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J. Antimicrob. Chemother. 67, 59-63. Disruption of the blaOXA-51-like gene by ISAba16 and activation of the blaOXA-58 gene leading to carbapenem resistance in Acinetobacter baumannii Ab244.

Lopes. B.S., Evans, B.A., and Amyes, S.G.B.

Notes: This study investigated the genetic basis of carbapenem resistance in the multidrug resistant Acinetobacter baumannii isolate, Ab244. Transposable elements are known to play an important role in multidrug resistance in A. baumannii. The authors used a multiplex PCR approach to detect the presence of known resistance genes and insertion elements, followed by RT-PCR to study expression of the genes identified. For RT-PCR, cDNA was synthesized from 100 ng of RNA using the AccessQuick™ RT-PCR System. Results of the study indicated that the blaOXA-132 gene was inactivated in A. Baumannii AB244 by insertion of ISAba16, and that carbapenem resistance in that isolate was due to an alternate resistance mechanism caused by overexpression of the blaOXA-58 gene. (4344)

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Sci. Signal. 4, ra51. Distinct Phosphorylation Sites on the β2-Adrenergic Receptor Establish a Barcode That Encodes Differential Functions of β-Arrestin 2011

Nobles, K.N., Xiao, K., Ahn, S., Shukla, A.K., Lam, C.M., Rajagopal, S., Strachan, R.T., Huang, T.Y., Bressler, E.A., Hara, M.R., Shenoy, S.K., Gygi, S.P. and Lefkowitz, R.J.

Notes: The authors created a stably transfected HEK293 cell line expressing a luminogenic cAMP-binding protein using GloSensor™ technology to quantify cAMP levels in live cells. The HEK293 cells express a beta2-adrenergic receptor (β2AR ), a Gs-coupled receptor, that when activated with an agonist, stimulates the production of cAMP. The cell line was used to demonstrate that the phosphorylation pattern (“barcode”) of the β2AR created by various G protein-coupled receptor kinases (GPKs) affects the binding and function of beta-arrestin and subsequent internalization of β2AR. GloSensor™ transfection and transcription were confirmed by stimulation of endogenous β2AR with isoproterenol. Endogenous β2ARs in HEK293 cells were prestimulated with either vehicle DMSO or isoproterenol, then washed and rechallenged with serially diluted isoproterenol. In cells transfected with control siRNA, pretreatment with isoproterenol induced a 50% loss of the maximal cAMP signal when rechallenged. Cells transfected with siRNA targeting GRK2, GRK6, or both GRK2 and GRK6 showed impairment of this desensitization. This GRK knockdown effect decreased the change observed in the maximal cAMP response (Emax) after isoproterenol pretreatment. (4138)

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mBio. 2(6), e00275-11. Epsilon-toxin production by Clostridium perfringens type D strain CN3718 is dependent upon the agr operon but not the VirS/VirR two-component regulatory system. 2011

Chen, J., Rood, J.I., and McClane, B.A.

Notes: These authors investigated whether ETX toxin production in C. perfringens type D is regulated by the Agr-like quorum sensing system, the VirS/VirR system, or both. They demonstrated that an agr mutant lacked ETX expression, and showed that lack of VirR had no effect on ETX production. The AccessQuick™ System was used in RT-PCR analysis of RNA isolated from mutant, wildtype and reconstituted (complemented) strains to confirm absence of agr transcripts in mutant strains. RT-PCR was also used to confirm the presence of etx transcripts in wild type strains, and their absence in the agr mutant. Previous studies had shown that toxin production is upregulated upon contact with enterocyte-like Caco-2 cells. This study showed that that the agr operon is required for such upregulation. (4289)

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Appl. Environ. Microbiol. 77, 2943–53. Evaluation of procedures for the collection, processing, and analysis of biomolecules from low-biomass surfaces. 2011

Kwan K., Cooper M., La Duc M.T., Vaishampayan P., Stam C., Benardini J.N., Scalzi G., Moissl-Eichinger C. and Venkateswaran K.

