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Anal. Biochem. 423(2), 224-228. A bioluminescence assay for DNA methyltransferase activity based on methylation-resistant cleavage. 2012

Jiang, C., Yan, C.Y., Huang, C., Jiang, J.H., and Yu, R.Q.

Notes: This paper describes a bioluminescence-based method for the detection of DNA methyltransferase activity based on methylation-resistant cleavage and protein expression. The authors used Dam methylase as a model enzyme, MboI as the methylation-resistant endonuclease, and luciferase reporter DNA (LR-DNA) as the target. LR-DNA was amplified by PCR and then used as substrate in a Dam methylation reaction. If the target sites in the LR-DNA were fully methylated, they were resistant to subsequent MboI cleavage and expressed luciferase upon in vitro transcription/translation. Incomplete methylation or the absence of methylation resulted in DNA digestion and diminished/absent luciferase activity. TNT® T7 Quick for PCR DNA was used for in vitro translation of the LR-DNA, and the Luciferase Assay System and GloMax® 20/20 Luminometer were used to measure luciferase activity. (4191)

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Proc. Natl. Acad. Sci. USA 109, 2654–9. A long noncoding RNA regulates photoperiod-sensitive male sterility, an essential component of hybrid rice 2012

Ding, J., Lu Q, Ouyang, Y., Mao, H., Zhang, P., Yao, J., Xu, C. Li, X., Xiao, J. and Zhang, Q.

Notes: Genomic DNA was isolated from rice plants and bisulfite treated. Target regions were specifically amplified using bisulfite primers, and the amplified products cloned into the pGEM®-T vector prior to large scale bisulfite sequencing. Sequencing analysis was performed using the Kismeth tool. pGEM®-T was also used as the vector for fragments amplified from cDNA clones of transcripts of interest prior to generating sense and antisense RNA probes for RNA in situ hybridization experiments. Apoptosis of tissue from anthers was assessed using the DeadEnd Fluorometric TUNEL System. (4558)

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mBio. 3(5), e00266–12. A multicenter blinded analysis indicates no association between chronic fatigue syndrome/myalgic encephalomyelitis and either xenotropic murine leukemia virus-related virus or polytropic murine leukemia virus. 2012

Alter, H.J., Mikovits, J.A., Switzer, W.M., Ruscetti, F.W., Lo, S.C., Klimas, N., Komaroff, A.L., Montoya, J.G., Bateman, L., Levine, S., Peterson, D., Levin, B., Hanson, M.R., Genfi, A., Bhat, M., Zheng, H., Wang, R., Li, B., Hung, G.C., Lee, L.L., Sameroff, S., Heneine, W., Coffin, J., Hornig, M. and Lipkin, W.I.

Notes: In this report, the original investigators who found XMRV and pMLV (polytropic murine leukemia virus) in blood of subjects with Chronic Fatigue Syndrome (CFS) report that this association is not confirmed in a blinded analysis of samples from rigorously characterized subjects.

The CDC performed nucleic acid testing assays. Plasma was centrifuged and RNA isolated from the pellet. Quantitative real-time RT-PCR assays (qRT-PCR) for generic pMLV/XMRV pro (protease) and gag detection were performed on RNA extracts, using the AccessQuick™ RT-PCR System and an AgPath one-step RT-PCR kit.

ArrayScript RT and AmpliTaq Gold DNA polymerase were used for cDNA synthesis and amplification in the pro and gag qRT-PCR assays, respectively. A third PCR was done using the primers XPOLOF and XPOLOR, followed by a nested PCR with the primers XPOLIF and XPOLIR for the generic detection of MLV/XMRV 216-bp pol sequences. For this reaction, cDNA synthesis and amplification of RNA was done using Promega AMV Reverse Transcriptase and a RobustI RT-PCR kit. Each PCR experiment included 20 water-only reactions to control for contamination. (4300)

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Cancer Prev. Res. 5(6), 822-33. A novel sulindac derivative that potently suppresses colon tumor cell growth by inhibiting cGMP phosphodiesterase and β-catenin transcriptional activity 2012

Whitt, J.D., Li, N., Tinsley, H.N., Chen, X., Zhang, W., Li, Y., Gary, B.D., Keeton, A.B., Xi, Y., Abadi, A.H., Grizzle, W.E., and Piazza, G.A.

