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J. Exp. Bot. 61, 191–202. Physiological and molecular changes in Oryza meridionalis Ng., a heat-tolerant species of wild rice. 2010

Scafaro, A.P., Haynes, P.A. and Atwell, B.J.

Notes: The authors compared seedling growth rates, photosynthesis rates and expression levels of heat-responsive genes in the heat-resistant wild rice Oryza meridionalis and the domesticated rice O. sativa when grown at optimal and elevated temperatures. Proteins that were up- or downregulated in response to heat were identified by two-dimensional gel electrophoresis coupled with nano liquid chromatography on line with tandem mass spectrometry (nanoLC-MS/MS). Trypsin was used to cleave proteins prior to nanoLC-MS/MS. Semi-quantitative RT-PCR was performed using the GoTaq® Green Master Mix to determine if the heat-responsive proteins were transcriptionally regulated. (4092)

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J. Biochem. 148, 721–32. A recombinant catalytic domain of matriptase induces detachment and apoptosis of small-intestinal epithelial IEC-6 cells cultured on laminin-coated surface. 2010

Mochida, S., Tsuzuki, S., Inouye, K. and Fushiki, T.

Notes: The authors determined that a recombinant catalytic domain of rat matriptase (His6t-S-CD) caused detachment of small-intestinal epithelial cells (IEC-6 cells) from laminin-coated plates. His6t-S-CD was expressed in the yeast P. pastoris and purified by ammonium sulfate precipitation, gel filtration through a PD-10 column, then Ni2+-chelating chromatography using HisLink™ Resin. The authors also treated IEC-6 cells with purified His6t-S-CD to determine if this domain induced apoptosis by monitoring annexin-V staining, DNA fragmentation and caspase-3 activity. For the DNA fragmentation analysis, IEC-6 cells were treated with His6t-S-CD, then harvested, and genomic DNA was purified using the Wizard® SV Gel and PCR Clean-Up System. DNA fragmentation was assessed by agarose gel electrophoresis and ethidium bromide staining. (4100)

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Appl. Environ. Microbiol. 76, 3590–9. Growth of bacteria on 3-nitropropionic acid as a sole source of carbon, nitrogen, and energy. 2010

Nishino, S.F., Shin, K.A., Payne, R.B. and Spain, J.C.

Notes: The authors identified a bacterial strain that can use the toxin 3-nitropropionic acid (3NPA) as its sole carbon and nitrogen sources and cloned the genes that encode the enzymes responsible for the initial steps in the 3NPA degradation pathway. The Pseudomonas library used to clone these genes was created using genomic DNA isolated using the Wizard® SV Genomic DNA Purification System. Closely related genes were amplified from other bacterial species for phylogenetic analysis. PCRs were performed using the GoTaq® Hot Start Polymerase. (4164)

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Am. J. Bot. 97, 1574–8. Clonal structure of wild populations and origins of horticultural stocks of Illicium parviflorum (Illiciaceae). 2010

Newell, D.L. and Morris, A.B.

Notes: The authors investigated genetic diversity in a Florida population of Illicium parviflorum, an endangered evergreen shrub, by amplifying intersimple sequence repeats (ISSRs). Amplifications were performed using GoTaq® Hot Start Polymerase. (4161)

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Nucl. Acids Res. 38, 6985-96. Targeted next-generation sequencing of DNA regions proximal to a conserved CXGXXG signaling motif enables systematic discovery of tyrosine kinase fusions in cancer 2010

Chmielecki,J., Peifer, M., Socci, N.D., Hutchinson, K., Viale, A., Zhao, Z., Thomas, R.K. and Pao, W.

Notes: Human Genomic DNA:Male was used as a negative control in standard PCR and Sanger Sequencing to confirm fusion genomic breakpoints identified by NGS experiments. (4534)

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Am. J. Pathol. 177, 2347–56. Development of sporadic microsatellite instability in colorectal tumors involves hypermethylation at methylated-in-tumor loci in adenoma. 2010

de Maat, M.F., Narita, N., Benard, A., Yoshimura, T., Kuo, C., Tollenaar, R.A., de Miranda, N.F., Turner, R.R., van de Velde, C.J., Morreau, H. and Hoon, D.S.

