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Antimicrob. Agents Chemother. 56, 1300–8. Impaired fitness and transmission of macrolide-resistant Campylobacter jejuni in its natural host. 2012

Luangtongkum, T. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Campylobacter jejuni. (4317)

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Virol. J. 9, 144. Inaccurate identification of rotavirus genotype G9 as genotype G3 strains due to primer mismatch. 2012

Mitui, M.T., Chandrasena, T.N., Chan, P.K., Rajindrajith, S., Nelson, E.A., Leung, T.F., Nishizono, A. and Ahmed, K.

Notes: This study examined how well primers developed in 1990 and 2004 for type A rotavirus (RVA) were able to genotype (G type) currently circulating RVAs in Asia. The VP7 gene from RVA was amplified using 2µl of dsRNA template with the AccessQuick™ RT-PCR System in a total volume of 50µl. The G type was determined using hemi-nested multiplex PCR using 1µl of the VP7 cDNA and PCR Master Mix in a final volume of 50µl. The final products were sequenced. (4340)

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FASEB J. 26, 1629-39. Influenza A virus inhibits cytoplasmic stress granule formation. 2012

Khaperskyy DA, Hatchette TF, McCormick C.

Notes: Fugene HD was used with HeLa-TetOff cells, seeded at a density of 100,000 cells/well of a 12-well culture plate. On day 2, cells were transfected with 0.5 μg total DNA/well and FuGene HD reagent with a DNA to FuGENE HD ratio of 1:3 (w/v). (4423)

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PLos ONE 7(1), e30061. Inhibition of firefly luciferase by general anesthetics: effect on in vitro and in vivo bioluminescence imaging. 2012

Keyaerts, M., Remory, I., Caveliers, V., Breckpot, K., Bos, T.J., Poelaert, J., Bossuyt, A., and Lahoutte, T.

Notes: These authors investigated the effects of various anesthetics on bioluminescence imaging with firefly luciferase. They observed decreases in luminescence with volatile anaethetics, and found increased luciferase expression with injectable anaethetics in intact cells, but not in cell lysates in vitro. They concluded that the decreases in luciferase activity observed with volatile anaesthesia were due to hemodynamic effects, and not due to a direct inhibitory effect on luciferase enzyme itself. The apparent enhancement of luciferase activity with certain injectable anaesthetics appeared to be due to cytotoxic effects that resulted in increased permeablity to luciferase, as the same enhancement was not observed in cell lysates. D-Luciferin was used for in vivo imaging experiments. The pGL4.10 vector (encoding firefly luciferase), Luciferase Assay Reagent, and the GloMax® 96 Microplate Luminometer were used for in vitro assays using cell lysates. (4190)

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Mol. Biol. Cell 23, 3499-510. Large G3BP-induced granules trigger eIF2α phosphorylation. 2012

Reineke, L.C., Dougherty, J.D., Pierre, P., and Lloyd, R.E.

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect HeLa and MEF cells. Transfection conditions were as follows: HeLa Tet-On cells were plated on glass coverslips at 1-1.3 X 10e5 cells/well in 12-well plates 1 day prior to transfection with 0.2µg plasmid DNA. Mouse embryonic fibroblast (MEF) cells were plated at 5x10e4 cells/well in 12-well plates 1 day prior to transfection with 2µg plasmid DNA.

 

  (4414)

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J. Bacteriol. 194, 1389-1400. Legionella pneumophila LidA affects nucleotide binding and activity of the host GTPase Rab1. 2012

Neunuebel, M.R., Mohammadi, S., Jarnik, M., and Machner, M.P.

Notes: In this study, the L. pneumophila protein Lem3 was expressed as a HaloTag® fusion protein and purified using HaloLink™ Resin. Lem3 was first cloned into the pFN22K HaloTag® Vector and the resultant HaloTag-Lem3 protein was expressed in Single-Step (KRX) competent cells before purification using the HaloTag® Protein Purification System. Lem3 was cleaved from the HaloLink™ Resin using TEV protease.

  (4350)

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Funct. Food Health Disease 2, 228–241. Lowbush blueberries, Vaccinium angustifolium, modulate the functional potential of nutrient utilization and DNA maintenance mechanisms in the rat proximal colon microbiota 2012

Lacombe, A., Li, R.W., Klimis-Zacas, D., Kristo, A.S., Tadepalli, S., Krauss, E., Young, R. and Wu, V.C.H.