Notes: These authors used the Maxwell® 16 System to extract DNA from multiple sample collection devices containing a model microbial community (MMC) comprised of 11 distinct species of bacterial, archaeal and fungal lineages associated with spacecraft or clean-room surfaces. The authors compared cotton swabs, polyester wipes and biological sampling kits to assess the success of recovering DNA of rRNA genes for species-specific PCR analysis. (4124)

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Virol. J. 8, 387. First report of multiple lineages of dengue viruses type 1 in Rio de Janeiro, Brazil. 2011

dos Santos, F.B., Nogueira, F.B., Castro, M.G., Nunes, P.C., de Filippis, A.M., Fariam, N.R., Simões, J.B., Sampaio, S.A., Santos, C.R. and Nogueira, R.M.

Notes: The authors examined the strains of Dengue virus serotype 1 (DENV-1) found in the State of Rio de Janeiro since it was introduced in 1986. Viral RNA was extracted from patient serum samples or cell culture and 5µl of RNA reverse transcribed and amplified using the AccessQuick™ RT-PCR System with primers for the 2,325bp C/prM/M/E region of DENV-1. The products were sequenced and aligned to examine how the virus had evolved over time. (4342)

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Arch. Med. Sci. 7, 501-507. Frequency of Firmicutes and Bacteroidetes in gut microbiota in obese and normal weight Egyptian children and adults. 2011

Ismail, N.A., Ragab, S.H., Elbaky, A.A, Shoeib, A.R., Alhosary, Y., and Fekry, D.

Notes: These authors investigated the differences in gut microbial flora between obese and normal-weight subjects. They used the Wizard® Genomic DNA Purification Kit to extract DNA from diluted fecal extracts. The extracted DNA was analyzed by PCR to identify Bacteroidetes and Firmicutes. Differences in distribution of these phyla was between the subject groups were identified. (4220)

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Appl. Environ. Microbiol. 77, 2113–21. General suppression of Escherichia coli O157:H7 in sand-based dairy livestock bedding. 2011

Westphal, A., Williams, M.L., Baysal-Gurel, F., LeJeune, J.T. and McSpadden Gardener, B.B.

Notes: The authors investigated the suppression of E. coli O157:H7 in sand-based livestock bedding and hypothesized that suppression of E. coli O157:H7 growth was mediated by an environmentally stable population of pathogen-suppressing bacteria. These bacteria were identified by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA gene sequences isolated from used bedding followed by cloning and sequencing of the most abundant terminal restriction fragments. Amplifications were performed using the GoTaq® Flexi DNA Polymerase, then PCR products were cloned into the pGEM®-T Easy Vector. The PureYield™ Plasmid Miniprep System was used to purify plasmids for sequencing. (4165)

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Cytokine 55, 79-89. GM-CSF plays a key role in zymosan-stimulated human dendritic cells for activation of Th1 and Th17 cells. 2011

Wei, W.C., Su, Y.H., Chen, S.S., Sheu, J.H., and Yang, N.S.

Notes: This study compared the effects of zymosan and LPS on human monocyte-derived dendritic cells (DCs). The  authors found that zymosan-activated DCs had a unique cytokine expression profile. In zymosan-activated DCs, high levels of GM-CSF and IL-27, rather than IL-12 p70, were involved in Th1 cell activation. As part of the study, RT-PCR was used to investigate the molecular basis of this failure to induce production of active IL-12 p70. Expression levels of the biologically inactive subunits of IL-12 p70 (p40 and p35) was assessed using the AccessQuick™ RT-PCR System. The results showed that zymosan induced expression of p40, but not p35 mRNA, indicating that lack of induction of p35 was the reason for failure to induce active IL-12 p70. (4348)

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Proc. Natl. Acad. Sci. USA 108, 17159–64. Identification of the bacterial protein FtsX as a unique target of chemokine-mediated antimicrobial activity against Bacillus anthracis. 2011

Crawford, M.A., Lowe, D.E., Fisher, D.J., Stibitz, S., Plaut, R.D., Beaber, J.W., Zemansky, J., Mehrad, B., Glomski, I.J., Strieter, R.M. and Hughes, M.A.