Notes: These authors characterized a novel sulindac derivative (SBA) that inhibits cell proliferation and induces apoptosis in human colon tumor cells. To investigate the effect of SBA  on intracellular cGMP levels, they transfected HEK293 cells with a GloSensor™ construct containing firefly luciferase fused to the human PDE5 GAF-A cGMP binding domain (GloSensor cGMP-40F plasmid). Binding of cGMP causes a conformational change that results in increased luminescence. Kinetic measurements of luminescence showed that intracellular cGMP levels increased upon treatment with SBA. (4524)

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Gene 493, 124-131. A preliminary sketch of horn cancer transcriptome in Indian zebu cattle. 2012

Tripathi ,A.K., Koringa, P.G., Jakhesara, S.J., Ahir, V.B., Ramani, U.V., Bhatt, V.D., Sajnani, M.R., Patel, D.A., Joshi, A.J., Shanmuga, S.J., Rank, D.N., and Joshi, C.G.

Notes: These authors used the Roche 454 next generation sequencing platform to sequence and compare cancerous and normal horn tissue transcripts. mRNA isolated from each sample was fragmented prior to cDNA synthesis. cDNA quality was verified using a high sensitivity DNA Chip kit on the  Bioanalyzer 2100 and the  QuantiFluor™-ST Fluorometer. The transcripts were compared and potential tumor associated genes identified. (4229)

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Proc. Natl. Acad. Sci. USA 109(21), 8067-8072. A short adaptive path from DNA to RNA polymerases. 2012

Cozens, C., Pinheirom V.B., Vaismanm, A., Woodgate, R., and Holligerm, P.

Notes: This paper describes a potential "specificity checkpoint" for nucleotide incorporation by DNA polymerases. Using Tgo DNA polymerase from Thermococcus gorgonarius as a model system, the authors identified a single key residue (E664) that influenced the ability to incorporate rNTPs. Mutation of E664 to lysine (K) enabled RNA polymerization in the context of another mutation (the steric-gate mutation (Y409G)). The paper describes characterization of the mutant phenotype. As part of the study, luciferase mRNA was generated from the luciferase control DNA using the mutant polymerase. Synthesis and in vitro translation of the synthesized RNA was also evaluated using ssDNA templates, New England Biolabs PURExpress™ protein synthesis kit, and the FluoroTect™ GreenLys in vitro translation labeling system. (4248)

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Int. J. Syst. Evol. Microbiol. 62, 2743–9. Algiphilus aromaticivorans gen. nov., sp. nov., an aromatic hydrocarbon-degrading bacterium isolated from a culture of the marine dinoflagellate Lingulodinium polyedrum, and proposal of Algiphilaceae fam. nov. 2012

Gutierrez, T. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Algiphilus aromaticivorans. (4297)

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Int. J. Syst. Evol. Microbiol. 62, 409–13. Algoriphagus jejuensis sp. nov., isolated from seawater. 2012

Lee, D.-H., Kahng, H.-Y. Lee, S.B.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Algoriphagus jejuensis. (4298)

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Hum. Mol. Genet. 21(3), 664-680. An aggregation sensing reporter identifies leflunomide and teriflunomide as polyglutamine aggregate inhibitors. 2012

Fuentealba, R.A., Marasa, J., Diamond, M.I., Piwnica-Worms, D., and Weihl, C.C.

Notes: These authors developed a protein-aggregation reporter that uses huntingtin exon 1 containing 72 glutamines fused to the N-terminal end of firefly luciferase (httQ72-Luc). This construct did not aggregate unless seeded by a non-luciferase-containing polyglutamine (polyQ) protein. Upon co-aggregation, httQ72-luc becomes insoluble and loses its enzymatic activity.  httQ72-Luc and a polyQ protein were used to screen a library for compounds that prevent polyQ aggregation. Leflunomide and its active metabolite teriflunomide inhibited protein aggregation independently of their known role in pyrimidine biosynthesis. This study demonstrated the usefulness of luciferase-based protein aggregate reporters for high-throughput screening applications. Luciferase activity was measured using the Luciferase Assay System and the GloMax®-Multi Detection System. (4188)

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Int. J. Syst. Evol. Microbiol. 62, 2469–74. Anaerosalibacter bizertensis gen. nov., sp. nov., a halotolerant bacterium isolated from sludge. 2012

Rezgui, R. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Anaerosalibacter bizertensis. (4308)

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LWT—Food Science and Technology 46, 428–36. Antibacterial potential of Enterococcus faecium strains isolated from ewes' milk and cheese. 2012

Rivas, F.P. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Enterococcus faecium. (4338)

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J. Wildl. Dis. 48, 416–24. Antibody prevalence and molecular identification of Babesia spp. in roe deer in France. 2012

Bastian, S. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Babesia spp. (4310)

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Int. J. Syst. Evol. Microbiol. 62, 2731–6. Arthrobacter cupressi sp. nov., an actinomycete isolated from the rhizosphere soil of Cupressus sempervirens. 2012

Zhang, J., Ma, Y. and Yu, H.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Arthrobacter cupressi. (4309)

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J. Biol. Chem. 287, 3217–3230. C5a-regulated CCAAT/enhancer-binding proteins β and δ are essential in Fcγ receptor-mediated inflammatory cytokine and chemokine production in macrophages. 2012

Yan, C., Zhu, M., Staiger, J., Johnson, P.F. and Gao, H.