Notes: The authors examined the methylation status of methylated-in-tumor (MINT) loci and the degree of microsatellite instability (MSI) in colorectal cancer to determine if methylation of MINT loci during the progression of adenoma to cancer was linked to MSI. They used on-slide sodium bisulfite modification and methylation-specific PCR to examine the methylation index in paraffin-embedded tissue blocks containing normal, adenoma and cancer tissues. MSI status was determined using the MSI Analysis System. Patients with instability at more than 4 markers were classified as MSI-high, and patients with instability at more than 1 marker were considered microsatellite-stable. (4106)

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Development 137, 901–11. SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. 2010

Hattori, T., Müller, C., Gebhard, S., Bauer, E., Pausch, F., Schlund, B., Bösl, M.R., Hess, A., Surmann-Schmitt, C., von der Mark, H., de Crombrugghe, B. and von der Mark, K.

Notes: To study the role of the transcription factor Sox9 in the transition from cartilage to bone in newborn mice, the authors performed chromatin immunoprecipitation using the HaloCHIP™ System. Primary rib chondrocytes were transfected with a vector expressing full-length Sox9 with a HaloTag® protein tag, then proteins and DNA were cross-linked. DNA was isolated and sonicated, and the Sox9:DNA complexes were precipitated using the HaloLink™ Resin. The precipitated DNA then was amplified by PCR to determine that SOX9 binds to the SRY sites in the Vegfa gene. (4054)

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Appl. Environ. Microbiol. 76, 2783–90. Revelation by single-nucleotide polymorphism genotyping that mutations leading to a premature stop codon in inlA are common among Listeria monocytogenes isolates from ready-to-eat foods but not human listeriosis cases. 2010

Van Stelten, A., Simpson, J.M., Ward, T.J. and Nightingale, K.K.

Notes: Listeria monocytogenes uses the internalin A protein (InlA) to cross the intestinal barrier and cause foodborne illness. Mutations in inlA can introduce a premature stop codon, producing a truncated InlA protein that is secreted rather than associated with the bacterial cell wall. Strains with these inlA mutations have reduced virulence. The authors describe an inlA single nucleotide polymorphism (SNP)-based genotyping assay to distinguish isolates with the inlA mutations and use this assay to screen >1,000 L. monocytogenes isolates from ready-to-eat foods and human listeriosis cases. The assay involves amplification of the full-length inlA gene using GoTaq® Colorless Master Mix, purification of amplified products, then single-base-pair extension reactions. (4099)

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Clin. Can. Res. 16, 1391–401. High-resolution array comparative genomic hybridization in sporadic and celiac disease-related small bowel adenocarcinomas. 2010

Diosdado, B., Buffart, T.E., Watkins, R., Carvalho, B., Ylstra, B., Tijssen, M., Bolijn, A.S., Lewis, F., Maude, K., Verbeke, C., Nagtegaal, I.D., Grabsch, H., Mulder, C.J., Quirke, P., Howdle, P. and Meijer, G.A.

Notes: To better examine the molecular mechanisms of small bowel adenocarcinomas, DNA was extracted from paraffin-embedded tissue and tested for microsatellite instability (MSI) using the MSI Analysis System, Version 1.2. The PCR products were run on the Applied Biosystems 3130 Genetic Analyzer and analyzed using GeneScan® software. If two or more of the the BAT-25, BAT-26, NR-21, NR-24 or MONO-27 monomorphic markers had altered lengths, the tumors were designated as MSI unstable. (4112)

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Clin. Chem. 55, 748–56. Coamplification at lower denaturation temperature-PCR increases mutation-detection selectivity of TaqMan-based real-time PCR. 2009

Li, J., Wang, L., Jänne, P.A. and Makrigiorgos, GM.

Notes: The authors describe a new form of PCR, co-amplification at lower denaturation temperature PCR (COLD-PCR), to detect low-level somatic mutations. This technique is based on the facts that a) each DNA sequence has a critical denaturation temperature (Tc), which is lower than the melting temperature (Tm) and below which PCR efficiency decreases dramatically and b) Tc depends on DNA sequence. The authors used GoTaq® Flexi DNA Polymerase and mutation-specific TaqMan® probes for tumor protein 53 (TP53) and epidermal growth factor receptor (EGFR) to detect low-level somatic mutations in a mixture of wildtype and mutant DNAs. Conventional TaqMan® technology can detect mutant alleles at an abundance of 10–20% of that of the wildtype allele; using COLD-PCR the authors were able to increase selectivity 15- to 30-fold, detecting as little as 0.8% mutuant alleles. (4038)

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Appl. Environ. Microbiol. 75, 2275–83. Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by gacA. 2009

Shi, X.Y., Dumenyo, C.K., Hernandez-Martinez, R., Azad, H. and Cooksey, D.A.

