasic composites

A novel RET rearrangement (ACBD5/RET) by pericentric inversion, inv(10)(p12.1;q11.2), in papillary thyroid cancer from an atomic bomb survivor exposed to high-dose radiation.

PubMed

Hamatani, Kiyohiro; Eguchi, Hidetaka; Koyama, Kazuaki; Mukai, Mayumi; Nakachi, Kei; Kusunoki, Yoichiro

2014-11-01

During analysis of RET/PTC rearrangements in papillary thyroid cancer (PTC) among atomic bomb survivors, a cDNA fragment of a novel type of RET rearrangement was identified in a PTC patient exposed to a high radiation dose using the improved 5' RACE method. This gene resulted from the fusion of the 3' portion of RET containing tyrosine kinase domain to the 5' portion of the acyl-coenzyme A binding domain containing 5 (ACBD5) gene, by pericentric inversion inv(10)(p12.1;q11.2); expression of the fusion gene was confirmed by RT-PCR. ACBD5 gene is ubiquitously expressed in various human normal tissues including thyroid. Full-length cDNA of the ACBD5-RET gene was constructed and then examined for tumorigenicity. Enhanced phosphorylation of ERK proteins in the MAPK pathway was observed in NIH3T3 cells transfected with expression vector encoding the full-length ACBD5/RET cDNA, while this was not observed in the cells transfected with empty expression vector. Stable NIH3T3 transfectants with ACBD5-RET cDNA induced tumor formation after their injection into nude mice. These findings suggest that the ACBD5-RET rearrangement is causatively involved in the development of PTC.

Integrated computational biology analysis to evaluate target genes for chronic myelogenous leukemia.

PubMed

Zheng, Yu; Wang, Yu-Ping; Cao, Hongbao; Chen, Qiusheng; Zhang, Xi

2018-06-05

Although hundreds of genes have been linked to chronic myelogenous leukemia (CML), many of the results lack reproducibility. In the present study, data across multiple modalities were integrated to evaluate 579 CML candidate genes, including literature‑based CML‑gene relation data, Gene Expression Omnibus RNA expression data and pathway‑based gene‑gene interaction data. The expression data included samples from 76 patients with CML and 73 healthy controls. For each target gene, four metrics were proposed and tested with case/control classification. The effectiveness of the four metrics presented was demonstrated by the high classification accuracy (94.63%; P<2x10‑4). Cross metric analysis suggested nine top candidate genes for CML: Epidermal growth factor receptor, tumor protein p53, catenin β 1, janus kinase 2, tumor necrosis factor, abelson murine leukemia viral oncogene homolog 1, vascular endothelial growth factor A, B‑cell lymphoma 2 and proto‑oncogene tyrosine‑protein kinase. In addition, 145 CML candidate pathways enriched with 485 out of 579 genes were identified (P<8.2x10‑11; q=0.005). In conclusion, weighted genetic networks generated using computational biology may be complementary to biological experiments for the evaluation of known or novel CML target genes.

Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAFV600E patient-derived papillary thyroid carcinoma preclinical model.

PubMed

Duquette, Mark; Sadow, Peter M; Husain, Amjad; Sims, Jennifer N; Antonello, Zeus A; Fischer, Andrew H; Song, Chen; Castellanos-Rizaldos, Elena; Makrigiorgos, G Mike; Kurebayashi, Junichi; Nose, Vania; Van Hummelen, Paul; Bronson, Roderick T; Vinco, Michelle; Giordano, Thomas J; Dias-Santagata, Dora; Pandolfi, Pier Paolo; Nucera, Carmelo

2015-12-15

BRAF(V600E) mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAF(V600E) inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAF(V600E) selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAF(V600E)-PTC, intrathyroidal BRAF(V600E)-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAF(V600E)-PTC orthotopic mouse. We find that metastatic BRAF(V600E)-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAF(WT)-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAF(V600E)-PTC cells but lesser in metastatic BRAF(V600E)-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment-associated pro-metastatic molecules, with no effects in BRAF(WT)-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAF(WT/V600E)-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAF(V600E)-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAF(V600E)-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for

Metastasis-associated MCL1 and P16 copy number alterations dictate resistance to vemurafenib in a BRAFV600E patient-derived papillary thyroid carcinoma preclinical model

PubMed Central

Duquette, Mark; Sadow, Peter M.; Fischer, Andrew H.; Song, Chen; Castellanos-Rizaldos, Elena; Makrigiorgos, G. Mike; Kurebayashi, Junichi; Nose, Vania; Van Hummelen, Paul; Bronson, Roderick T.; Vinco, Michelle; Giordano, Thomas J.; Dias-Santagata, Dora; Pandolfi, Pier Paolo; Nucera, Carmelo

2015-01-01

BRAFV600E mutation exerts an essential oncogenic function in many tumors, including papillary thyroid carcinoma (PTC). Although BRAFV600E inhibitors are available, lack of response has been frequently observed. To study the mechanism underlying intrinsic resistance to the mutant BRAFV600E selective inhibitor vemurafenib, we established short-term primary cell cultures of human metastatic/recurrent BRAFV600E-PTC, intrathyroidal BRAFV600E-PTC, and normal thyroid (NT). We also generated an early intervention model of human BRAFV600E-PTC orthotopic mouse. We find that metastatic BRAFV600E-PTC cells elicit paracrine-signaling which trigger migration of pericytes, blood endothelial cells and lymphatic endothelial cells as compared to BRAFWT-PTC cells, and show a higher rate of invasion. We further show that vemurafenib therapy significantly suppresses these aberrant functions in non-metastatic BRAFV600E-PTC cells but lesser in metastatic BRAFV600E-PTC cells as compared to vehicle treatment. These results concur with similar folds of down-regulation of tumor microenvironment–associated pro-metastatic molecules, with no effects in BRAFWT-PTC and NT cells. Our early intervention preclinical trial shows that vemurafenib delays tumor growth in the orthotopic BRAFWT/V600E-PTC mice. Importantly, we identify high copy number gain of MCL1 (chromosome 1q) and loss of CDKN2A (P16, chromosome 9p) in metastatic BRAFV600E-PTC cells which are associated with resistance to vemurafenib treatment. Critically, we demonstrate that combined vemurafenib therapy with BCL2/MCL1 inhibitor increases metastatic BRAFV600E-PTC cell death and ameliorates response to vemurafenib treatment as compared to single agent treatment. In conclusion, short-term PTC and NT cultures offer a predictive model for evaluating therapeutic response in patients with PTC. Our PTC pre-clinical model suggests that combined targeted therapy might be an important therapeutic strategy for metastatic and refractory BRAFV600

Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells.

PubMed

Byrne, Susan M; Church, George M

2015-01-01

CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem or induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe an optimized protocol for genome engineering of human iPSCs using a simple transient transfection of plasmids and/or single-stranded oligonucleotides. With this protocol, we achieve transfection efficiencies greater than 60%, with gene disruption efficiencies from 1-25% and gene insertion/replacement efficiencies from 0.5-10% without any further selection or enrichment steps. We also describe how to design and assess optimal sgRNA target sites and donor targeting vectors; cloning individual iPSC by single cell FACS sorting, and genotyping successfully edited cells.

Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

PubMed Central

Li, Minghui; Yang, Huihui; Zhao, Jiue; Fang, Lingling; Shi, Hongjuan; Li, Mengru; Sun, Yunlv; Zhang, Xianbo; Jiang, Dongneng; Zhou, Linyan; Wang, Deshou

2014-01-01

Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F1. Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species. PMID:24709635

Specific genetic modifications of domestic animals by gene targeting and animal cloning

PubMed Central

Wang, Bin; Zhou, Jiangfeng

2003-01-01

The technology of gene targeting through homologous recombination has been extremely useful for elucidating gene functions in mice. The application of this technology was thought impossible in the large livestock species until the successful creation of the first mammalian clone "Dolly" the sheep. The combination of the technologies for gene targeting of somatic cells with those of animal cloning made it possible to introduce specific genetic mutations into domestic animals. In this review, the principles of gene targeting in somatic cells and the challenges of nuclear transfer using gene-targeted cells are discussed. The relevance of gene targeting in domestic animals for applications in bio-medicine and agriculture are also examined. PMID:14614774

Novel β-catenin target genes identified in thalamic neurons encode modulators of neuronal excitability

PubMed Central

2012-01-01

Background LEF1/TCF transcription factors and their activator β-catenin are effectors of the canonical Wnt pathway. Although Wnt/β-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by β-catenin in neurons. We recently showed that β-catenin accumulates specifically in thalamic neurons where it activates Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new β-catenin targets in the adult thalamus. Results We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear β-catenin from thalamic neurons in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between β-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of β-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca2+-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by β-catenin, although the binding of β-catenin to the

Oncogenic mutations in KEAP1 disturbing inhibitory Nrf2-Keap1 interaction: Activation of antioxidative pathway in papillary thyroid carcinoma.

PubMed

Danilovic, Debora Lucia Seguro; de Mello, Evandro Sobroza; Frazzato, Eliana Salgado Turri; Wakamatsu, Alda; de Lima Jorge, Alexander Augusto; Hoff, Ana Oliveira; Marui, Suemi

2018-06-01

Nuclear factor erythroid 2-like 2 (NFE2L2) encodes Nrf2, transcription factor of antioxidative genes. In the presence of reactive oxygen species, Keap1 (Kelch-ECH-associating protein-1) inhibitor complex undergoes conformational changes disrupting Keap1-Nrf2 binding and Nrf2 translocates into nucleus. We evaluated the presence of mutations in NFE2L2 and KEAP1 in papillary thyroid carcinomas (PTCs) and correlated them with clinical presentation. Coding regions of NFE2L2 and KEAP1 were sequenced in 131 patients with PTC. Clinical and histopathological features were analyzed. Immunohistochemical analysis of Nrf2 expression was performed in mutated carcinomas. Although no mutations were found in NFE2L2, missense mutations in KEAP1 were observed in 6 patients with PTC (4.6%). Immunohistochemistry showed increased Nrf2 expression in nuclei of all mutated carcinomas, which presented poor prognostic features in histopathology. We identified mutations in KEAP1 associated with Nrf2 overexpression in PTC. Mutations favored disruption of inhibitory interaction Nrf2-Keap1 to enable increased antioxidant Nrf2 activity, possibly with prognostic consequences. © 2018 Wiley Periodicals, Inc.

Targeted gene disruption in Koji mold Aspergillus oryzae.

PubMed

Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

2011-01-01

Filamentous fungi have received attentions as hosts for heterologous protein production because of their high secretion capability and eukaryotic post-translational modifications. One of the safest hosts for heterologous protein production is Koji mold Aspergillus oryzae since it has been used in the production of Japanese fermented foods for over 1,000 years. The production levels of proteins from higher eukaryotes are much lower than those of homologous (fungal) proteins. Bottlenecks in the heterologous protein production are suggested to be proteolytic degradation of the produced protein in the medium and the secretory pathway. For construction of excellent host strains, many genes causing the bottlenecks should be disrupted rapidly and efficiently. We developed a marker recycling system with the highly efficient gene-targeting background in A. oryzae. By employing this technique, we performed multiple gene disruption of the ten protease genes. The decuple protease gene disruptant showed fourfold production level of a heterologous protein compared with the wild-type strain.

Id-1 and Id-2 genes and products as therapeutic targets for treatment of breast cancer and other types of carcinoma

DOEpatents

Desprez, Pierre-Yves; Campisi, Judith

2014-09-30

A method for treatment and amelioration of breast, cervical, ovarian, endometrial, squamous cells, prostate cancer and melanoma in a patient comprising targeting Id-1 or Id-2 gene expression with a delivery vehicle comprising a product which modulates Id-1 or Id-2 expression.

New target genes in endometrial tumors show a role for the estrogen-receptor pathway in microsatellite-unstable cancers.

PubMed

Ferreira, Ana M; Tuominen, Iina; Sousa, Sónia; Gerbens, Frans; van Dijk-Bos, Krista; Osinga, Jan; Kooi, Krista A; Sanjabi, Bahram; Esendam, Chris; Oliveira, Carla; Terpstra, Peter; Hardonk, Menno; van der Sluis, Tineke; Zazula, Monika; Stachura, Jerzy; van der Zee, Ate G; Hollema, Harry; Sijmons, Rolf H; Aaltonen, Lauri A; Seruca, Raquel; Hofstra, Robert M W; Westers, Helga

2014-12-01

Microsatellite instability (MSI) in tumors results in an accumulation of mutations in (target) genes. Previous studies suggest that the profile of target genes differs according to tumor type. This paper describes the first genome-wide search for target genes for mismatch repair-deficient endometrial cancers. Genes expressed in normal endometrium containing coding repeats were analyzed for mutations in tumors. We identified 44 possible genes of which seven are highly mutated (>15%). Some candidates were also found mutated in colorectal and gastric tumors. The most frequently mutated gene, NRIP1 encoding nuclear receptor-interacting protein 1, was silenced in an endometrial tumor cell line and expression microarray experiments were performed. Silencing of NRIP1 was associated with differences in the expression of several genes in the estrogen-receptor network. Furthermore, an enrichment of genes related to cell cycle (regulation) and replication was observed. We present a new profile of target genes, some of them tissue specific, whereas others seem to play a more general role in MSI tumors. The high-mutation frequency combined with the expression data suggest, for the first time, an involvement of NRIP1 in endometrial cancer development. © 2014 WILEY PERIODICALS, INC.