Notes: The authors of this study sought to determine how a diet enriched with low bush blueberries would affect the metabolic potential of gut microbes. For this study, they fed rats either a control diet or the control diet plus 8% weight/volume powder derived from low bush blueberries. After six weeks, rats were sacrificed and DNA was extracted from stool. Genomic DNA concentration was determined using the Quantus™ Fluorometer prior to being subjected to next generation sequencing (NGS). (4502)

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Funct. Food Health Disease 2, 228–41. Lowbush blueberries, Vaccinium angustifolium, modulate the functional potential of nutrient utilization and DNA maintenance mechanisms in the rat proximal colon microbiota. 2012

Lacombe, A., Li, R.W., Klimis-Zacas, D., Kristo, A.S., Tadepalli, S., Krauss, E., Young, R. and Wu, V.C.H

Notes: The authors characterized the gut microbiota in two populations of Sprague Dawley rats—one that was fed the AIN93 diet and the other was fed the AIN93 diet with lowbush wild blueberry powder. After 6 weeks, rats were sacrificed and DNA was purified from the contents of the proximal colon and subjected to whole genome sequencing to identify the resident bacterial species. The DNA concentration was determined using the QuantiFluor™-ST Fluorometer prior to Illumina next-generation sequencing. (4239)

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Proc. Natl. Acad. Sci. USA 109(41) , 16540-16545. . Massively parallel measurements of molecular interaction kinetics on a microfluidic platform. 2012

Geertz, M., Shore, D., and Maerkl, S.J.

Notes: These authors describe use of a microfluidic device based on mechanically induced trapping of molecular interactions (k-MITOMI) for kinetic characterization of 768 biomolecular interactions in parallel. Using this approach, they measured the association and dissociation kinetics of numerous transcription factors with their associated consensus sequences. The TNT® T7 Quick Coupled Transcription/Translation System was used to express the transcription factors from linear templates, either on-chip or in bulk reactions. (4247)

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DNA Research 19, 395-406. Mate pair sequencing of whole-genome-amplified DNA following laser capture microdissection of prostate cancer 2012

Murphy, S.J., Cheville, J.C., Zarei, S., Johnson, S.H., Sikkink, R.A., Kosari, F., Feldman, A.L., Eckloff, B.W., Karnes, R.J. and Vasmatzis, G.

Notes: Genomic rearrangements detected by NGS were mapped and confirmed by PCR. Human Genomic DNA: Male was used as a control for the confirmatory experiments. (4539)

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J. Biol. Chem. 287, 14052–68. MDM2 protein-mediated ubiquitination of NUMB protein: Identification of a second physiological substrate of MDM2 that employs a dual-site docking mechanism. 2012

Sczaniecka, M., Gladstone, K., Pettersson, S., McLaren, L., Huart, A.S. and Wallace, M.

Notes: The E3 ubiquitin ligase murine double minute 2 (MDM2) regulates transcriptional activity and expression levels of the p53 tumor suppressor protein. In this article, the authors show that the cell fate determinant NUMB interacts with MDM2, disrupting the interaction of MDM2 and p53 and preventing the ubiquitination of p53. To study protein-protein interactions between NUMB and MDM2, the authors used the pFN2A (GST) Flexi® Vector to express full-length and partial NUMB sequences as GST fusion proteins for protein pull-down experiments. To examine their hypothesis that the interaction between NUMB and MDM2 increases p53 levels and thus apoptosis in cells, they treated ZR75 human breast carcinoma cells and A375 human melanoma cells with NUMB-derived peptides and small molecule ligands that block the interaction between NUMB and MDM2. These treatments resulted in increased levels of p53 protein, Annexin V staining as well as caspase-3/7 activity, as determined using the Caspase-Glo® 3/7 Assay System. (4351)

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Bioorg. Med. Chem. 20, 6134–43. Minutissamides E-L, antiproliferative cyclic lipodecapeptides from the cultured freshwater cyanobacterium cf. Anabaena sp. 2012

Kang, H.-S. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Anabaena sp. (4299)

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Biochim. Biophys. Acta 1822, 248-260. MiR-21 is involved in cervical squamous cell tumorigenesis and regulates CCL20. 2012

Yao, T., and Lin, Z.

Notes: This study identified that miR-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines, and showed that the level of miR-21 correlates with tumor differentiation, and regulates proliferation, apoptosis, and migration of HPV16-positive cells. The authors used gene expression profiling and luciferase reporter assay to identify candidate target genes for miR-21. Wildtype and mutant 3′-UTR sequences from the target gene CCL20 containing putative binding sites for miR-21 were subcloned into the psiCHECK-2 Vector and used to transfect HEK293 cells. The Dual-Luciferase® Reporter Assay System and GloMax® Multi Luminometer were used to measure luciferase activity from both constructs. (4192)

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Int. J. Infect. Dis. 16(1), e53–9. Molecular detection and characterization of West Nile virus associated with multifocal retinitis in patients from southern India. 2012

Shukla, J., Saxena, D., Rathinam, S., Lalitha, P., Joseph, C.R., Sharma, S., Soni, M., Rao, P.V. and Parida, M.