Notes: RAW 264.7 mouse macrophage cells were plated in 12-well plates at 1.5 × 105 cells/well for transient transfection with 0.5μg of DNA and 1.5μl of FuGENE® 6 Transfection Reagent in 50µl of Opti-MEM I medium. After 24 hours, the cells were treated with IgG IC for 5 hours and luciferase activity analyzed using the Dual-Luciferase® Reporter Assay System. (4402)

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Proc. Natl. Acad. Sci. USA 109, 13428-13433. CAG expansion induces nucleolar stress in polyglutamine diseases. 2012

Tsoi, H., Lau, T.C., Tsang, S.Y., Lau, K.F., and Chan, H.Y.

Notes: Polyglutamine diseases are neurodegenerative disorders associated with the presence of proteins containing polyglutamine repeats. These authors studied the mechanism of polygluatmine toxicity. Mutant RNAs carrying an expanded CAG repeat were shown to activate the nucleolar stress pathway and induce apoptosis. Expanded CAG RNAs were shown to interact with nucleolin, preventing it from binding to an upstream control element of the rRNA promoter and causing decreased rRNA transcription, which in turn induced apoptosis. Perturbations in rRNA transcription were identified by real-time PCR, and fluorescence in situ hybridization was used to determine that expanded CAG RNAs localized to the nucleolus. Hybridization solutions were supplemented with RNasin® Ribonuclease Inhibitor. ImProm-II™ Reverse Transcriptase was used in RT-qPCR assays. (4228)

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Mol. Biol. Cell 23, 864–80. Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content. 2012

Mundy, D.I., Li, W.P., Luby-Phelps, K. and Anderson, R.G.

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect SV589 Human Fibroblast cells. Transfection conditions were as follows: Cells were grown in 35mm plates and transfected with 2µg DNA at a 3:1 FuGENE® reagent:DNA ratio. (4422)

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J. Virol. 86, 8198–8209. Cellular GCN5 is a Novel Regulator of Human Adenovirus E1A-Conserved Region 3 Transactivation 2012

Ablack, J.N.G., Cohen,M., Thillainadesan, G., Fonseca, G.J., Pelka, P., Torchia, J. and Mymryk, J.S.

Notes: FuGENE HD reagent was used for transient transfection of A549 cells and wild type mouse embryonic fibroblasts. A 3:1 ratio of reagent to DNA was used in the transfection reactions (9µl reagent, 3µg DNA). (4410)

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Proc. Natl. Acad. Sci. USA 109, 12662-12667. Circadian clock protein cryptochrome regulates the expression of proinflammatory cytokines. 2012

Narasimamurthy, R., Hatori, M., Nayak, S.K., Liu, F., Panda, S., and Verma, I.M.

Notes: Sleep deprivation increases susceptibility to diseases such as diabetes and cancer. Because chronic inflammation is a feature of these diseases, the authors of this paper investigated whether the circadian oscillator component Cryptochrome (CRY) has a role in regulating the immune response. They found constitutive NF-κB and protein kinase A (PKA) activation in Cry1(-/-);Cry2(-/-) knockout mice, and high basal levels of cAMP. As part of the study, the effect of CRY overexpression on intracellular cAMP levels was evaluated in 293T cells using the bioluminescence-based GloSensor™ cAMP Assay. Overexpression of CRY1 reduced the cAMP production induced by forskolin, prostaglandin E2 or isoproterenol. Immunoprecipitation analysis with Flag-tagged CRY1 showed that CRY1 binds to adenylyl cyclase and limits cAMP production. The authors suggest that reduced CRY expression in chronic sleep deprivation may result in elevated cAMP levels, leading to increased PKA and NF-κB activation and offering a potential link between sleep deprivation, circadian rhythm disruption and chronic inflammation. (4224)

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Viruses 4, 200–10. Clinical characteristics and genetic variability of human rhinovirus in Mexico. 2012

Landa-Cardeña, A., Morales-Romero, J., García-Roman, R., Cobián-Güemes, A.G., Méndez, E., Ortiz-Leon, C., Pitalúa-Cortés, F., Mora, S.I. and Montero, H.