The tumor suppressor TERE1 (UBIAD1) prenyltransferase regulates the elevated cholesterol phenotype in castration resistant prostate cancer by controlling a program of ligand dependent SXR target genes

PubMed Central

Fredericks, William J.; Sepulveda, Jorge; Lal, Priti; Tomaszewski, John E.; Lin, Ming-Fong; McGarvey, Terry; Rauscher, Frank J; Malkowicz, S. Bruce

2013-01-01

Castrate-Resistant Prostate Cancer (CRPC) is characterized by persistent androgen receptor-driven tumor growth in the apparent absence of systemic androgens. Current evidence suggests that CRPC cells can produce their own androgens from endogenous sterol precursors that act in an intracrine manner to stimulate tumor growth. The mechanisms by which CRPC cells become steroidogenic during tumor progression are not well defined. Herein we describe a novel link between the elevated cholesterol phenotype of CRPC and the TERE1 tumor suppressor protein, a prenyltransferase that synthesizes vitamin K-2, which is a potent endogenous ligand for the SXR nuclear hormone receptor. We show that 50% of primary and metastatic prostate cancer specimens exhibit a loss of TERE1 expression and we establish a correlation between TERE1 expression and cholesterol in the LnCaP-C81 steroidogenic cell model of the CRPC. LnCaP-C81 cells also lack TERE1 protein, and show elevated cholesterol synthetic rates, higher steady state levels of cholesterol, and increased expression of enzymes in the de novo cholesterol biosynthetic pathways than the non-steroidogenic prostate cancer cells. C81 cells also show decreased expression of the SXR nuclear hormone receptor and a panel of directly regulated SXR target genes that govern cholesterol efflux and steroid catabolism. Thus, a combination of increased synthesis, along with decreased efflux and catabolism likely underlies the CRPC phenotype: SXR might coordinately regulate this phenotype. Moreover, TERE1 controls synthesis of vitamin K-2, which is a potent endogenous ligand for SXR activation, strongly suggesting a link between TERE1 levels, K-2 synthesis and SXR target gene regulation. We demonstrate that following ectopic TERE1 expression or induction of endogenous TERE1, the elevated cholesterol levels in C81 cells are reduced. Moreover, reconstitution of TERE1 expression in C81 cells reactivates SXR and switches on a suite of SXR target genes that

Cyclic voltammetric analysis of C 1-C 4 alcohol electrooxidations with Pt/C and Pt-Ru/C microporous electrodes

NASA Astrophysics Data System (ADS)

Lee, Choong-Gon; Umeda, Minoru; Uchida, Isamu

The effect of temperature on methanol, ethanol, 2-propanol, and 2-butanol electrooxidation is investigated with Pt/C and Pt-Ru/C microporous electrodes. Cyclic voltammetry is employed in temperatures ranging from 25 to 80 °C to provide quantitative and qualitative information on the kinetics of alcohol oxidation. Methanol displays the greatest activity atom alcohols. The addition of ruthenium reduces the poisoning effect, although it is ineffective with secondary alcohols. Secondary alcohols undergo a different oxidation mechanism at higher temperatures. Microporous electrodes provide detailed information on alcohol oxidation.

The human RHOX gene cluster: target genes and functional analysis of gene variants in infertile men.

PubMed

Borgmann, Jennifer; Tüttelmann, Frank; Dworniczak, Bernd; Röpke, Albrecht; Song, Hye-Won; Kliesch, Sabine; Wilkinson, Miles F; Laurentino, Sandra; Gromoll, Jörg

2016-11-15

The X-linked reproductive homeobox (RHOX) gene cluster encodes transcription factors preferentially expressed in reproductive tissues. This gene cluster has important roles in male fertility based on phenotypic defects of Rhox-mutant mice and the finding that aberrant RHOX promoter methylation is strongly associated with abnormal human sperm parameters. However, little is known about the molecular mechanism of RHOX function in humans. Using gene expression profiling, we identified genes regulated by members of the human RHOX gene cluster. Some genes were uniquely regulated by RHOXF1 or RHOXF2/2B, while others were regulated by both of these transcription factors. Several of these regulated genes encode proteins involved in processes relevant to spermatogenesis; e.g. stress protection and cell survival. One of the target genes of RHOXF2/2B is RHOXF1, suggesting cross-regulation to enhance transcriptional responses. The potential role of RHOX in human infertility was addressed by sequencing all RHOX exons in a group of 250 patients with severe oligozoospermia. This revealed two mutations in RHOXF1 (c.515G > A and c.522C > T) and four in RHOXF2/2B (-73C > G, c.202G > A, c.411C > T and c.679G > A), of which only one (c.202G > A) was found in a control group of men with normal sperm concentration. Functional analysis demonstrated that c.202G > A and c.679G > A significantly impaired the ability of RHOXF2/2B to regulate downstream genes. Molecular modelling suggested that these mutations alter RHOXF2/F2B protein conformation. By combining clinical data with in vitro functional analysis, we demonstrate how the X-linked RHOX gene cluster may function in normal human spermatogenesis and we provide evidence that it is impaired in human male fertility.

Cancer gene therapy with targeted adenoviruses.

PubMed

Bachtarzi, Houria; Stevenson, Mark; Fisher, Kerry

2008-11-01

Clinical experience with adenovirus vectors has highlighted the need for improved delivery and targeting. This manuscript aims to provide an overview of the techniques currently under development for improving adenovirus delivery to malignant cells in vivo. Primary research articles reporting improvements in adenoviral gene delivery are described. Strategies include genetic modification of viral coat proteins, non-genetic modifications including polymer encapsulation approaches and pharmacological interventions. Reprogramming adenovirus tropism in vitro has been convincingly demonstrated using a range of genetic and physical strategies. These studies have provided new insights into our understanding of virology and the field is progressing. However, there are still some limitations that need special consideration before adenovirus-targeted cancer gene therapy emerges as a routine treatment in the clinical setting.

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92

PubMed Central

Jin, Hyun Yong; Oda, Hiroyo; Chen, Pengda; Kang, Seung Goo; Valentine, Elizabeth; Liao, Lujian; Zhang, Yaoyang; Gonzalez-Martin, Alicia; Shepherd, Jovan; Head, Steven R.; Kim, Pyeung-Hyeun; Fu, Guo; Liu, Wen-Hsien; Han, Jiahuai

2017-01-01

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes. PMID:28241004

MicroRNA expression, target genes, and signaling pathways in infants with a ventricular septal defect.

PubMed

Chai, Hui; Yan, Zhaoyuan; Huang, Ke; Jiang, Yuanqing; Zhang, Lin

2018-02-01

This study aimed to systematically investigate the relationship between miRNA expression and the occurrence of ventricular septal defect (VSD), and characterize the miRNA target genes and pathways that can lead to VSD. The miRNAs that were differentially expressed in blood samples from VSD and normal infants were screened and validated by implementing miRNA microarrays and qRT-PCR. The target genes regulated by differentially expressed miRNAs were predicted using three target gene databases. The functions and signaling pathways of the target genes were enriched using the GO database and KEGG database, respectively. The transcription and protein expression of specific target genes in critical pathways were compared in the VSD and normal control groups using qRT-PCR and western blotting, respectively. Compared with the normal control group, the VSD group had 22 differentially expressed miRNAs; 19 were downregulated and three were upregulated. The 10,677 predicted target genes participated in many biological functions related to cardiac development and morphogenesis. Four target genes (mGLUR, Gq, PLC, and PKC) were involved in the PKC pathway and four (ECM, FAK, PI3 K, and PDK1) were involved in the PI3 K-Akt pathway. The transcription and protein expression of these eight target genes were significantly upregulated in the VSD group. The 22 miRNAs that were dysregulated in the VSD group were mainly downregulated, which may result in the dysregulation of several key genes and biological functions related to cardiac development. These effects could also be exerted via the upregulation of eight specific target genes, the subsequent over-activation of the PKC and PI3 K-Akt pathways, and the eventual abnormal cardiac development and VSD.

Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma.

PubMed

Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

2016-04-01

Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P < 0.05), reduced expression of proliferation marker (PCNA) (P < 0.05), induced expression of apoptotic protein, c-PARP-1, (P < 0.05) and inhibited expression of extracellular matrix-related genes (TGF beta 3) and angiogenesis-related genes (VEGF & IGF-1) (P < 0.01). There were no detectable adenovirus in tested tissues other than leiomyoma lesions with both targeted and untargeted adenovirus. Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. © The Author(s) 2016.

Target mimics: an embedded layer of microRNA-involved gene regulatory networks in plants.

PubMed

Meng, Yijun; Shao, Chaogang; Wang, Huizhong; Jin, Yongfeng

2012-05-21

MicroRNAs (miRNAs) play an essential role in gene regulation in plants. At the same time, the expression of miRNA genes is also tightly controlled. Recently, a novel mechanism called "target mimicry" was discovered, providing another layer for modulating miRNA activities. However, except for the artificial target mimics manipulated for functional studies on certain miRNA genes, only one example, IPS1 (Induced by Phosphate Starvation 1)-miR399 was experimentally confirmed in planta. To date, few analyses for comprehensive identification of natural target mimics have been performed in plants. Thus, limited evidences are available to provide detailed information for interrogating the questionable issue whether target mimicry was widespread in planta, and implicated in certain biological processes. In this study, genome-wide computational prediction of endogenous miRNA mimics was performed in Arabidopsis and rice, and dozens of target mimics were identified. In contrast to a recent report, the densities of target mimic sites were found to be much higher within the untranslated regions (UTRs) when compared to those within the coding sequences (CDSs) in both plants. Some novel sequence characteristics were observed for the miRNAs that were potentially regulated by the target mimics. GO (Gene Ontology) term enrichment analysis revealed some functional insights into the predicted mimics. After degradome sequencing data-based identification of miRNA targets, the regulatory networks constituted by target mimics, miRNAs and their downstream targets were constructed, and some intriguing subnetworks were further exploited. These results together suggest that target mimicry may be widely implicated in regulating miRNA activities in planta, and we hope this study could expand the current understanding of miRNA-involved regulatory networks.

Regulation of p53 Target Gene Expression by Peptidylarginine Deiminase 4 ▿ †

PubMed Central

Li, Pingxin; Yao, Hongjie; Zhang, Zhiqiang; Li, Ming; Luo, Yuan; Thompson, Paul R.; Gilmour, David S.; Wang, Yanming

2008-01-01

Histone Arg methylation has been correlated with transcriptional activation of p53 target genes. However, whether this modification is reversed to repress the expression of p53 target genes is unclear. Here, we report that peptidylarginine deiminase 4, a histone citrullination enzyme, is involved in the repression of p53 target genes. Inhibition or depletion of PAD4 elevated the expression of a subset of p53 target genes, including p21/CIP1/WAF1, leading to cell cycle arrest and apoptosis. Moreover, the induction of p21, cell cycle arrest, and apoptosis by PAD4 depletion is p53 dependent. Protein-protein interaction studies showed an interaction between p53 and PAD4. Chromatin immunoprecipitation assays showed that PAD4 is recruited to the p21 promoter in a p53-dependent manner. RNA polymerase II (Pol II) activities and the association of PAD4 are dynamically regulated at the p21 promoter during UV irradiation. Paused RNA Pol II and high levels of PAD4 were detected before UV treatment. At early time points after UV treatment, an increase of histone Arg methylation and a decrease of citrullination were correlated with a transient activation of p21. At later times after UV irradiation, a loss of RNA Pol II and an increase of PAD4 were detected at the p21 promoter. The dynamics of RNA Pol II activities after UV treatment were further corroborated by permanganate footprinting. Together, these results suggest a role of PAD4 in the regulation of p53 target gene expression. PMID:18505818

Detection of Genes Regulated by Lmx1b During Limb Dorsalization

PubMed Central

Feenstra, Jennifer M.; Kanaya, Kohei; Pira, Charmaine U; Hoffman, Sarah E.; Eppey, Richard J.; Oberg, Kerby C.

2012-01-01

Lmx1b is a homeodomain transcription factor that regulates dorsal identity during limb development. Lmx1b knockout (KO) mice develop distal ventral-ventral limbs. Although induction of Lmx1b is linked to Wnt7a expression in the dorsal limb ectoderm, the downstream targets of Lmx1b that accomplish limb dorsalization are unknown. To identify genes targeted by Lmx1b, we compared gene arrays from Lmx1b KO and wildtype mouse limbs during limb dorsalization, i.e., 11.5, 12.5, and 13.5 days post coitum. We identified 54 target genes differentially expressed in all three stages. Several skeletal targets, including Emx2, Matrilin1 and Matrilin4, demonstrated a loss of scapular expression in the Lmx1b KO mice, supporting a role for Lmx1b in scapula development. Furthermore, the relative abundance of extracellular matrix-related soft tissue targets regulated by Lmx1b, such as collagens and proteoglycans, suggests a mechanism which includes changes in the extracellular matrix composition to accomplish limb dorsalization. Our study provides the most comprehensive characterization of genes regulated by Lmx1b during limb development to-date and provides targets for further investigation. PMID:22417325

RET/PTC and PAX8/PPARγ chromosomal rearrangements in post-Chernobyl thyroid cancer and their association with I-131 radiation dose and other characteristics

PubMed Central

Leeman-Neill, Rebecca J.; Brenner, Alina V.; Little, Mark P.; Bogdanova, Tetiana I.; Hatch, Maureen; Zurnadzy, Liudmyla Y.; Mabuchi, Kiyohiko; Tronko, Mykola D.; Nikiforov, Yuri E.