Notes: This study describes the clinical observations and laboratory investigations performed on 170 of the 2,000 suspected West Niles Virus (WNV) cases. These cases were admitted to Aravind Eye Hospital, Madurai, Tamil Nadu with ocular complications. Conventional reverse transcription PCR (RT-PCR) and real-time RT-PCR assays were used to detect WNV infection. In addition, reverse transcription loop-mediated isothermal gene amplification (RT-LAMP) was performed to determine the feasibility of using this method as an alternative cost-effective tool to the real-time RT-PCR.

After proving negative for DENV- and CHIKV, samples were tested for the presence of WNV-specific RNA by RT-PCR, real-time RT-PCR and RT-LAMP assays. RNA was extracted from the patient serum, plasma and infected culture supernatant. The RNA was then eluted in 50µl of nuclease-free water and used as template in the AccessQuick™ RT-PCR System, with primer pairs targeting the env gene.

Amplification was performed in a 50µl total reaction volume with the AccessQuick™ RT-PCR System, using 50pmol of each forward and reverse primer and 2µl of extracted viral RNA. (4331)

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Veterinary Microbiology 160(3-4), 463–467. Molecular detection of murine noroviruses in laboratory and wild mice. 2012

Farkas, T., Fey, B., Keller, G., Martella, V. and Egyed, L.

Notes: Mice RNA samples were converted to cDNA using an oligo-dT primer with the Reverse Transcription System, ethanol precipitated, vacuum dried and transferred to another lab. There they were reconstituted in 20μl of molecular biology grade water.

Detection of caliciviruses in the wild mice samples was attempted using generic calicivirus primers targeting sequences encoding conserved amino acid motifs in the RNA-dependent RNA polymerase (RdRp) region of ORF1. Two microliters of cDNA was used in 25μl PCR reactions using the GoTaq® Green Master Mix. Laboratory mouse RNA samples were tested only with MNV-specific primers in the AccessQuick™ RT-PCR System using 2μl RNA as template.

PCR products were cloned into pGEM-T® Vector and sequenced using M13 forward and reverse primers on an ABI PRISM® 3730 DNA Analyzer. (4330)

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Anaerobe 18, 566–75. Morphological, biochemical, physiological and molecular aspects of the response of Fusobacterium nucleatum exposed to subinhibitory concentrations of antimicrobials. 2012

de Souza Filho, J.A. et al.

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Fusobacterium nucleatum. (4335)

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PLoS Genet. 8, e1002941. MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells. 2012

Goodier, J.L., Cheung, L.E. and Kazazian, H.H. Jr.

Notes: To better understand the role MOV10 protein, a putative RNA helicase and component of the RNA–induced silencing complex (RISC), in reducing retrotransposon activity, 293T human embryonic kidney cells expressing MOV10 and one of three retrotransposons (LINE1 (L1), Alu or SVA) were lysed in a buffer that included RNasin® Ribonuclease Inhibitor, and FLAG-tagged L1 complexes immunoprecipitated and analyzed by mass spectrometry. Several retrotransposon assays were conducted using FuGENE® HD Transfection Reagent for transfected constructs into 293T, HeLa and HeLa-HA cells. (4255)

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Development 139, 3986-96. Pancortins interact with amyloid precursor protein and modulate cortical cell migration. 2012

Rice, H.C., Townsend, M., Bai, J., Suth, S., Cavanaugh, W., Selkoe, D.J., and Young-Pearse, T.L.

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect HEK293 cells. HEK293 cells were plated at 1 X 10e6 cells/well in 6-well plates prior to transfection. (4413)

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J. Neurooncol. epub ahead of print. Phenytoin reduces 5-aminoleuvulinic acid-induced protoporphyrin IX accumulation in malignant glioma cells. 2012

Hefti, M., Albert, I., and Luginbuehl, V.