Notes: To gain a better understanding of how Xylella fastidiosa causes diseases in grapes, the authors mutated conserved regulatory genes, including gacA, that affect expression of virulence-related factors in other species. The relative expression levels of gacA in wildtype and mutated strains were examined using RT-PCR. The authors also identified and quantified a number of genes that were regulated by GacA by microarray analysis. Microarray results were confirmed using RT-PCR. RT-PCR was performed using the AccessQuick™ RT-PCR System. (4052)

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Neuropsychopharmacology Feb. 11, (epub ahead of print). Nucleus accumbens CREB activity is necessary for nicotine conditioned place preference. 2009

Brunzell, D.H., Mineur, Y.S, Neve, R.L. and Picciotto, M.R.

Notes: The authors of this study used the HRE-CRE-luciferase reporter cell line (Glo-Response™ Cells) to test HSV constructs for activity. Cells were infected with HSV-CREB, HSV-mCREB (dominant negative) or HSV-LacZ control vector. Comparisons indicated that cells transfected with HSV-CREB showed increase in CRE-mediated activity, while those transfected with HSV-mCREB showed attenuation of CRE-mediated cellular activity. (3956)

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Clin. Can. Res. 15, 7562–70. Smoking-related gene expression in laser capture-microdissected human lung. 2009

Tan, X.L., Wang, T., Xiong, S., Kumar, S.V., Han, W. and Spivack, S.D.

Notes: The authors characterized differential expression of several carcinogen metabolism genes in human alveolar compartment (AC) and bronchial epithelial compartment (BEC) lung tissues in smokers, former smokers and people who have never smoked. They combined laser capture microdissection (LCM) and quantitative RT-PCR. RNA was isolated from paired microdissected malignant and nonmalignant lung tissue, 100ng of total RNA was reverse transcribed in a 20µl reaction, then 1µl of cDNA was amplified by real-time PCR using an ABI PRISM® 7500HT sequence detection system, GoTaq® Flexi DNA Polymerase and gene-specific primers. The expression level for each gene was normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results showed that expression of cytochrome P450 1B1 and glutathione-S-transferase P1 in AC, but not BEC, tissue was strongly associated with exposure to tobacco. (4095)

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Otolaryngol. Head Neck Surg. 140, 55–60. Microsatellite instability analysis of sinonasal carcinomas. 2009

Martínez, J.G., Pérez-Escuredo, J., López, F., Suárez, C., Alvarez-Marcos, C., Llorente, J.L. and Hermsen, M.A.

Notes: Because intestinal-type sinonasal adenocarcinoma (ITAC) and squamous cell carcinoma of the nasal cavity (SCCNC) are histopathologically similar to microsatellite-unstable colorectal adenocarcinoma or laryngeal squamous cell carcinoma, respectively, the microsatellite instability (MSI) state of the nasal tumors were of interest to researchers. Two nanograms of purified DNA from 41 ITACs and 24 SCCNCs were amplified for shifts in five mononucleotide microsatellite loci using the MSI Analysis System, Version 1.2. The multiplex PCR products were analyzed by capillary electrophoresis and noted as MSI positive if there was a size shift of at least one marker. (4117)

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J. Biol. Chem. 284, 19402–11. Structural Determinants of G-protein α Subunit Selectivity by Regulator of G-protein Signaling 2 (RGS2) 2009

Kimple, A.J., Soundararajan, M., Hutsell, S.Q., Roos, A.K., Urban, D.J., Setola, V., Temple, B.R.S., Roth, B.L., Knapp, S.K., Willard, F.S. and Siderovsk, D.P.

Notes: The authors created a triple mutant of Regulator of G-Protein Signaling Protein 2 (RGS2) to characterize the structural features responsible for its selectivity in binding to the Gαq or Gαi/o subunits of GTPase-accelerating protein (GAP). The RGS2 enhances the termination of G-protein coupled signaling by enhancing GAP. RGS proteins are considered key modulators of G Protein-Coupled Receptor (GPCR) signaling based on their ability to accelerate GTP hydrolysis. The GloSensor™ cAMP assay was used to assess the level of GPCR activity and indicate which structural determinants of RGS2 affect binding to Gα subunits of GAP.