2012-01-01

Background Childhood exposure to I-131 from the 1986 Chernobyl accident led to a sharp increase in papillary thyroid carcinoma (PTC) incidence in regions surrounding the reactor. Data concerning the association between genetic mutations in PTCs and individual radiation doses are limited. Methods We performed mutational analysis of 62 PTCs diagnosed in a Ukrainian cohort of patients who were <18 y.o. in 1986 and received 0.008-8.6 Gy of I-131 to the thyroid and explored associations between mutation types and I-131 dose and other characteristics. Results RET/PTC rearrangements were most common (35%), followed by BRAF (15%) and RAS (8%) point mutations. Two tumors carrying PAX8/PPARγ rearrangement were identified. We found a significant negative association with I-131 dose for BRAF and RAS point mutations and a significant concave association with I-131 dose, with an inflection point at 1.6 Gy and odds ratio 2.1, based on a linear-quadratic model for RET/PTC and PAX8/PPARγ rearrangements. The trends with dose were significantly different between tumors with point mutations and rearrangements. Compared to point mutations, rearrangements were associated with residence in the relatively iodine deficient Zhytomyr region, younger age at exposure or surgery, and male gender. Conclusions Our results provide the first demonstration of PAX8/PPARγ rearrangements in post-Chernobyl tumors and show different associations for point mutations and chromosomal rearrangements with I-131 dose and other factors. These data support the relationship between chromosomal rearrangements, but not point mutations, and I-131 exposure and point to a possible role of iodine deficiency in generation of RET/PTC rearrangements in these patients. PMID:23436219

CRISPR-mediated HDAC2 disruption identifies two distinct classes of target genes in human cells.

PubMed

Somanath, Priyanka; Herndon Klein, Rachel; Knoepfler, Paul S

2017-01-01

The transcriptional functions of the class I histone deacetylases (HDACs) HDAC1 and HDAC2 are mainly viewed as both repressive and redundant based on murine knockout studies, but they may have additional independent roles and their physiological functions in human cells are not as clearly defined. To address the individual epigenomic functions of HDAC2, here we utilized CRISPR-Cas9 to disrupt HDAC2 in human cells. We find that while HDAC2 null cells exhibited signs of cross-regulation between HDAC1 and HDAC2, specific epigenomic phenotypes were still apparent using RNA-seq and ChIP assays. We identified specific targets of HDAC2 repression, and defined a novel class of genes that are actively expressed in a partially HDAC2-dependent manner. While HDAC2 was required for the recruitment of HDAC1 to repressed HDAC2-gene targets, HDAC2 was dispensable for HDAC1 binding to HDAC2-activated targets, supporting the notion of distinct classes of targets. Both active and repressed classes of gene targets demonstrated enhanced histone acetylation and methylation in HDAC2-null cells. Binding of the HDAC1/2-associated SIN3A corepressor was altered at most HDAC2-targets, but without a clear pattern. Overall, our study defines two classes of HDAC2 targets in human cells, with a dependence of HDAC1 on HDAC2 at one class of targets, and distinguishes unique functions for HDAC2.

Targeted gene deletion of miRNAs in mice by TALEN system.

PubMed

Takada, Shuji; Sato, Tempei; Ito, Yoshiaki; Yamashita, Satoshi; Kato, Tomoko; Kawasumi, Miyuri; Kanai-Azuma, Masami; Igarashi, Arisa; Kato, Tomomi; Tamano, Moe; Asahara, Hiroshi

2013-01-01

Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.

Mitochondrial Gene Therapy: Advances in Mitochondrial Gene Cloning, Plasmid Production, and Nanosystems Targeted to Mitochondria.

PubMed

Coutinho, Eduarda; Batista, Cátia; Sousa, Fani; Queiroz, João; Costa, Diana

2017-03-06

Mitochondrial gene therapy seems to be a valuable and promising strategy to treat mitochondrial disorders. The use of a therapeutic vector based on mitochondrial DNA, along with its affinity to the site of mitochondria, can be considered a powerful tool in the reestablishment of normal mitochondrial function. In line with this and for the first time, we successfully cloned the mitochondrial gene ND1 that was stably maintained in multicopy pCAG-GFP plasmid, which is used to transform E. coli. This mitochondrial-gene-based plasmid was encapsulated into nanoparticles. Furthermore, the functionalization of nanoparticles with polymers, such as cellulose or gelatin, enhances their overall properties and performance for gene therapy. The fluorescence arising from rhodamine nanoparticles in mitochondria and a fluorescence microscopy study show pCAG-GFP-ND1-based nanoparticles' cell internalization and mitochondria targeting. The quantification of GFP expression strongly supports this finding. This work highlights the viability of gene therapy based on mitochondrial DNA instigating further in vitro research and clinical translation.

MicroRNA-320 suppresses colorectal cancer by targeting SOX4, FOXM1, and FOXQ1

PubMed Central

Vishnubalaji, Radhakrishnan; Hamam, Rimi; Shijun, Yue; Al-Obeed, Omar; Kassem, Moustapha; Liu, Fei-Fei; Aldahmash, Abdullah; Alajez, Nehad M.

2016-01-01

Colorectal cancer (CRC) is the third most common cancer causing high mortality rates world-wide. Delineating the molecular mechanisms leading to CRC development and progression, including the role of microRNAs (miRNAs), are currently being unravelled at a rapid rate. Here, we report frequent downregulation of the microRNA miR-320 family in primary CRC tissues and cell lines. Lentiviral-mediated re-expression of miR-320c (representative member of the miR-320 family) inhibited HCT116 CRC growth and migration in vitro, sensitized CRC cells to 5-Fluorouracil (5-FU), and inhibited tumor formation in SCID mice. Global gene expression analysis in CRC cells over-expressing miR-320c, combined with in silico prediction identified 84 clinically-relevant potential gene targets for miR-320 in CRC. Using a series of biochemical assays and functional validation, SOX4, FOXM1, and FOXQ1 were validated as novel gene targets for the miR-320 family. Inverse correlation between the expression of miR-320 members with SOX4, FOXM1, and FOXQ1 was observed in primary CRC patients' specimens, suggesting that these genes are likely bona fide targets for the miR-320 family. Interestingly, interrogation of the expression levels of this gene panel (SOX4, FOXM1, and FOXQ1) in The Cancer Genome Atlas (TCGA) colorectal cancer data set (319 patients) revealed significantly poor disease-free survival in patients with elevated expression of this gene panel (P-Value: 0.0058). Collectively, our data revealed a novel role for the miR-320/SOX4/FOXM1/FOXQ1 axes in promoting CRC development and progression and suggest targeting those networks as potential therapeutic strategy for CRC. PMID:27119506

HEAT-INDUCED TAS1 TARGET1 Mediates Thermotolerance via HEAT STRESS TRANSCRIPTION FACTOR A1a–Directed Pathways in Arabidopsis[C][W

PubMed Central

Li, Shuxia; Liu, Jinxin; Liu, Zhongyuan; Li, Xiaorong; Wu, Feijie; He, Yuke

2014-01-01

Many heat stress transcription factors (Hsfs) and heat shock proteins (Hsps) have been identified to play important roles in the heat tolerance of plants. However, many of the key factors mediating the heat response pathways remain unknown. Here, we report that two genes, which are targets of TAS1 (trans-acting siRNA precursor 1)–derived small interfering RNAs that we named HEAT-INDUCED TAS1 TARGET1 (HTT1) and HTT2, are involved in thermotolerance. Microarray analysis revealed that the HTT1 and HTT2 genes were highly upregulated in Arabidopsis thaliana seedlings in response to heat shock. Overexpression of TAS1a, whose trans-acting small interfering RNAs target the HTT genes, elevated accumulation of TAS1-siRNAs and reduced expression levels of the HTT genes, causing weaker thermotolerance. By contrast, overexpression of HTT1 and HTT2 upregulated several Hsf genes, leading to stronger thermotolerance. In heat-tolerant plants overexpressing HsfA1a, the HTT genes were upregulated, especially at high temperatures. Meanwhile, HsfA1a directly activated HTT1 and HTT2 through binding to their promoters. HTT1 interacted with the heat shock proteins Hsp70-14 and Hsp40 and NUCLEAR FACTOR Y, SUBUNIT C2. Taken together, these results suggest that HTT1 mediates thermotolerance pathways because it is targeted by TAS1a, mainly activated by HsfA1a, and acts as cofactor of Hsp70-14 complexes. PMID:24728648

Genome-wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ueda, Kohei; Fujiki, Katsunori; Shirahige, Katsuhiko

Highlights: • We define a target gene of MR as that with MR-binding to the adjacent region of DNA. • We use ChIP-seq analysis in combination with microarray. • We, for the first time, explore the genome-wide binding profile of MR. • We reveal 5 genes as the direct target genes of MR in the renal epithelial cell-line. - Abstract: Background and objective: Mineralocorticoid receptor (MR) is a member of nuclear receptor family proteins and contributes to fluid homeostasis in the kidney. Although aldosterone-MR pathway induces several gene expressions in the kidney, it is often unclear whether the gene expressionsmore » are accompanied by direct regulations of MR through its binding to the regulatory region of each gene. The purpose of this study is to identify the direct target genes of MR in a murine distal convoluted tubular epithelial cell-line (mDCT). Methods: We analyzed the DNA samples of mDCT cells overexpressing 3xFLAG-hMR after treatment with 10{sup −7} M aldosterone for 1 h by chromatin immunoprecipitation with deep-sequence (ChIP-seq) and mRNA of the cell-line with treatment of 10{sup −7} M aldosterone for 3 h by microarray. Results: 3xFLAG-hMR overexpressed in mDCT cells accumulated in the nucleus in response to 10{sup −9} M aldosterone. Twenty-five genes were indicated as the candidate target genes of MR by ChIP-seq and microarray analyses. Five genes, Sgk1, Fkbp5, Rasl12, Tns1 and Tsc22d3 (Gilz), were validated as the direct target genes of MR by quantitative RT-qPCR and ChIP-qPCR. MR binding regions adjacent to Ctgf and Serpine1 were also validated. Conclusions: We, for the first time, captured the genome-wide distribution of MR in mDCT cells and, furthermore, identified five MR target genes in the cell-line. These results will contribute to further studies on the mechanisms of kidney diseases.« less

RNAi targeting GPR4 influences HMEC-1 gene expression by microarray analysis

PubMed Central

Ren, Juan; Zhang, Yuelang; Cai, Hui; Ma, Hongbing; Zhao, Dongli; Zhang, Xiaozhi; Li, Zongfang; Wang, Shufeng; Wang, Jiangsheng; Liu, Rui; Li, Yi; Qian, Jiansheng; Wei, Hongxia; Niu, Liying; Liu, Yan; Xiao, Lisha; Ding, Muyang; Jiang, Shiwen

2014-01-01

G-protein coupled receptor 4 (GPR4) belongs to a protein family comprised of 3 closely related G protein-coupled receptors. Recent studies have shown that GPR4 plays important roles in angiogenesis, proton sensing, and regulating tumor cells as an oncogenic gene. How GPR4 conducts its functions? Rare has been known. In order to detect the genes related to GPR4, microarray technology was employed. GPR4 is highly expressed in human vascular endothelial cell HMEC-1. Small interfering RNA against GPR4 was used to knockdown GPR4 expression in HMEC-1. Then RNA from the GPR4 knockdown cells and control cells were analyzed through genome microarray. Microarray results shown that among the whole genes and expressed sequence tags, 447 differentially expressed genes were identified, containing 318 up-regulated genes and 129 down-regulated genes. These genes whose expression dramatically changed may be involved in the GPR4 functions. These genes were related to cell apoptosis, cytoskeleton and signal transduction, cell proliferation, differentiation and cell-cycle regulation, gene transcription and translation and cell material and energy metabolism. PMID:24753754

The role of two families of bacterial enzymes in putrescine synthesis from agmatine via agmatine deiminase.

PubMed

Landete, José M; Arena, Mario E; Pardo, Isabel; Manca de Nadra, María C; Ferrer, Sergi

2010-12-01

Putrescine, one of the main biogenic amines associated to microbial food spoilage, can be formed by bacteria from arginine via ornithine decarboxylase (ODC), or from agmatine via agmatine deiminase (AgDI). This study aims to correlate putrescine production from agmatine to the pathway involving N-carbamoylputrescine formation via AdDI (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), or putrescine carbamoyltransferase (the ptcA product) in bacteria. PCR methods were developed to detect the two genes involved in putrescine production from agmatine. Putrescine production from agmatine could be linked to the aguA and ptcA genes in Lactobacillus hilgardii X1B, Enterococcus faecalis ATCC 11700, and Bacillus cereus ATCC 14579. By contrast Lactobacillus sakei 23K was unable to produce putrescine, and although a fragment of DNA corresponding to the gene aguA was amplified, no amplification was observed for the ptcA gene. Pseudomonas aeruginosa PAO1 produces putrescine and is reported to harbour aguA and aguB genes, responsible for agmatine deiminase and N-carbamoylputrescine amidohydrolase activities. The enzyme from P. aeruginosa PAO1 that converts N-carbamoylputrescine to putrescine (the aguB product) is different from other microorganisms studied (the ptcA product). Therefore, the aguB gene from P. aeruginosa PAO1 could not be amplified with ptcA-specific primers. The aguB and ptcA genes have frequently been erroneously annotated in the past, as in fact these two enzymes are neither homologous nor analogous. Furthermore, the aguA, aguB and ptcA sequences available from GenBank were subjected to phylogenetic analysis, revealing that gram-positive bacteria harboured ptcA, whereas gram-negative bacteria harbour aguB. This paper also discusses the role of the agmatine deiminase system (AgDS) in acid stress resistance.

Acute morphine effects on respiratory activity in mice with target deletion of the tachykinin 1 gene (Tac1-/-).