Notes: 5-aminolevulinic acid (5-ALA)-induced protoprophyrin IX (PpIX) fluorescence is often used for diagnostics and therapy in malignant glioma surgery; however changes in the ability of malignant glioma cells to accumulate PpIX can result in erroneous interpretation of the fluorescent signal and affect the safety of fluorescence-guided surgery. Such changes can result from several factors, including drug interactions. The authors of this paper investigated the effects of antiepiletic drugs (AED) commonly given to glioma patients on the ability of glioma cells to accumulate PpIX. They looked at cell health and metabolism in glioma cells treated with these drugs, including glutathione metabolism. The GSH/GSSG-Glo™ Assay was used to generate a ratio of reduced (GSH) and oxidized (GSSG) glutathione in glioma cell lines (U-87 MG and U373 MG). Cells were seeded at 5 × 103 cells/well in 96-well plates and treated with either 47µg/ml phenytoin, 104µg/ml levetiracetam or DMSO-only for three days, and the assay was performed as described in the technical manual. Neither test compound effected the GSH/GSSG ratio.  (4211)

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J. Biol. Chem. 287, 22969-22987. Pink1 kinase and its membrane potential (Deltaψ)-dependent cleavage product both localize to outer mitochondrial membrane by unique targeting mode. 2012

Becker, D., Richter, J., Tocilescu, M,A., Przedborski, S., and Voos, W.

Notes: The authors of this paper studied the targeting mode of the Parkinson disease-associated kinase Pink1. They constructed a number of truncation and deletion mutants in the pGEM®-4Z Vector, and verified the identity of these clones using next-generation sequencing. The authors then expressed radiolabeled versions of the various Pink1 constructs using the TNT® Coupled Rabbit Reticulocyte Lysate System. These labeled proteins were used in mitochondrial localization studies.   (4563)

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J. Immunol. 188(4), 1896–1904. Plac8-dependent and inducible NO synthase-dependent mechanisms clear Chlamydia muridarum infections from the genital tract. 2012

Johnson, R.M., Kerr, M.S. and Slaven, J.E.

Notes: The authors previously showed that there are two independent mechanisms by which Chlamydia-specific CD4 T cells clear infection in epithelial cells; an iNOS-dependent mechanism and a Plac8-dependent mechanism. To further identify the Plac8 mechanism, they used microarrays to identify a second mechanism dependent on Plac8 for terminating Chlamydia replication in epithelial cells.

Several Chlamydia-specific CD4 T cell clones were purified at the end of their culture cycle and grown for 3 days in their usual culture media plus growth factors, without Ag stimulation. Total RNA was isolated from each clone using a protocol that included an RNase-free DNase I treatment step. Specific mRNA gene reverse transcription and amplification were performed using the AccessQuick™ RT-PCR System. (4324)

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Dev. Biol. 361(2), 392-402. Porcupine-mediated lipidation is required for Wnt recognition by Wls. 2012

Herr, P. and Basler. K.

Notes: These authors performed a comprehensive analysis of all Drosophila Wnt (DWnt) family members. During their study they used FuGENE® HD reagent to transfect various constructs into Kc or S2R+ cells. They also used various firefly and Renilla luciferase reporter constructs and the GloMax® Multi Detection system luminometer module to investigate and the ability of the DWnts to activate the canonical Wnt signaling pathway. (4198)

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Genome Res. 22, 125-133. Preparation of high-quality next-generation sequencing libraries from picogram quantities of target DNA 2012

Parkinson, N.J., Maslau, S., Ferneyhough, B., Zhang, G., Gregory, L, Buck, D., Ragoussis, J., Ponting, C.P. and Fischer, M.D.

Notes: The 1kb DNA Ladder and 1TE, 1X, Molecular Biology Grade were used during library preparation for next-generation sequencing. (4540)

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Human Pathology in press. Proliferative glomerulonephritis with monoclonal immunoglobulin G3κ deposits in association with parvovirus B19 infection 2012

Fujita, E., Shimizu, A., Kaneko, T., Masuda, Y., Ishihara, C., Mii, A., Higo, S., Kajimoto, Y., Kanzaki, G., Nagasaka, S., Iino, Y., Katayama, Y. and Fukuda, Y.

Notes: The authors used the ReliaPrep™ FFPE gDNA Miniprep System to isolate DNA from parafin-embedded kidney sections for use in real-time PCR to detect parvovirus B19. (4235)

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J. Biol. Chem. 287, 21599-21614. Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis. 2012

Culver, B.P., Savas, J.N., Park, S.K., Choi, J.H., Zheng, S., Zeitlin, S.O., Yates, J.R., and Tanese, N.

Notes: These authors analyzed and compared affinity-purified protein complexes from brain homogenates of wild type and huntingtin (Htt) mutant mice by mass spectrometry. Brain tissue from FLAG-tagged wild-type and Htt mice was homogenized in HEPES buffer supplemented with protease inhibitors and RNasin®. After affinity purification, protein complexes were digested using Sequencing-Grade Modified Trypsin and ProteaseMAX™ Trypsin Enhancer prior to mass-spectrometry analysis. (4227)

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