HEK293T cells were transiently co-transfected with expression vectors for the GloSensor™ cAMP biosensor and the Gi-coupled dopamine D2-receptor with empty vector, wild type RGS2, or the RGS2(triple) mutant. Treatment of transfected cells with forskolin produced an increase in luminescence from the cAMP sensor, reflecting direct activation of adenylyl cyclase by forskolin. Quinpirole, a dopamine D2 receptor agonist, produced a dose-dependent inhibition of cAMP production. Inhibition of forskolin-stimulated cAMP production was assessed after activation of the D2 receptor with various concentrations of quinpirole to compare IC50 values for the empty vector, wild type RGS2 and triple mutant RGS2. Cellular expression of the triple mutant resulted in a significantly higher IC50 for quinpirole (762nM versus 18 nM for empty vector), indicating that the three point mutations weaken Gαi subunit binding responsible for enhanced GTPase activity.

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Nucl. Acids Res. 37, 78–95. Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage. 2009

Wilson, P.M., Fazzone, W., LaBonte, M.J., Lenz, H.J. and Ladner, R.D.

Notes: The authors examined the role of p53 in modulating dUTPase promoter activity. Base substitution mutations of Sp1- and E2F-binding sites in the dUTPase promoter were performed using the GeneEditor™ in vitro Site-Directed Mutagenesis System. Each mutant was confirmed by DNA sequencing. To determine growth inhibition, HCT116 human colon cancer cells were seeded in 96-well plates at 3 × 103 cells/well and treated with 5-fluorouracil (5-FU), fluorodeoxyuridine (FUdR), oxaliplatin or in combination. After 72 hours, the CellTiter® 96 AQueous One Solution was dispensed into each well and absorbance measured. RNA was isolated from HCT116 p53+/+ and HCT116 p53-/- cells. cDNA was reverse transcribed from 200ng total RNA followed by multiplex qPCR using the Plexor™ qPCR System to amplify dUTPase, thymidylate synthase and GAPDH, a housekeeping gene. The 1.2 kb region of the dUTPase promoter upstream of the transcriptional start site was amplified by PCR and the fragment cloned into the pGL3-Basic Vector. Truncated promoters were also generated by PCR and cloned into the same vector. Drosophila SL-2 cells and HCT116 cell lines were seeded in a 24-well plate and transfected with dUTPase pGL3 promoter constructs or with pCI-Neo:p53WT, pCI-Neo:p53MUT and the empty pCI-neo Mammalian Expression Vector; all transfections included the pRL-TK Vector at a ratio of 1:10. After six hours, the cells were incubated in either fresh medium or medium containing a cytotoxic agent at the appropriate concentration. Thirty hours later, the cells were lysed, quantitated by Western blotting and 20µl of lysate analyzed with the Dual-Luciferase® Reporter Assay System. Electrophoretic mobility shift analyses (EMSA) were performed using –64 to –91 of the dUTPase-nuclear isoform transcriptional start site in the Gel Shift Assay System. (4031)

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ISME J. 3, 498–502. Malassezia furfur fingerprints as possible markers for human phylogeography. 2009

Gaitanis, G., Velegraki, A., Alexopoulos, E.C., Kapsanaki-Gotsi, E., Zisova, L., Ran, Y., Zhang, H., Arsenis, G., Bassukas, I.D. and Faergemann, J.

Notes: The authors examined Malassezia furfur strains isolated from Scandinavians permanently residing in Greece and clinical isolates from Greece, Bulgaria and China to determine if there was an association between strain and the host’s geographical origin and any underlying skin conditions. Prior to strain identification by PCR fingerprinting, M. furfur DNA was isolated using the Maxwell® 16 Instrument and a modified Maxwell® 16 Cell DNA Purification protocol. A single 2–3mm diameter yeast colony yielded 20ng of DNA. (3959)

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Methods in Mol. Biol. 577, 25-39. High-Throughput Construction of ORF Clones for Production of the Recombinant Proteins 2009

Yamakawa, Hisashi

Notes: The authors use the Flexi® Cloning System to convert their cDNA clones to expression-ready clones. They wanted clones that could be used for comprehensive analysis with the HaloTag® Technology. They also describe a method of transferring ORFs between Flexi® Vectors in a 96-well plate format. They also used Wizard® SV 96 Plasmid DNA Purification, Wizard® SV PCR Clean-Up, and Wizard® SV Gel and PCR Clean-Up Systems. (4056)

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Genetics 182, 133–144. A proximal centriole-like structure is present in Drosophila spermatids and can serve as a model to study centriole duplication. 2009

Blachon, S., Cai, X., Roberts, K.A., Yang, K., Polyanovsky, A., Church, A. and Avidor-Reiss, T.