PubMed

Shvarev, Yuri; Berner, Jonas; Bilkei-Gorzo, Andras; Lagercrantz, Hugo; Wickström, Ronny

2010-01-01

Search for physiological mechanisms which could antagonize the opioid-induced respiratory depression is of important clinical value. In this study, we investigated the acute effects of morphine on respiratory activity in genetically modified newborn (P2) mice with target deletion of the (Tac1 -/-) gene lacking substance P (SP) and neurokinin A (NKA). In vivo, as shown with whole-body flow barometric plethysmography technique, morphine induced significantly attenuated minute ventilation during intermittent hypoxia in control animals. In contrast, knockout mice revealed significant increase in minute ventilation. In vitro, in brainstem preparation, knockout mice demonstrated greater changes in burst frequency during intermittent anoxia challenge. The data suggest that hereditary deficiency in tachykinins, SP and NKA results in more robust hypoxic response in newborn Tac1-/- mice during respiratory depression induced by morphine.

RNA-guided genome editing for target gene mutations in wheat.

PubMed

Upadhyay, Santosh Kumar; Kumar, Jitesh; Alok, Anshu; Tuli, Rakesh

2013-12-09

The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas-mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3' end of the target site abolished the cleavage activity completely. The mismatches at the 5' end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3' end. This approach provides a powerful method for genome engineering in plants.

GTSE1: a novel TEAD4-E2F1 target gene involved in cell protrusions formation in triple-negative breast cancer cell models

PubMed Central

Stelitano, Debora; Leticia, Yamila Peche; Dalla, Emiliano; Monte, Martin; Piazza, Silvano; Schneider, Claudio

2017-01-01

GTSE1 over-expression has been reported as a potential marker for metastasis in various types of malignancies, including breast cancer. Despite this, the transcriptional regulation of this protein and the causes of its misregulation in tumors remain largely unknown. The aims of this work were to elucidate how GTSE1 is regulated at the transcriptional level and to clarify the mechanism underlying GTSE1-dependent cell functions in triple-negative breast cancer (TNBC). Here, we identified GTSE1 as a novel target gene of the TEAD4 transcription factor, highlighting a role for the YAP and TAZ coactivators in the transcriptional regulation of GTSE1. Moreover, we found that TEAD4 controls the formation of cell protrusions required for cell migration through GTSE1, unveiling a relevant effector role for this protein in the TEAD-dependent cellular functions and confirming TEAD4 role in promoting invasion and metastasis in breast cancer. Finally, we highlighted a role for the pRb-E2F1 pathway in the control of GTSE1 transcription and observed that treatment with drugs targeting the pRb-E2F1 or YAP/TAZ-TEAD pathways dramatically downregulated the expression levels of GTSE1 and of other genes involved in the formation of metastasis, suggesting their potential use in the treatment of TNBC. PMID:28978043

Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

PubMed

Tolmachov, Oleg E

2015-01-01

Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo

Nrf2 target genes are induced under marginal selenium-deficiency

PubMed Central

Müller, Mike; Banning, Antje; Brigelius-Flohé, Regina

2010-01-01

A suboptimal selenium supply appears to prevail in Europe. The current study, therefore, was focused on the changes in gene expression under a suboptimal selenium intake. Previous microarray analyses in the colon of mice fed either a selenium-adequate or a moderately deficient diet revealed a change in genes of several pathways. Severe selenium-deficiency has been found previously to influence Nrf2-regulated genes of the adaptive response. Since the previous pathway analyses were done with a program not searching for Nrf2 target genes, respective genes were manually selected and confirmed by qPCR. qPCR revealed an induction of phase II (Nqo1, Gsts, Sult1b1 and Ugt1a6) and antioxidant enzymes (Hmox1, Mt2, Prdx1, Srxn1, Sod1 and Gclc) under the selenium-poor diet, which is considered to compensate for the loss of selenoproteins. The strongest effects were observed in the duodenum where preferentially genes for antioxidant enzymes were up-regulated. These also include the mRNA of the selenoproteins TrxR1 and GPx2 that would enable their immediate translation upon selenium refeeding. The down-regulation of Gsk3β in moderate selenium-deficiency observed in the previous paper provides a possible explanation for the activation of the Nrf2 pathway, because inhibition of GSK3β results in the nuclear accumulation of Nrf2. PMID:21189866

Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma

PubMed Central

Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P.; Al-Hendy, Ayman

2016-01-01

Background: Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. Study design: An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Materials and methods: Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. Results: In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P < 0.05), reduced expression of proliferation marker (PCNA) (P < 0.05), induced expression of apoptotic protein, c-PARP-1, (P < 0.05) and inhibited expression of extracellular matrix-related genes (TGF beta 3) and angiogenesis-related genes (VEGF & IGF-1) (P < 0.01). There were no detectable adenovirus in tested tissues other than leiomyoma lesions with both targeted and untargeted adenovirus. Conclusion: Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. PMID:26884457

Taste Quality Confusions: Influences of Age, Smoking, PTC Taster Status, and other Subject Characteristics.

PubMed

Doty, Richard L; Chen, Jonathan H; Overend, Jane

2017-01-01

Many persons misidentify the quality of taste stimuli, a phenomenon termed "taste confusion." In this study of 1000 persons, we examined the influences of age, sex, causes of chemosensory disturbances, and genetically determined phenylthiocarbamide (PTC) taster status on taste quality confusions for four tastants (sucrose, citric acid, sodium chloride, caffeine). Overall, sour-bitter confusions were most common (19.3%), followed by bitter-sour (11.4%), salty-bitter (7.3%), salty-sour (7.0%), bitter-salty (3.5%), bitter-sweet (3.4), and sour-salty (2.4%) confusions. Confusions for sweet were <1%. Asymmetries were common (e.g., bitter-sour confusions were less frequent than sour-bitter confusions). Women had fewer salty-bitter confusions than did men (5.7% vs. 11.4%). Overall, PTC tasters had fewer confusions than non-tasters except for salty-bitter confusions. Confusions typically increased monotonically with age. Current smokers exhibited more sour-bitter confusions than never smokers (48.9% vs. 32.2%), whereas past smokers had more bitter-sour confusions than never smokers (23.8% vs. 14.2%). Previous head trauma was associated with higher bitter-salty and salty-bitter confusions relative to those of some other etiologies. This study demonstrates, for the first time, that multiple subject factors influence taste confusions and, along with literature accounts, supports the view that there are both biological and psychological determinants of taste quality confusions.

Inhibition of HSV-1 Replication by Gene Editing Strategy

PubMed Central

Roehm, Pamela C.; Shekarabi, Masoud; Wollebo, Hassen S.; Bellizzi, Anna; He, Lifan; Salkind, Julian; Khalili, Kamel

2016-01-01

HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases. PMID:27064617

Role of structural H 2O in TiO 2 nanotubes in enhancing Pt/C direct ethanol fuel cell anode electro-catalysts

NASA Astrophysics Data System (ADS)

Song, Huanqiao; Qiu, Xinping; Guo, Daojun; Li, Fushen

TiO 2 nanotubes (TNTs) with different structural water were obtained by heat treatment under different temperatures. The role of the structural water in TNTs co-catalyzing ethanol oxidation with Pt/C catalyst was studied systematically. Electrochemical studies using cyclic voltammetry and CO stripping voltammetry indicated that more structural water in TNTs was favorable for improving the tolerance of Pt/C to poisoning species; while chronoamperometry curves and repeated cyclic voltammograms showed that slightly less structural water in TNTs actually led to higher catalytic activity and better stability of Pt/C catalysts for ethanol oxidation. This strange result has been analyzed and was ascribed to the appropriate balance of bi-functional mechanism and ethanol transfer in the catalyst layer with less structural water.

Insulin gene therapy for type 1 diabetes mellitus.

PubMed

Handorf, Andrew M; Sollinger, Hans W; Alam, Tausif

2015-04-01

Type 1 diabetes mellitus is an autoimmune disease resulting from the destruction of pancreatic β cells. Current treatments for patients with type 1 diabetes mellitus include daily insulin injections or whole pancreas transplant, each of which are associated with profound drawbacks. Insulin gene therapy, which has shown great efficacy in correcting hyperglycemia in animal models, holds great promise as an alternative strategy to treat type 1 diabetes mellitus in humans. Insulin gene therapy refers to the targeted expression of insulin in non-β cells, with hepatocytes emerging as the primary therapeutic target. In this review, we present an overview of the current state of insulin gene therapy to treat type 1 diabetes mellitus, including the need for an alternative therapy, important features dictating the success of the therapy, and current obstacles preventing the translation of this treatment option to a clinical setting. In so doing, we hope to shed light on insulin gene therapy as a viable option to treat type 1 diabetes mellitus.

Identification of regulatory targets of tissue-specific transcription factors: application to retina-specific gene regulation

PubMed Central

Qian, Jiang; Esumi, Noriko; Chen, Yangjian; Wang, Qingliang; Chowers, Itay; Zack, Donald J.

2005-01-01

Identification of tissue-specific gene regulatory networks can yield insights into the molecular basis of a tissue's development, function and pathology. Here, we present a computational approach designed to identify potential regulatory target genes of photoreceptor cell-specific transcription factors (TFs). The approach is based on the hypothesis that genes related to the retina in terms of expression, disease and/or function are more likely to be the targets of retina-specific TFs than other genes. A list of genes that are preferentially expressed in retina was obtained by integrating expressed sequence tag, SAGE and microarray datasets. The regulatory targets of retina-specific TFs are enriched in this set of retina-related genes. A Bayesian approach was employed to integrate information about binding site location relative to a gene's transcription start site. Our method was applied to three retina-specific TFs, CRX, NRL and NR2E3, and a number of potential targets were predicted. To experimentally assess the validity of the bioinformatic predictions, mobility shift, transient transfection and chromatin immunoprecipitation assays were performed with five predicted CRX targets, and the results were suggestive of CRX regulation in 5/5, 3/5 and 4/5 cases, respectively. Together, these experiments strongly suggest that RP1, GUCY2D, ABCA4 are novel targets of CRX. PMID:15967807

Synthesis of galactosyl compounds for targeted gene delivery.

PubMed

Ren, T; Zhang, G; Liu, D

2001-11-01

Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.

Detection of genes regulated by Lmx1b during limb dorsalization.

PubMed

Feenstra, Jennifer M; Kanaya, Kohei; Pira, Charmaine U; Hoffman, Sarah E; Eppey, Richard J; Oberg, Kerby C

2012-05-01

Lmx1b is a homeodomain transcription factor that regulates dorsal identity during limb development. Lmx1b knockout (KO) mice develop distal ventral-ventral limbs. Although induction of Lmx1b is linked to Wnt7a expression in the dorsal limb ectoderm, the downstream targets of Lmx1b that accomplish limb dorsalization are unknown. To identify genes targeted by Lmx1b, we compared gene arrays from Lmx1b KO and wild type mouse limbs during limb dorsalization, i.e., 11.5, 12.5, and 13.5 days post coitum. We identified 54 target genes that were differentially expressed in all three stages. Several skeletal targets, including Emx2, Matrilin1 and Matrilin4, demonstrated a loss of scapular expression in the Lmx1b KO mice, supporting a role for Lmx1b in scapula development. Furthermore, the relative abundance of extracellular matrix-related soft tissue targets regulated by Lmx1b, such as collagens and proteoglycans, suggests a mechanism that includes changes in the extracellular matrix composition to accomplish limb dorsalization. Our study provides the most comprehensive characterization of genes regulated by Lmx1b during limb development to-date and provides targets for further investigation. © 2012 The Authors. Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.

Identification of early target genes of aflatoxin B1 in human hepatocytes, inter-individual variability and comparison with other genotoxic compounds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Josse, Rozenn; Dumont, Julie; Fautrel, Alain

Gene expression profiling has recently emerged as a promising approach to identify early target genes and discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens. However, early gene changes induced by genotoxic compounds in human liver remain largely unknown. Primary human hepatocytes and differentiated HepaRG cells were exposed to aflatoxin B1 (AFB1) that induces DNA damage following enzyme-mediated bioactivation. Gene expression profile changes induced by a 24 h exposure of these hepatocyte models to 0.05 and 0.25 μM AFB1 were analyzed by using oligonucleotide pangenomic microarrays. The main altered signaling pathway was the p53 pathway and related functions such as cellmore » cycle, apoptosis and DNA repair. Direct involvement of the p53 protein in response to AFB1 was verified by using siRNA directed against p53. Among the 83 well-annotated genes commonly modulated in two pools of three human hepatocyte populations and HepaRG cells, several genes were identified as altered by AFB1 for the first time. In addition, a subset of 10 AFB1-altered genes, selected upon basis of their function or tumor suppressor role, was tested in four human hepatocyte populations and in response to other chemicals. Although they exhibited large variable inter-donor fold-changes, several of these genes, particularly FHIT, BCAS3 and SMYD3, were found to be altered by various direct and other indirect genotoxic compounds and unaffected by non-genotoxic compounds. Overall, this comprehensive analysis of early gene expression changes induced by AFB1 in human hepatocytes identified a gene subset that included several genes representing potential biomarkers of genotoxic compounds. -- Highlights: ► Gene expression profile changes induced by aflatoxin B1 in human hepatocytes. ► AFB1 modulates various genes including tumor suppressor genes and proto-oncogenes. ► Important inter-individual variations in the response to AFB1. ► Some genes also altered

Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants.

PubMed

Luo, Ming; Gilbert, Brian; Ayliffe, Michael

2016-07-01

Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described.

Gene expression analysis of murine cells producing amphotropic mouse leukaemia virus at a cultivation temperature of 32 and 37 degrees C.