Notes: This study examined the prevalence of strains of human rhinovirus (HRV) that may be causing respiratory infections in Mexican children. Nucleic acids were purified from nasal swabs of two-year-old children, and screened for the presence of HRV by amplifying 20ng of HRV-RNA using the AccessQuick™ RT-PCR System with primers for the 5´ nontranslated region. Products were sequenced and aligned with sequences found in GenBank. (4343)

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Clin. Chem. 58, 580–9. COLD-PCR enrichment of rare cancer mutations prior to targeted amplicon resequencing. 2012

Milbury, C.A. et al.

Notes: Human Genomic DNA: Male was used to dilute experimental genomic DNA from cell lines and tumor samples for PCR before sequencing. (4543)

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Appl. Environ. Microbiol. 78, 420–8. Cultured representatives of two major phylogroups of human colonic Faecalibacterium prausnitzii can utilize pectin, uronic acids, and host-derived substrates for growth. 2012

Lopez-Siles, M. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Faecalibacterium prausnitzii. (4333)

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Cancer Res. 72, 335-345. Curcumin analogue CDF inhibits pancreatic tumor growth by switching on suppressor microRNAs and attenuating EZH2 expression.
2012

Bao, B., Ali, S., Banerjee, S., Wang, Z., Logna, F., Azmi, A.S., Kong, D., Ahmad, A., Li, Y., Padhye, S., and Sarkar, F.H.

Notes: EZH2 is an epigenetic regulator of cell proliferation that is known to be overexpressed in certain cancers. These authors evaluated the effect of the curcumin analogue CDF on expression of EZH2 in pancreatic cancer cells. The identity of the human pancreatic cancer cell lines used in the study was verified using the PowerPlex® 16 System. (4223)

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Anti-Cancer Drugs 23, 51–64. Cytotoxicity of human recombinant arginase I (Co)-PEG5000 in the presence of supplemental L-citrulline is dependent on decreased argininosuccinate synthetase expression in human cells
2012

Agrawal, V., Woo, J. H., Mauldin, J.P., Jo, C., Stone, E.M., Georgiou, G. and Frankel, A.E.

Notes: The authors used the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) as a third assay (in addition to 3H thymidine and 3H leucine assays) to assess the response of cells to CoArgIPEG and demonstrated not only a decline in cell survival with treatment but also a decline in proliferation and protein synthesis in most cases.

The CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was also used to evaluate the effect of recovery in cancer cells. The authors determined that prolonged treatment resulted in cytotoxicity; however, assessment at 24 hours showed that some cells survived in the short term. Because the authors knew proliferation/protein synthesis inhibition was occurring, evaluation with a cell survival assay like the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was required for a more complete understanding of cellular health and response. (4511)

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Food Control 23, 400-404. Development and validation of fast Real-Time PCR assays for species identification in raw and cooked meat mixtures. 2012

Cammà, C., Di Domenico, M., and Monaco, F.

Notes: These authors used the Maxwell® 16 Tissue DNA Purification Kit to extract DNA from 200µl samples of raw meat homogenate from beef, pork, poultry and lamb samples. They also used the Wizard® Genomic DNA Isolation System to extract DNA from cooked meat samples. The extracted DNA was used in real-time, quantitative PCR assays to identify species-specific DNA spiked at 1% in mixed DNA samples. (4353)

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J. Biomol. Scr. 17, 993–8. Development of a novel ectonucleotidase assay suitable for high-throughput screening. 2012

Sachsenmeier, K.F., Hay, C., Brand, E., Clarke, L., Rosenthal, K., Guillard, S., Rust, S., Minter, R. and Hollingsworth, R.

Notes: The authors of this paper describe the development of a high-throughput assay to screen for antibody inhibitors of 5´-ectonucleotidase activity (converts AMP to adeonsine plus free phosphate). They used the CellTiter-Glo® Cell Viability Assay to screen for activity. The CellTiter-Glo® luciferase reaction is inhibited by AMP; activity of 5´-ectonucleotidase eliminates AMP, thereby relieving the reaction from inhibition, producing light. The authors demonstrate release of the luciferase reaction from inhibition as a result of 5´-ectonucleotidase activity, and that they were able to show inhibition of 5´-ectonucleotidase activity by anti-5´-ectonucleotidase antibodies. (4261)

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