Notes: These authors studied the formation of centrioles in Drosophila spermatids. Genomic DNA was extracted from whole flies using the Wizard® SV Genomic DNA Purification System. The DNA was then subjected to PCR, purified and sequenced. RNA was purified from whole flies using the SV Total RNA Isolation System. After RNA extraction, the samples were used in RT-PCR. (4064)

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J. Clin. Endocrinol. Metab. 94, 2594-2601. KLF15 Is a transcriptional regulator of the human 17beta-hydroxysteroid dehydrogenase type 5 gene. A potential link between regulation of testosterone production and fat stores in women 2009

Du, X., Rosenfield, R. and Qin, K.

Notes: The authors used a HaloTag® vector and the HaloCHIP™ System to identify a KLF15 binding site in the HSD17B5 promoter. (4059)

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Vet. Rec. 164, 44–47. Prevalence of thermophilic Campylobacter species in household cats and dogs in Ireland. 2009

Acke, E., McGill, K., Golden, O., Jones, B.R., Fanning, S. and Whyte, P.

Notes: To examine the prevalence of Campylobacter species in asymtomatic carriers that can pass the bacteria onto humans, rectal swabs were collected from 147 dogs and 35 cats in Ireland and cultured on various diagnostic plates. The Wizard® Genomic DNA Purification System was used to isolate DNA from any suspect Campylobacter cultures. The purified DNA was used in multiplex PCR and RFLP to determine which species of Campylobacter was present. (4016)

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Proc. Natl. Acad. Sci. USA 106, 21306–11. Human cancers converge at the HIF-2alpha oncogenic axis. 2009

Franovic, A., Holterman, C.E., Payette, J. and Lee, S.

Notes: These authors used short heteronuclear RNAs (shRNA) in multiple cancer cell lines to silence hypoxia-inducing factor-2α (HIF-2α), a gene that many cancers exploit to increase angiogenesis and activate fundamental receptor tyrosine kinase signaling pathways to gain growth signal autonomy. Western blotting and RT-PCR were used to monitor the level of HIF-2α silencing. RT-PCR was performed using the AccessQuick™ RT-PCR System. The authors also examined the level of tyrosine kinase activation in shRNA-treated cells by Western blot analysis. To examine levels of ERK1/2 phopshorylation, the authors used the Anti-ERK 1/2 pAb to compare levels of activated and unactivated ERK1 and 2. (4050)

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Drug Metab. Dispos. 37, 1759–1768. Quantitative analysis of UDP-glucuronosyltransferase (UGT) 1A and UGT2B expression levels in human livers. 2009

Izukawa, T., Nakajima, M., Fujiwara, R., Yamanaka, H., Fukami, T., Takamiya, M., Aoki, Y., Ikushiro, S., Sakaki, T. and Yokoi, T.

Notes: This study examined the expression levels of each UGT isoform in human liver and evaluated the variability between individuals. Total RNA from appropriate human tissues or various cell lines was used for RT-PCR of various human UDP-glucuronosyltransferases (UGT) cDNAs. The amplimers were cloned into the pTARGET™ Mammalian Expression Vector and verified by sequencing. The UGT vectors were linearized by restriction enzyme digestion and used for standards in real-time RT-PCR analysis. (4034)

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J. Biol. Chem. 284, 29526–35. Escherichia coli unsaturated fatty acid synthesis: complex transcription of the fabA gene and in vivo identification of the essential reaction catalyzed by FabB. 2009

Feng, Y. and Cronan, J.E.

Notes: The authors examined the role of two promoters in the regulation of fabA, an enzyme involved in unsaturated fatty acid synthesis. fabA transcript levels were quantified using real-time quantitative RT-PCR using ImProm-II™ Reverse Transcriptase, followed by a SYBR® Green method. (4053)

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Drug Metab. Dispos. 37, 1726–1732. Characterizing the effects of common UDP glucuronosyltransferase (UGT) 1A6 and UGT1A1 polymorphisms on cis- and trans-resveratrol glucuronidation. 2009

Iwuchukwu, O.F., Ajetunmobi, J., Ung, D. and Nagar, S.

Notes: This study examined the genotype-phenotype correlation of the two major UGT isoforms, UGT1A1 and UGT1A6, involved in resveratrol metabolism. Genomic DNA was isolated from 30mg human liver tissue samples (normal and metastatic) using the Wizard® SV Genomic DNA Purification System. The purified DNA was eluted with 65°C water and 200–400ng of eluted DNA was used in a PCR-RFLP UGT1A6 genotyping assay. Amplification was carried out using PCR Master Mix in a final volume of 50µl, and the amplimers digested with appropriate restriction enzymes. (4018)

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