PubMed

Beer, Christiane; Buhr, Petra; Hahn, Heidi; Laubner, Daniela; Wirth, Manfred

2003-07-01

Cultivation of retrovirus packaging cells at 32 degrees C represents a common procedure to achieve high titres in mouse retrovirus production. Gene expression profiling of mouse NIH 3T3 cells producing amphotropic mouse leukaemia virus 4070A revealed that 10 % of the 1176 cellular genes investigated were regulated by temperature shift (37/32 degrees C), while 5 % were affected by retrovirus infection. Strikingly, retrovirus production at 32 degrees C activated the cholesterol biosynthesis/transport pathway and caused an increase in plasma membrane cholesterol levels. Furthermore, these conditions resulted in transcriptional activation of smoothened (smo), patched (ptc) and gli-1; Smo, Ptc and Gli-1, as well as cholesterol, are components of the Sonic hedgehog (Shh) signalling pathway, which directs pattern formation, diversification and tumourigenesis in mammalian cells. These findings suggest a link between cultivation at 32 degrees C, production of MLV-A and the Shh signalling pathway.

Lmx1b-targeted cis-regulatory modules involved in limb dorsalization.

PubMed

Haro, Endika; Watson, Billy A; Feenstra, Jennifer M; Tegeler, Luke; Pira, Charmaine U; Mohan, Subburaman; Oberg, Kerby C

2017-06-01

Lmx1b is a homeodomain transcription factor responsible for limb dorsalization. Despite striking double-ventral (loss-of-function) and double-dorsal (gain-of-function) limb phenotypes, no direct gene targets in the limb have been confirmed. To determine direct targets, we performed a chromatin immunoprecipitation against Lmx1b in mouse limbs at embryonic day 12.5 followed by next-generation sequencing (ChIP-seq). Nearly 84% ( n =617) of the Lmx1b-bound genomic intervals (LBIs) identified overlap with chromatin regulatory marks indicative of potential cis -regulatory modules (PCRMs). In addition, 73 LBIs mapped to CRMs that are known to be active during limb development. We compared Lmx1b-bound PCRMs with genes regulated by Lmx1b and found 292 PCRMs within 1 Mb of 254 Lmx1b-regulated genes. Gene ontological analysis suggests that Lmx1b targets extracellular matrix production, bone/joint formation, axonal guidance, vascular development, cell proliferation and cell movement. We validated the functional activity of a PCRM associated with joint-related Gdf5 that provides a mechanism for Lmx1b-mediated joint modification and a PCRM associated with Lmx1b that suggests a role in autoregulation. This is the first report to describe genome-wide Lmx1b binding during limb development, directly linking Lmx1b to targets that accomplish limb dorsalization. © 2017. Published by The Company of Biologists Ltd.

A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila.

PubMed Central

Weill, Mylène; Fort, Philippe; Berthomieu, Arnaud; Dubois, Marie Pierre; Pasteur, Nicole; Raymond, Michel

2002-01-01

Acetylcholinesterase (AChE) is the target of two major insecticide families, organophosphates (OPs) and carbamates. AChE insensitivity is a frequent resistance mechanism in insects and responsible mutations in the ace gene were identified in two Diptera, Drosophila melanogaster and Musca domestica. However, for other insects, the ace gene cloned by homology with Drosophila does not code for the insensitive AChE in resistant individuals, indicating the existence of a second ace locus. We identified two AChE loci in the genome of Anopheles gambiae, one (ace-1) being a new locus and the other (ace-2) being homologous to the gene previously described in Drosophila. The gene ace-1 has no obvious homologue in the Drosophila genome and was found in 15 mosquito species investigated. In An. gambiae, ace-1 and ace-2 display 53% similarity at the amino acid level and an overall phylogeny indicates that they probably diverged before the differentiation of insects. Thus, both genes are likely to be present in the majority of insects and the absence of ace-1 in Drosophila is probably due to a secondary loss. In one mosquito (Culex pipiens), ace-1 was found to be tightly linked with insecticide resistance and probably encodes the AChE OP target. These results have important implications for the design of new insecticides, as the target AChE is thus encoded by distinct genes in different insect groups, even within the Diptera: ace-2 in at least the Drosophilidae and Muscidae and ace-1 in at least the Culicidae. Evolutionary scenarios leading to such a peculiar situation are discussed. PMID:12396499

The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation.

PubMed

Zhang, Hui; Zhang, Jinshan; Wei, Pengliang; Zhang, Botao; Gou, Feng; Feng, Zhengyan; Mao, Yanfei; Yang, Lan; Zhang, Heng; Xu, Nanfei; Zhu, Jian-Kang

2014-08-01

The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9-induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9-induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large-scale off-targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off-target sites of a large number of T0 plants, low-frequency mutations were found in only one off-target site where the sequence had 1-bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis.

PubMed

Cheng, Feixiong; Murray, James L; Zhao, Junfei; Sheng, Jinsong; Zhao, Zhongming; Rubin, Donald H

2016-09-01

Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap) host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase). Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B) identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline) that may be potential for antiviral indication (e.g. anti-Ebola). In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.

New Wnt/β-catenin target genes promote experimental metastasis and migration of colorectal cancer cells through different signals.

PubMed

Qi, Jingjing; Yu, Yong; Akilli Öztürk, Özlem; Holland, Jane D; Besser, Daniel; Fritzmann, Johannes; Wulf-Goldenberg, Annika; Eckert, Klaus; Fichtner, Iduna; Birchmeier, Walter

2016-10-01

We have previously identified a 115-gene signature that characterises the metastatic potential of human primary colon cancers. The signature included the canonical Wnt target gene BAMBI, which promoted experimental metastasis in mice. Here, we identified three new direct Wnt target genes from the signature, and studied their functions in epithelial-mesenchymal transition (EMT), cell migration and experimental metastasis. We examined experimental liver metastases following injection of selected tumour cells into spleens of NOD/SCID mice. Molecular and cellular techniques were used to identify direct transcription target genes of Wnt/β-catenin signals. Microarray analyses and experiments that interfered with cell migration through inhibitors were performed to characterise downstream signalling systems. Three new genes from the colorectal cancer (CRC) metastasis signature, BOP1, CKS2 and NFIL3, were identified as direct transcription targets of β-catenin/TCF4. Overexpression and knocking down of these genes in CRC cells promoted and inhibited, respectively, experimental metastasis in mice, EMT and cell motility in culture. Cell migration was repressed by interfering with distinct signalling systems through inhibitors of PI3K, JNK, p38 mitogen-activated protein kinase and/or mTOR. Gene expression profiling identified a series of migration-promoting genes, which were induced by BOP1, CKS2 and NFIL3, and could be repressed by inhibitors that are specific to these pathways. We identified new direct Wnt/β-catenin target genes, BOP1, CKS2 and NFIL3, which induced EMT, cell migration and experimental metastasis of CRC cells. These genes crosstalk with different downstream signalling systems, and activate migration-promoting genes. These pathways and downstream genes may serve as therapeutic targets in the treatment of CRC metastasis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Identification of Homeotic Target Genes in Drosophila Melanogaster Including Nervy, a Proto-Oncogene Homologue

PubMed Central

Feinstein, P. G.; Kornfeld, K.; Hogness, D. S.; Mann, R. S.

1995-01-01

In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes. PMID:7498738

RET/PTC and PAX8/PPARγ chromosomal rearrangements in post-Chernobyl thyroid cancer and their association with iodine-131 radiation dose and other characteristics.

PubMed

Leeman-Neill, Rebecca J; Brenner, Alina V; Little, Mark P; Bogdanova, Tetiana I; Hatch, Maureen; Zurnadzy, Liudmyla Y; Mabuchi, Kiyohiko; Tronko, Mykola D; Nikiforov, Yuri E

2013-05-15

Childhood exposure to iodine-131 from the 1986 nuclear accident in Chernobyl, Ukraine, led to a sharp increase in papillary thyroid carcinoma (PTC) incidence in regions surrounding the reactor. Data concerning the association between genetic mutations in PTCs and individual radiation doses are limited. Mutational analysis was performed on 62 PTCs diagnosed in a Ukrainian cohort of patients who were < 18 years old in 1986 and received 0.008 to 8.6 Gy of (131) I to the thyroid. Associations between mutation types and (131) I dose and other characteristics were explored. RET/PTC (ret proto-oncogene/papillary thyroid carcinoma) rearrangements were most common (35%), followed by BRAF (15%) and RAS (8%) point mutations. Two tumors carrying PAX8/PPARγ (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement were identified. A significant negative association with (131) I dose for BRAF and RAS point mutations and a significant concave association with (131) I dose, with an inflection point at 1.6 Gy and odds ratio of 2.1, based on a linear-quadratic model for RET/PTC and PAX8/PPARγ rearrangements were found. The trends with dose were significantly different between tumors with point mutations and rearrangements. Compared with point mutations, rearrangements were associated with residence in the relatively iodine-deficient Zhytomyr region, younger age at exposure or surgery, and male sex. These results provide the first demonstration of PAX8/PPARγ rearrangements in post-Chernobyl tumors and show different associations for point mutations and chromosomal rearrangements with (131) I dose and other factors. These data support the relationship between chromosomal rearrangements, but not point mutations, and (131) I exposure and point to a possible role of iodine deficiency in generation of RET/PTC rearrangements in these patients. Copyright © 2013 American Cancer Society.

Multifunctional Nucleus-targeting Nanoparticles with Ultra-high Gene Transfection Efficiency for In Vivo Gene Therapy

PubMed Central

Li, Ling; Li, Xia; Wu, Yuzhe; Song, Linjiang; Yang, Xi; He, Tao; Wang, Ning; Yang, Suleixin; Zeng, Yan; Wu, Qinjie; Qian, Zhiyong; Wei, Yuquan; Gong, Changyang

2017-01-01

Cancer stem cell-like cells (CSCL) are responsible for tumor recurrence associated with conventional therapy (e.g. surgery, radiation, and chemotherapy). Here, we developed a novel multifunctional nucleus-targeting nanoparticle-based gene delivery system which is capable of targeting and eradicating CSCL. These nanoparticles can facilitate efficient endosomal escape and spontaneously penetrate into nucleus without additional nuclear localization signal. They also induced extremely high gene transfection efficiency (>95%) even in culture medium containing 30% serum, which significantly surpassed that of some commercial transfection reagents, such as Lipofectamine 2000 and Lipofectamine 3000 etc. Especially, when loaded with the TRAIL gene, this system mediated remarkable depletion of CSCL. Upon systemic administration, the nanoparticles accumulated in tumor sites while sparing the non-cancer tissues and significantly inhibited the growth of tumors with no evident systemic toxicity. Taken together, our results suggest that these novel multifunctional, nucleus-targeting nanoparticles are a very promising in vivo gene delivery system capable of targeting CSCL and represent a new treatment candidate for improving the survival of cancer patients. PMID:28529641

Targeted gene flow and rapid adaptation in an endangered marsupial.

PubMed

Kelly, Ella; Phillips, Ben L

2018-06-13

Targeted gene flow is an emerging conservation strategy. It involves translocating individuals with favorable genes to areas where they will have a conservation benefit. The applications for targeted gene flow are wide-ranging, but include pre-adapting natives to the arrival of invasive species. The endangered carnivorous marsupial, the northern quoll, has declined rapidly since the introduction of the cane toad, which fatally poisons quolls that attack them. There are, however, a few remaining toad-invaded quoll populations in which the quolls survive because they know not to eat cane toads. It is this "toad-smart" behavior that we hope to promote through targeted gene flow. For targeted gene flow to be feasible, however, toad-smarts must have a genetic basis. To assess this, we used a common garden experiment and found offspring from toad-exposed populations were substantially less likely to eat toads than those with toad-naïve parents. Hybrid offspring showed similar responses to quolls with two toad-exposed parents, indicating the trait may be dominant. Together, these results suggest a heritable trait and rapid adaptive response in small number of toad-impacted populations. Although questions remain about outbreeding depression, our results are encouraging for targeted gene flow: suggesting it should be possible to introduce toad-smart behavior into soon to be impacted quoll populations. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Novel mutations in CRB1 gene identified in a chinese pedigree with retinitis pigmentosa by targeted capture and next generation sequencing

PubMed Central

Lo, David; Weng, Jingning; Liu, xiaohong; Yang, Juhua; He, Fen; Wang, Yun; Liu, Xuyang

2016-01-01

PURPOSE To detect the disease-causing gene in a Chinese pedigree with autosomal-recessive retinitis pigmentosa (ARRP). METHODS All subjects in this family underwent a complete ophthalmic examination. Targeted-capture next generation sequencing (NGS) was performed on the proband to detect variants. All variants were verified in the remaining family members by PCR amplification and Sanger sequencing. RESULTS All the affected subjects in this pedigree were diagnosed with retinitis pigmentosa (RP). The compound heterozygous c.138delA (p.Asp47IlefsX24) and c.1841G>T (p.Gly614Val) mutations in the Crumbs homolog 1 (CRB1) gene were identified in all the affected patients but not in the unaffected individuals in this family. These mutations were inherited from their parents, respectively. CONCLUSION The novel compound heterozygous mutations in CRB1 were identified in a Chinese pedigree with ARRP using targeted-capture next generation sequencing. After evaluating the significant heredity and impaired protein function, the compound heterozygous c.138delA (p.Asp47IlefsX24) and c.1841G>T (p.Gly614Val) mutations are the causal genes of early onset ARRP in this pedigree. To the best of our knowledge, there is no previous report regarding the compound mutations. PMID:27806333

Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis

PubMed Central

Marques Howarth, Michelle; Simpson, David; Ngok, Siu P.; Nieves, Bethsaida; Chen, Ron; Siprashvili, Zurab; Vaka, Dedeepya; Breese, Marcus R.; Crompton, Brian D.; Alexe, Gabriela; Hawkins, Doug S.; Jacobson, Damon; Brunner, Alayne L.; West, Robert; Mora, Jaume; Stegmaier, Kimberly; Khavari, Paul; Sweet-Cordero, E. Alejandro

2014-01-01

Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma–associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes. PMID:25401475

Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide

PubMed Central

Karnan, Sivasundaram; Ota, Akinobu; Konishi, Yuko; Wahiduzzaman, Md; Hosokawa, Yoshitaka; Konishi, Hiroyuki

2016-01-01

The adeno-associated virus (AAV)-based targeting vector has been one of the tools commonly used for genome modification in human cell lines. It allows for relatively efficient gene targeting associated with 1–4-log higher ratios of homologous-to-random integration of targeting vectors (H/R ratios) than plasmid-based targeting vectors, without actively introducing DNA double-strand breaks. In this study, we sought to improve the efficiency of AAV-mediated gene targeting by introducing a 2A-based promoter-trap system into targeting constructs. We generated three distinct AAV-based targeting vectors carrying 2A for promoter trapping, each targeting a GFP-based reporter module incorporated into the genome, PIGA exon 6 or PIGA intron 5. The absolute gene targeting efficiencies and H/R ratios attained using these vectors were assessed in multiple human cell lines and compared with those attained using targeting vectors carrying internal ribosome entry site (IRES) for promoter trapping. We found that the use of 2A for promoter trapping increased absolute gene targeting efficiencies by 3.4–28-fold and H/R ratios by 2–5-fold compared to values obtained with IRES. In CRISPR-Cas9-assisted gene targeting using plasmid-based targeting vectors, the use of 2A did not enhance the H/R ratios but did upregulate the absolute gene targeting efficiencies compared to the use of IRES. PMID:26657635

Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells.

PubMed

Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

2017-03-16

Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 ( FGF1 and FGF12 ), bone morphogenetic factor-1 ( BMP1 ), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 ( SMARCA4 ), Matrix extracellular phosphoglycoprotein ( MEPE ), Integrin, β4 ( ITGBP4 ), Matrix Metalloproteinase -1, -28 ( MMP1 and MMP28 ), and signal transducer and activator of transcription-4 ( STAT4 ) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1 , MMP28 and kallikrein related peptidase-7 ( KLK7 ), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology.

Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

PubMed Central

Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

2017-01-01

Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12), bone morphogenetic factor-1 (BMP1), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4), Matrix extracellular phosphoglycoprotein (MEPE), Integrin, β4 (ITGBP4), Matrix Metalloproteinase -1, -28 (MMP1 and MMP28), and signal transducer and activator of transcription-4 (STAT4) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology. PMID:28300755

A comparative study of disease genes and drug targets in the human protein interactome

PubMed Central

2015-01-01

Background Disease genes cause or contribute genetically to the development of the most complex diseases. Drugs are the major approaches to treat the complex disease through interacting with their targets. Thus, drug targets are critical for treatment efficacy. However, the interrelationship between the disease genes and drug targets is not clear. Results In this study, we comprehensively compared the network properties of disease genes and drug targets for five major disease categories (cancer, cardiovascular disease, immune system disease, metabolic disease, and nervous system disease). We first collected disease genes from genome-wide association studies (GWAS) for five disease categories and collected their corresponding drugs based on drugs' Anatomical Therapeutic Chemical (ATC) classification. Then, we obtained the drug targets for these five different disease categories. We found that, though the intersections between disease genes and drug targets were small, disease genes were significantly enriched in targets compared to their enrichment in human protein-coding genes. We further compared network properties of the proteins encoded by disease genes and drug targets in human protein-protein interaction networks (interactome). The results showed that the drug targets tended to have higher degree, higher betweenness, and lower clustering coefficient in cancer Furthermore, we observed a clear fraction increase of disease proteins or drug targets in the near neighborhood compared with the randomized genes. Conclusions The study presents the first comprehensive comparison of the disease genes and drug targets in the context of interactome. The results provide some foundational network characteristics for further designing computational strategies to predict novel drug targets and drug repurposing. PMID:25861037

A comparative study of disease genes and drug targets in the human protein interactome.

PubMed

Sun, Jingchun; Zhu, Kevin; Zheng, W; Xu, Hua

2015-01-01

Disease genes cause or contribute genetically to the development of the most complex diseases. Drugs are the major approaches to treat the complex disease through interacting with their targets. Thus, drug targets are critical for treatment efficacy. However, the interrelationship between the disease genes and drug targets is not clear. In this study, we comprehensively compared the network properties of disease genes and drug targets for five major disease categories (cancer, cardiovascular disease, immune system disease, metabolic disease, and nervous system disease). We first collected disease genes from genome-wide association studies (GWAS) for five disease categories and collected their corresponding drugs based on drugs' Anatomical Therapeutic Chemical (ATC) classification. Then, we obtained the drug targets for these five different disease categories. We found that, though the intersections between disease genes and drug targets were small, disease genes were significantly enriched in targets compared to their enrichment in human protein-coding genes. We further compared network properties of the proteins encoded by disease genes and drug targets in human protein-protein interaction networks (interactome). The results showed that the drug targets tended to have higher degree, higher betweenness, and lower clustering coefficient in cancer Furthermore, we observed a clear fraction increase of disease proteins or drug targets in the near neighborhood compared with the randomized genes. The study presents the first comprehensive comparison of the disease genes and drug targets in the context of interactome. The results provide some foundational network characteristics for further designing computational strategies to predict novel drug targets and drug repurposing.

Fe3O4 Nanoparticles in Targeted Drug/Gene Delivery Systems

PubMed Central

Shen, Lazhen; Li, Bei; Qiao, Yongsheng

2018-01-01

Fe3O4 nanoparticles (NPs), the most traditional magnetic nanoparticles, have received a great deal of attention in the biomedical field, especially for targeted drug/gene delivery systems, due to their outstanding magnetism, biocompatibility, lower toxicity, biodegradability, and other features. Naked Fe3O4 NPs are easy to aggregate and oxidize, and thus are often made with various coatings to realize superior properties for targeted drug/gene delivery. In this review, we first list the three commonly utilized synthesis methods of Fe3O4 NPs, and their advantages and disadvantages. In the second part, we describe coating materials that exhibit noticeable features that allow functionalization of Fe3O4 NPs and summarize their methods of drug targeting/gene delivery. Then our efforts will be devoted to the research status and progress of several different functionalized Fe3O4 NP delivery systems loaded with chemotherapeutic agents, and we present targeted gene transitive carriers in detail. In the following section, we illuminate the most effective treatment systems of the combined drug and gene therapy. Finally, we propose opportunities and challenges of the clinical transformation of Fe3O4 NPs targeting drug/gene delivery systems. PMID:29473914

miR-203 inhibits melanoma invasive and proliferative abilities by targeting the polycomb group gene BMI1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Xiao; Sun, Yong; Han, Siqi

2015-01-02

Highlights: • First reported deregulation of miR-203 and up-regulation of BMI1 in metastatic melanoma. • miR-203 decreased BMI1 expression by directly binding to 3′UTR. • Further found miR-203 overexpression suppressed cell invasion and stemness. • Re-expression of BMI1 rescued miR-203-mediated suppression. • miR-203-BMI1 axis may be potential therapeutic targets of melanoma metastasis. - Abstract: Metastasis is the major problem in malignant melanoma, posing a therapeutic challenge to clinicians. The investigation of the underlying mechanism driving this progress remains a large unmet need. In this study, we revealed a miR-203-BMI1 axis that regulated melanoma metastasis. We found significantly deregulation of miR-203more » and up-regulation of BMI1 in melanoma, particularly in metastatic melanoma. An inverse correlation between the levels of miR-203 and BMI1 was further observed in melanoma tissues and cell lines. We also identified BMI1 as a downstream target gene of miR-203, which bound to the 3′UTR of BMI1. Overexpression of miR-203 was associated with decreased BMI1 expression and impaired cell invasion and tumor sphere formation activities. Re-expression of BMI1 markedly rescued miR-203-mediated suppression of these events. Taken together, our results demonstrated that miR-203 regulated melanoma invasive and proliferative abilities in part by targeting BMI1, providing new insights into potential mechanisms of melanoma metastasis.« less

Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohkawa, Mayumi; Ohno, Yoshiya; Masuko, Kazue

Highlights: {yields} We established LAT1 amino-acid transporter-disrupted DT40 cells. {yields} LAT1-disrupted cells showed slow growth and lost the oncogenicity. {yields} siRNA and mAb inhibited human tumor growth in vitro and in vivo. {yields} LAT1 is a promising target molecule for cancer therapy. -- Abstract: L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1{supmore » -/-}) cell clones, derived from a heterozygous LAT1{sup +/-} clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1{sup -/-} DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1{sup -/-} cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1{sup -/-} DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1{sup -/-} DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1{sup +/-} DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.« less

Bacteriophages and medical oncology: targeted gene therapy of cancer.

PubMed

Bakhshinejad, Babak; Karimi, Marzieh; Sadeghizadeh, Majid

2014-08-01

Targeted gene therapy of cancer is of paramount importance in medical oncology. Bacteriophages, viruses that specifically infect bacterial cells, offer a variety of potential applications in biomedicine. Their genetic flexibility to go under a variety of surface modifications serves as a basis for phage display methodology. These surface manipulations allow bacteriophages to be exploited for targeted delivery of therapeutic genes. Moreover, the excellent safety profile of these viruses paves the way for their potential use as cancer gene therapy platforms. The merge of phage display and combinatorial technology has led to the emergence of phage libraries turning phage display into a high throughput technology. Random peptide libraries, as one of the most frequently used phage libraries, provide a rich source of clinically useful peptide ligands. Peptides are known as a promising category of pharmaceutical agents in medical oncology that present advantages such as inexpensive synthesis, efficient tissue penetration and the lack of immunogenicity. Phage peptide libraries can be screened, through biopanning, against various targets including cancer cells and tissues that results in obtaining cancer-homing ligands. Cancer-specific peptides isolated from phage libraries show huge promise to be utilized for targeting of various gene therapy vectors towards malignant cells. Beyond doubt, bacteriophages will play a more impressive role in the future of medical oncology.

Identification of key target genes and pathways in laryngeal carcinoma

PubMed Central

Liu, Feng; Du, Jintao; Liu, Jun; Wen, Bei

2016-01-01

The purpose of the present study was to screen the key genes associated with laryngeal carcinoma and to investigate the molecular mechanism of laryngeal carcinoma progression. The gene expression profile of GSE10935 [Gene Expression Omnibus (GEO) accession number], including 12 specimens from laryngeal papillomas and 12 specimens from normal laryngeal epithelia controls, was downloaded from the GEO database. Differentially expressed genes (DEGs) were screened in laryngeal papillomas compared with normal controls using Limma package in R language, followed by Gene Ontology (GO) enrichment analysis and pathway enrichment analysis. Furthermore, the protein-protein interaction (PPI) network of DEGs was constructed using Cytoscape software and modules were analyzed using MCODE plugin from the PPI network. Furthermore, significant biological pathway regions (sub-pathway) were identified by using iSubpathwayMiner analysis. A total of 67 DEGs were identified, including 27 up-regulated genes and 40 down-regulated genes and they were involved in different GO terms and pathways. PPI network analysis revealed that Ras association (RalGDS/AF-6) domain family member 1 (RASSF1) was a hub protein. The sub-pathway analysis identified 9 significantly enriched sub-pathways, including glycolysis/gluconeogenesis and nitrogen metabolism. Genes such as phosphoglycerate kinase 1 (PGK1), carbonic anhydrase II (CA2), and carbonic anhydrase XII (CA12) whose node degrees were >10 were identified in the disease risk sub-pathway. Genes in the sub-pathway, such as RASSF1, PGK1, CA2 and CA12 were presumed to serve critical roles in laryngeal carcinoma. The present study identified DEGs and their sub-pathways in the disease, which may serve as potential targets for treatment of laryngeal carcinoma. PMID:27446427

A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets

PubMed Central

Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-01-01

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways. PMID:25475013

A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

PubMed

Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-12-05

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.

Recurrent Targeted Genes of Hepatitis B Virus in the Liver Cancer Genomes Identified by a Next-Generation Sequencing–Based Approach

PubMed Central

Ding, Dong; Lou, Xiaoyan; Hua, Dasong; Yu, Wei; Li, Lisha; Wang, Jun; Gao, Feng; Zhao, Na; Ren, Guoping; Li, Lanjuan; Lin, Biaoyang

2012-01-01

Integration of the viral DNA into host chromosomes was found in most of the hepatitis B virus (HBV)–related hepatocellular carcinomas (HCCs). Here we devised a massive anchored parallel sequencing (MAPS) method using next-generation sequencing to isolate and sequence HBV integrants. Applying MAPS to 40 pairs of HBV–related HCC tissues (cancer and adjacent tissues), we identified 296 HBV integration events corresponding to 286 unique integration sites (UISs) with precise HBV–Human DNA junctions. HBV integration favored chromosome 17 and preferentially integrated into human transcript units. HBV targeted genes were enriched in GO terms: cAMP metabolic processes, T cell differentiation and activation, TGF beta receptor pathway, ncRNA catabolic process, and dsRNA fragmentation and cellular response to dsRNA. The HBV targeted genes include 7 genes (PTPRJ, CNTN6, IL12B, MYOM1, FNDC3B, LRFN2, FN1) containing IPR003961 (Fibronectin, type III domain), 7 genes (NRG3, MASP2, NELL1, LRP1B, ADAM21, NRXN1, FN1) containing IPR013032 (EGF-like region, conserved site), and three genes (PDE7A, PDE4B, PDE11A) containing IPR002073 (3′, 5′-cyclic-nucleotide phosphodiesterase). Enriched pathways include hsa04512 (ECM-receptor interaction), hsa04510 (Focal adhesion), and hsa04012 (ErbB signaling pathway). Fewer integration events were found in cancers compared to cancer-adjacent tissues, suggesting a clonal expansion model in HCC development. Finally, we identified 8 genes that were recurrent target genes by HBV integration including fibronectin 1 (FN1) and telomerase reverse transcriptase (TERT1), two known recurrent target genes, and additional novel target genes such as SMAD family member 5 (SMAD5), phosphatase and actin regulator 4 (PHACTR4), and RNA binding protein fox-1 homolog (C. elegans) 1 (RBFOX1). Integrating analysis with recently published whole-genome sequencing analysis, we identified 14 additional recurrent HBV target genes, greatly expanding the HBV recurrent

A system for the measurement of gene targeting efficiency in human cell lines using an antibiotic resistance-GFP fusion gene.

PubMed

Konishi, Yuko; Karnan, Sivasundaram; Takahashi, Miyuki; Ota, Akinobu; Damdindorj, Lkhagvasuren; Hosokawa, Yoshitaka; Konishi, Hiroyuki

2012-09-01

Gene targeting in a broad range of human somatic cell lines has been hampered by inefficient homologous recombination. To improve this technology and facilitate its widespread application, it is critical to first have a robust and efficient research system for measuring gene targeting efficiency. Here, using a fusion gene consisting of hygromycin B phosphotransferase and 3'-truncated enhanced GFP (HygR-5' EGFP) as a reporter gene, we created a molecular system monitoring the ratio of homologous to random integration (H/R ratio) of targeting vectors into the genome. Cell clones transduced with a reporter vector containing HygR-5' EGFP were efficiently established from two human somatic cell lines. Established HygR-5' EGFP reporter clones retained their capacity to monitor gene targeting efficiency for a longer duration than a conventional reporter system using an unfused 5' EGFP gene. With the HygR-5' EGFP reporter system, we reproduced previous findings of gene targeting frequency being up-regulated by the use of an adeno-associated viral (AAV) backbone, a promoter-trap system, or a longer homology arm in a targeting vector, suggesting that this system accurately monitors H/R ratio. Thus, our HygR-5' EGFP reporter system will assist in the development of an efficient AAV-based gene targeting technology.

Immuno-Oncology-The Translational Runway for Gene Therapy: Gene Therapeutics to Address Multiple Immune Targets.

PubMed

Weß, Ludger; Schnieders, Frank

2017-12-01

Cancer therapy is once again experiencing a paradigm shift. This shift is based on extensive clinical experience demonstrating that cancer cannot be successfully fought by addressing only single targets or pathways. Even the combination of several neo-antigens in cancer vaccines is not sufficient for successful, lasting tumor eradication. The focus has therefore shifted to the immune system's role in cancer and the striking abilities of cancer cells to manipulate and/or deactivate the immune system. Researchers and pharma companies have started to target the processes and cells known to support immune surveillance and the elimination of tumor cells. Immune processes, however, require novel concepts beyond the traditional "single-target-single drug" paradigm and need parallel targeting of diverse cells and mechanisms. This review gives a perspective on the role of gene therapy technologies in the evolving immuno-oncology space and identifies gene therapy as a major driver in the development and regulation of effective cancer immunotherapy. Present challenges and breakthroughs ranging from chimeric antigen receptor T-cell therapy, gene-modified oncolytic viruses, combination cancer vaccines, to RNA therapeutics are spotlighted. Gene therapy is recognized as the most prominent technology enabling effective immuno-oncology strategies.

Zooplankton community analysis in the Changjiang River estuary by single-gene-targeted metagenomics

NASA Astrophysics Data System (ADS)

Cheng, Fangping; Wang, Minxiao; Li, Chaolun; Sun, Song

2014-07-01

DNA barcoding provides accurate identification of zooplankton species through all life stages. Single-gene-targeted metagenomic analysis based on DNA barcode databases can facilitate longterm monitoring of zooplankton communities. With the help of the available zooplankton databases, the zooplankton community of the Changjiang (Yangtze) River estuary was studied using a single-gene-targeted metagenomic method to estimate the species richness of this community. A total of 856 mitochondrial cytochrome oxidase subunit 1 (cox1) gene sequences were determined. The environmental barcodes were clustered into 70 molecular operational taxonomic units (MOTUs). Forty-two MOTUs matched barcoded marine organisms with more than 90% similarity and were assigned to either the species (similarity>96%) or genus level (similarity<96%). Sibling species could also be distinguished. Many species that were overlooked by morphological methods were identified by molecular methods, especially gelatinous zooplankton and merozooplankton that were likely sampled at different life history phases. Zooplankton community structures differed significantly among all of the samples. The MOTU spatial distributions were influenced by the ecological habits of the corresponding species. In conclusion, single-gene-targeted metagenomic analysis is a useful tool for zooplankton studies, with which specimens from all life history stages can be identified quickly and effectively with a comprehensive database.

MicroRNA-targeted transcription factor gene RDD1 promotes nutrient ion uptake and accumulation in rice.

PubMed

Iwamoto, Masao; Tagiri, Akemi

2016-02-01

Fertilizers are often potential environmental pollutants, therefore increasing productivity and the efficiency of nutrient uptake to boost crop yields without the risk of environmental pollution is a desirable goal. Here, we show that the transcription factor encoding gene RDD1 plays a role in improving the uptake and accumulation of various nutrient ions in rice. RDD1 was found to be targeted by the microRNA miR166. An RDD1 transgene driven by a strong constitutive promoter exhibited a diurnally oscillating expression similar to that of the endogenous RDD1, and nucleotide substitution within the miR166 recognition site to prevent miR166-RDD1 mRNA pairing resulted in constitutive RDD1 expression. The RDD1 protein was localized to vascular tissue because miR166 repressed RDD1 expression in the mesophyll. The overexpression of RDD1 induced the expression of genes associated with the transport of several nutrients such as NH4(+), Na(+), SO4(2-), Cl(-), PO4(3-) and sucrose, and the uptake and accumulation of various nutrient ions under low-nutrient conditions. Moreover, the overexpression of RDD1 increased nitrogen responsiveness and grain productivity. Our results suggest that RDD1 can contribute to the increased grain productivity of rice via inducing the efficient uptake and accumulation of various nutrient ions. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Preference for tap, bottled, and recycled water: Relations to PTC taste sensitivity and personality.

PubMed

Harmon, Daniel; Gauvain, Mary; Z Reisz; Arthur, Isaac; Story, S Drew

2018-02-01

This study investigated people's preferences for different water sources and factors that predict such preferences using a blind taste test. Water preferences of 143 participants for one name-brand bottled water, one groundwater-sourced tap water, and one indirect potable reuse (IDR) water were assessed. For predictors of water preference, we measured each participant's PTC taste sensitivity and assessed two personality traits (Neuroticism, Openness to Experience). We also explored participants' descriptions of each water source. Results indicate a preference for water treated with Reverse Osmosis (RO) (bottled and IDR water) over groundwater-sourced water, which had higher pH levels and lower concentrations of Ca and HCO 3 - . PTC taste sensitivity did not predict preferences, while Openness to Experience and Neuroticism predicted preference for IDR water. Positive relations between Openness to Experience and preferences for bottled and IDR water were moderated by gender and were stronger among females. Participants described water primarily by its taste and texture. Findings suggest that (1) tap water treated by RO is equally preferable to some bottled water, (2) personality traits may affect water preferences, and (3) prior findings of gender differences in preferences for bottled water may reflect personality characteristics. Efforts to increase acceptance for sustainable water alternatives, such as IDR, may be more successful by assuring consumers about taste and addressing personality traits that encourage or inhibit use. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evidence of post-transcriptional readthrough regulation in FGF5 gene of alpaca.

PubMed

Pallotti, Stefano; Pediconi, Dario; Subramanian, Dharaneedharan; Molina, María Gabriela; Antonini, Marco; Morelli, Maria Beatrice; Renieri, Carlo; La Terza, Antonietta

2018-03-20

Two different phenotypes are described in alpaca, identified as suri and huacaya, which differ in the type of fleece. The huacaya fleece is characterized by compact, soft and highly crimped fibers, while the suri fleece is longer, straight, less-crimped and lustrous. In our study, the Fibroblast growth factor 5 (FGF5) was investigated as a possible candidate gene for hair length in alpaca (Vicugna pacos). As previously identified in other mammals, our results show that the alpaca FGF5 gene gives rise to a short (FGF5S) and a long (FGF5) isoform. Interestingly, in the long isoform, we observed a point mutation (i.e., a transition C>T at position 499 downstream of the ATG codon) that is able to generate a premature termination codon (PTC). The highly conserved nucleotide and amino acid sequence after PTC suggested a readthrough event (RT) that was confirmed by western blot analysis. The analysis of cDNA sequence revealed motifs and structures of mRNA undergoing RT. In fact, the event is positively influenced by particular signals harbored by the transcript. To the best of our knowledge, this is the first case of a readthrough event on PTC reported for the FGF5 gene and the first case of this translational mechanism in alpaca. Copyright © 2018 Elsevier B.V. All rights reserved.

Integrative proteomics and biochemical analyses define Ptc6p as the Saccharomyces cerevisiae pyruvate dehydrogenase phosphatase.

PubMed

Guo, Xiao; Niemi, Natalie M; Coon, Joshua J; Pagliarini, David J

2017-07-14

The pyruvate dehydrogenase complex (PDC) is the primary metabolic checkpoint connecting glycolysis and mitochondrial oxidative phosphorylation and is important for maintaining cellular and organismal glucose homeostasis. Phosphorylation of the PDC E1 subunit was identified as a key inhibitory modification in bovine tissue ∼50 years ago, and this regulatory process is now known to be conserved throughout evolution. Although Saccharomyces cerevisiae is a pervasive model organism for investigating cellular metabolism and its regulation by signaling processes, the phosphatase(s) responsible for activating the PDC in S. cerevisiae has not been conclusively defined. Here, using comparative mitochondrial phosphoproteomics, analyses of protein-protein interactions by affinity enrichment-mass spectrometry, and in vitro biochemistry, we define Ptc6p as the primary PDC phosphatase in S. cerevisiae Our analyses further suggest additional substrates for related S. cerevisiae phosphatases and describe the overall phosphoproteomic changes that accompany mitochondrial respiratory dysfunction. In summary, our quantitative proteomics and biochemical analyses have identified Ptc6p as the primary-and likely sole- S. cerevisiae PDC phosphatase, closing a key knowledge gap about the regulation of yeast mitochondrial metabolism. Our findings highlight the power of integrative omics and biochemical analyses for annotating the functions of poorly characterized signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

HABP2 p.G534E variant in patients with family history of thyroid and breast cancer

PubMed Central

Pinheiro, Maisa; Drigo, Sandra Aparecida; Tonhosolo, Renata; Andrade, Sonia C.S.; Marchi, Fabio Albuquerque; Jurisica, Igor; Kowalski, Luiz Paulo; Achatz, Maria Isabel; Rogatto, Silvia Regina

2017-01-01

Familial Papillary Thyroid Carcinoma (PTC) has been described as a hereditary predisposition cancer syndrome associated with mutations in candidate genes including HABP2. Two of 20 probands from families with history of PTC and breast carcinoma (BC) were evaluated by whole exome sequencing (WES) revealing HABP2 p.G534E. Sanger sequencing was used to confirm the involvement of this variant in three families (F1: 7 relatives; F2: 3 and F3: 3). The proband and his sister (with no malignant tumor so far) from F1 were homozygous for the variant whereas one relative with PTC from F2 was negative for the variant. Although the proband of the F3 with PTC was HABP2 wild type, three relatives presented the variant. Five of 170 healthy Brazilian individuals with no family history of BC or PTC and three of 50 sporadic PTC presented the p.G534E. These findings suggested no association of this variant with our familial PTC cases. Genes potentially associated with deregulation of the extracellular matrix organization pathway (CTSB, TNXB, COL4A3, COL16A1, COL24A1, COL5A2, NID1, LOXL2, MMP11, TRIM24 and MUSK) and DNA repair function (NBN and MSH2) were detected by WES, suggesting that other cancer-associated genes have pathogenic effects in the risk of familial PTC development. PMID:28402931

Predicting essential genes for identifying potential drug targets in Aspergillus fumigatus.

PubMed

Lu, Yao; Deng, Jingyuan; Rhodes, Judith C; Lu, Hui; Lu, Long Jason

2014-06-01

Aspergillus fumigatus (Af) is a ubiquitous and opportunistic pathogen capable of causing acute, invasive pulmonary disease in susceptible hosts. Despite current therapeutic options, mortality associated with invasive Af infections remains unacceptably high, increasing 357% since 1980. Therefore, there is an urgent need for the development of novel therapeutic strategies, including more efficacious drugs acting on new targets. Thus, as noted in a recent review, "the identification of essential genes in fungi represents a crucial step in the development of new antifungal drugs". Expanding the target space by rapidly identifying new essential genes has thus been described as "the most important task of genomics-based target validation". In previous research, we were the first to show that essential gene annotation can be reliably transferred between distantly related four Prokaryotic species. In this study, we extend our machine learning approach to the much more complex Eukaryotic fungal species. A compendium of essential genes is predicted in Af by transferring known essential gene annotations from another filamentous fungus Neurospora crassa. This approach predicts essential genes by integrating diverse types of intrinsic and context-dependent genomic features encoded in microbial genomes. The predicted essential datasets contained 1674 genes. We validated our results by comparing our predictions with known essential genes in Af, comparing our predictions with those predicted by homology mapping, and conducting conditional expressed alleles. We applied several layers of filters and selected a set of potential drug targets from the predicted essential genes. Finally, we have conducted wet lab knockout experiments to verify our predictions, which further validates the accuracy and wide applicability of the machine learning approach. The approach presented here significantly extended our ability to predict essential genes beyond orthologs and made it possible to

Transcriptome Analysis and Screening for Potential Target Genes for RNAi-Mediated Pest Control of the Beet Armyworm, Spodoptera exigua.

PubMed

Li, Hang; Jiang, Weihua; Zhang, Zan; Xing, Yanru; Li, Fei

2013-01-01

The beet armyworm, Spodoptera exigua (Hübner), is a serious pest worldwide that causes significant losses in crops. Unfortunately, genetic resources for the beet armyworm is extremely scarce. To improve these resources we sequenced the transcriptome of S. exigua representing all stages including eggs, 1(st) to 5(th) instar larvae, pupae, male and female adults using the Illumina Solexa platform. We assembled the transcriptome with Trinity that yielded 31,414 contigs. Of these contigs, 18,592 were annotated as protein coding genes by Blast searches against the NCBI nr database. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step towards developing an efficient RNAi-mediated pest control technique is to find suitable target genes. To screen for effective target genes in the beet armyworm, we selected nine candidate genes. The sequences of these genes were amplified using the RACE strategy. Then, siRNAs were designed and chemically synthesized. We injected 2 µl siRNA (2 µg/µl) into the 4(th) instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (∼20-94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and arf1 caused a significantly high level of mortality compared to the negative control (P<0.05). About 80% of the surviving insects in the siRNA-treated group of five genes (PGCP, chitinase1, tubulin1, tubulin2 and helicase) showed retarded development. In chitinase1-siRNA and chitinase7-siRNA administered groups, 12.5% survivors exhibited "half-ecdysis". In arf1-siRNA and arf2-siRNA groups, the body color of 15% became black 48 h after injections. In summary, the transcriptome could be a valuable genetic resource for identification of genes in S. exigua and this study provided putative targets for RNAi pest control.

Transcriptome Analysis and Screening for Potential Target Genes for RNAi-Mediated Pest Control of the Beet Armyworm, Spodoptera exigua

PubMed Central

Zhang, Zan; Xing, Yanru; Li, Fei

2013-01-01

The beet armyworm, Spodoptera exigua (Hübner), is a serious pest worldwide that causes significant losses in crops. Unfortunately, genetic resources for the beet armyworm is extremely scarce. To improve these resources we sequenced the transcriptome of S. exigua representing all stages including eggs, 1st to 5th instar larvae, pupae, male and female adults using the Illumina Solexa platform. We assembled the transcriptome with Trinity that yielded 31,414 contigs. Of these contigs, 18,592 were annotated as protein coding genes by Blast searches against the NCBI nr database. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step towards developing an efficient RNAi-mediated pest control technique is to find suitable target genes. To screen for effective target genes in the beet armyworm, we selected nine candidate genes. The sequences of these genes were amplified using the RACE strategy. Then, siRNAs were designed and chemically synthesized. We injected 2 µl siRNA (2 µg/µl) into the 4th instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (∼20–94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and arf1 caused a significantly high level of mortality compared to the negative control (P<0.05). About 80% of the surviving insects in the siRNA-treated group of five genes (PGCP, chitinase1, tubulin1, tubulin2 and helicase) showed retarded development. In chitinase1-siRNA and chitinase7-siRNA administered groups, 12.5% survivors exhibited “half-ecdysis”. In arf1-siRNA and arf2-siRNA groups, the body color of 15% became black 48 h after injections. In summary, the transcriptome could be a valuable genetic resource for identification of genes in S. exigua and this study provided putative targets for RNAi pest control. PMID

The Prx1 limb enhancers: targeted gene expression in developing zebrafish pectoral fins.

PubMed

Hernández-Vega, Amayra; Minguillón, Carolina

2011-08-01

Limbs represent an excellent model to study the induction, growth, and patterning of several organs. A breakthrough to study gene function in various tissues has been the characterization of regulatory elements that allow tissue-specific interference of gene function. The mouse Prx1 promoter has been used to generate limb-specific mutants and overexpress genes in tetrapod limbs. Although zebrafish possess advantages that favor their use to study limb morphogenesis, there is no driver described suitable for specifically interfering with gene function in developing fins. We report the generation of zebrafish lines that express enhanced green fluorescent protein (EGFP) driven by the mouse Prx1 enhancer in developing pectoral fins. We also describe the expression pattern of the zebrafish prrx1 genes and identify three conserved non-coding elements (CNEs) that we use to generate fin-specific EGFP reporter lines. Finally, we show that the mouse and zebrafish regulatory elements may be used to modify gene function in pectoral fins. Copyright © 2011 Wiley-Liss, Inc.

Pet-1 Switches Transcriptional Targets Postnatally to Regulate Maturation of Serotonin Neuron Excitability.

PubMed

Wyler, Steven C; Spencer, W Clay; Green, Noah H; Rood, Benjamin D; Crawford, LaTasha; Craige, Caryne; Gresch, Paul; McMahon, Douglas G; Beck, Sheryl G; Deneris, Evan

2016-02-03

Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known about the regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1(-/-) neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. Five-HT1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturation through secondary postmitotic regulatory factors. The early postnatal switch in Pet-1 targets uncovers a distinct neonatal stage-specific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability. The regulatory mechanisms

New target genes of MITF-induced microRNA-211 contribute to melanoma cell invasion.

PubMed

Margue, Christiane; Philippidou, Demetra; Reinsbach, Susanne E; Schmitt, Martina; Behrmann, Iris; Kreis, Stephanie

2013-01-01

The non-coding microRNAs (miRNA) have tissue- and disease-specific expression patterns. They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes. Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context. Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined. MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF). By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211. Expression of this miRNA is often reduced in melanoma samples. Here, we investigated functional roles of miR-211 by identifying and studying new target genes. We show that MITF-correlated miR-211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively. MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion. These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma.

Seamless Genome Editing in Rice via Gene Targeting and Precise Marker Elimination.

PubMed

Nishizawa-Yokoi, Ayako; Saika, Hiroaki; Toki, Seiichi

2016-01-01

Positive-negative selection using hygromycin phosphotransferase (hpt) and diphtheria toxin A-fragment (DT-A) as positive and negative selection markers, respectively, allows enrichment of cells harboring target genes modified via gene targeting (GT). We have developed a successful GT system employing positive-negative selection and subsequent precise marker excision via the piggyBac transposon derived from the cabbage looper moth to introduce desired modifications into target genes in the rice genome. This approach could be applied to the precision genome editing of almost all endogenous genes throughout the genome, at least in rice.

RNA interference can target pre-mRNA: consequences for gene expression in a Caenorhabditis elegans operon.

PubMed Central

Bosher, J M; Dufourcq, P; Sookhareea, S; Labouesse, M

1999-01-01

In nematodes, flies, trypanosomes, and planarians, introduction of double-stranded RNA results in sequence-specific inactivation of gene function, a process termed RNA interference (RNAi). We demonstrate that RNAi against the Caenorhabditis elegans gene lir-1, which is part of the lir-1/lin-26 operon, induced phenotypes very different from a newly isolated lir-1 null mutation. Specifically, lir-1(RNAi) induced embryonic lethality reminiscent of moderately strong lin-26 alleles, whereas the lir-1 null mutant was viable. We show that the lir-1(RNAi) phenotypes resulted from a severe loss of lin-26 gene expression. In addition, we found that RNAi directed against lir-1 or lin-26 introns induced similar phenotypes, so we conclude that lir-1(RNAi) targets the lir-1/lin-26 pre-mRNA. This provides direct evidence that RNA interference can prevent gene expression by targeting nuclear transcripts. Our results highlight that caution may be necessary when interpreting RNA interference without the benefit of mutant alleles. PMID:10545456

Targeted systemic gene therapy and molecular imaging of cancer contribution of the vascular-targeted AAVP vector.

PubMed

Hajitou, Amin

2010-01-01

Gene therapy and molecular-genetic imaging have faced a major problem: the lack of an efficient systemic gene delivery vector. Unquestionably, eukaryotic viruses have been the vectors of choice for gene delivery to mammalian cells; however, they have had limited success in systemic gene therapy. This is mainly due to undesired uptake by the liver and reticuloendothelial system, broad tropism for mammalian cells causing toxicity, and their immunogenicity. On the other hand, prokaryotic viruses such as bacteriophage (phage) have no tropism for mammalian cells, but can be engineered to deliver genes to these cells. However, phage-based vectors have inherently been considered poor vectors for mammalian cells. We have reported a new generation of vascular-targeted systemic hybrid prokaryotic-eukaryotic vectors as chimeras between an adeno-associated virus (AAV) and targeted bacteriophage (termed AAV/phage; AAVP). In this hybrid vector, the targeted bacteriophage serves as a shuttle to deliver the AAV transgene cassette inserted in an intergenomic region of the phage DNA genome. As a proof of concept, we assessed the in vivo efficacy of vector in animal models of cancer by displaying on the phage capsid the cyclic Arg-Gly-Asp (RGD-4C) ligand that binds to alphav integrin receptors specifically expressed on the angiogenic blood vessels of tumors. The ligand-directed vector was able to specifically deliver imaging and therapeutic transgenes to tumors in mice, rats, and dogs while sparing the normal organs. This chapter reviews some gene transfer strategies and the potential of the vascular-targeted AAVP vector for enhancing the effectiveness of existing systemic gene delivery and genetic-imaging technologies. Copyright (c) 2010 Elsevier Inc. All rights reserved.

STAT3 Target Genes Relevant to Human Cancers

PubMed Central

Carpenter, Richard L.; Lo, Hui-Wen

2014-01-01

Since its discovery, the STAT3 transcription factor has been extensively studied for its function as a transcriptional regulator and its role as a mediator of development, normal physiology, and pathology of many diseases, including cancers. These efforts have uncovered an array of genes that can be positively and negatively regulated by STAT3, alone and in cooperation with other transcription factors. Through regulating gene expression, STAT3 has been demonstrated to play a pivotal role in many cellular processes including oncogenesis, tumor growth and progression, and stemness. Interestingly, recent studies suggest that STAT3 may behave as a tumor suppressor by activating expression of genes known to inhibit tumorigenesis. Additional evidence suggested that STAT3 may elicit opposing effects depending on cellular context and tumor types. These mixed results signify the need for a deeper understanding of STAT3, including its upstream regulators, parallel transcription co-regulators, and downstream target genes. To help facilitate fulfilling this unmet need, this review will be primarily focused on STAT3 downstream target genes that have been validated to associate with tumorigenesis and/or malignant biology of human cancers. PMID:24743777

Mechanisms of epigenetic and cell-type specific regulation of Hey target genes in ES cells and cardiomyocytes.

PubMed

Weber, David; Heisig, Julia; Kneitz, Susanne; Wolf, Elmar; Eilers, Martin; Gessler, Manfred

2015-02-01

Hey bHLH transcription factors are critical effectors of Notch signaling. During mammalian heart development they are expressed in atrial and ventricular cardiomyocytes and in the developing endocardium. Hey knockout mice suffer from lethal cardiac defects, such as ventricular septum defects, valve defects and cardiomyopathy. Despite this functional relevance, little is known about the regulation of downstream targets in relevant cell types. The objective of this study was to elucidate the regulatory mechanisms by which Hey proteins affect gene expression in a cell type specific manner. We used an in vitro cardiomyocyte differentiation system with inducible Hey1 or Hey2 expression to study target gene regulation in cardiomyocytes (CM) generated from murine embryonic stem cells (ESC). The effects of Hey1 and Hey2 are largely redundant, but cell type specific. The number of regulated genes is comparable between ESC and CM, but the total number of binding sites is much higher, especially in ESC, targeting mainly genes involved in transcriptional regulation and developmental processes. Repression by Hey proteins generally correlates with the extent of Hey-binding to target promoters, Hdac recruitment and lower histone acetylation. Functionally, treatment with the Hdac inhibitor TSA abolished Hey target gene regulation. However, in CM the repressive effect of Hey-binding is lost for a subset of genes. These also lack Hey-dependent histone deacetylation in CM and are enriched for binding sites of cardiac specific activators like Srf, Nkx2-5, and Gata4. Ectopic Nkx2-5 overexpression in ESC blocks Hey-mediated repression of these genes. Thus, Hey proteins mechanistically repress target genes via Hdac recruitment and histone deacetylation. In CM Hey-repression is counteracted by cardiac activators, which recruit histone acetylases and prevent Hey mediated deacetylation and subsequent repression for a subset of genes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

NASA Astrophysics Data System (ADS)

Yuan, Chenyan; An, Yanli; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng

2014-08-01

Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression.

Targeted expression of suicide gene by tissue-specific promoter and microRNA regulation for cancer gene therapy.

PubMed

Danda, Ravikanth; Krishnan, Gopinath; Ganapathy, Kalaivani; Krishnan, Uma Maheswari; Vikas, Khetan; Elchuri, Sailaja; Chatterjee, Nivedita; Krishnakumar, Subramanian

2013-01-01

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.

HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development.

PubMed

Laurent, Frédéric; Girdziusaite, Ausra; Gamart, Julie; Barozzi, Iros; Osterwalder, Marco; Akiyama, Jennifer A; Lincoln, Joy; Lopez-Rios, Javier; Visel, Axel; Zuniga, Aimée; Zeller, Rolf

2017-05-23

The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost from Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.