Notes: The authors identified three genetic loci involved in chemokine-mediated antimicrobial effects against Bacillus anthracis using a transposon mutant library in which a transposon is randomly inserted into the B. anthracis genome, then treating the mutant cells with chemokine to select for resistant cells. To identify the transposon insertion site, and thus the resistance-conferring loci, the authors amplified regions flanking the transposon by PCR using the GoTaq® Green Master Mix. (4167)

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PLos ONE 6(7), E22438. Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes. 2011

Racca A.C., Camolotto S.A., Ridano M.E., Bocco J.L., Genti-Raimondi S., and Panzetta-Dutari, G.M.

Notes: These authors studied KLF6 expression during human trophoblast cell differentiation, and its role in the regulation of genes associated with placental development and pregnancy maintenance. They used immunofluorescence microscopy, RT-qPCR and luciferase reporter assays to investigate cellular localization, mRNA expression, and transcriptional activation. Reporter assays were performed using various luciferase reporter constructs, the Dual-Luciferase® Assay, and the GloMax®-Multi Detection System. KLF6 was shown to play a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG. (4197)

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J. Gen. Mol. Virol. 3, 18–26. Molecular epidemiology of human enterovirus71 (HEV71) strains isolated in Peninsular Malaysia and Sabah from year 2001 to 2009 2011

Yusof, M.A., Rais, F., Abdullah, M.A., Zamri, L.A., Ali, H.M., Kassim, F.M. and Saat, Z.

Notes: This study characterized the strains of hand, foot and mouth disease (HFMD) that circulated in Peninsular Malaysia and Sabah during the last ten years. Viral RNA was extracted from vesicle, throat and rectal swabs, and 5µl of purified RNA was amplified in a 50µl reaction using the AccessQuick™ RT-PCT System with primers for VP4 or VP1. The amplified products were then sequenced and compared to known HFMD sequences. (4341)

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Proc. Natl. Acad. Sci. USA 108, 745-750. Nanoparticle-mediated delivery of siRNA targeting Parp1 extends survival of mice bearing tumors derived from Brca1-deficient ovarian cancer cells.

Goldberg, M.S., Xing, D., Ren, Y., Orsulic, S., Bhatia, S.N., and Sharp, P.A.

Notes: These authors evaluated the effect of knockdown of Parp1 on survival of tumor cells after in vivo delivery of anti-Parp siRNA in a lipid-based nanoparticle format. Inhibition of Parp1 expression in mouse tumors was confirmed by qPCR analysis. Total RNA was extracted from the tumors using TRIzol® reagent (Life Technologies) and reverse transcribed using the ImProm-II™ Reverse Transcription System prior to qPCR using SYBR Green PCR Master Mix (Applied Biosystems) . (4222)

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J. Clin. Microbiol. 49, 1628–30. Novel nested direct PCR technique for malaria diagnosis using filter paper samples. 2011

Fuehrer, H.P., Fally, M.A., Habler, V.E., Starzengruber, P., Swoboda, P. and Noedl, H.

Notes: The authors developed a direct-amplification, nested PCR protocol to amplify Plasmodium DNA from 903 filter paper punches containing whole blood. The second round of nested PCR was performed using 2.5µl of PCR products from the first round and GoTaq® PCR Core System. Parallel PCRs were performed using purified DNA from blood, and both rounds of nested PCR were performed using the GoTaq® PCR Core System. (4166)

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J. Cell Sci. 124, 745–54. PERP regulates enamel formation via effects on cell-cell adhesion and gene expression. 2011

Jheon, A.H., Mostowfi, P., Snead, M.L., Ihrie, R.A., Sone, E., Pramparo, T., Attardi, L.D. and Klein, O.D.

Notes: The authors determined that PERP, a tetraspan membrane protein, is required for enamel formation during tooth development in mice. Using microarray analysis, they then identified genes that were differentially expressed in wildtype and Perp-null mice and might be involved in enamel formation. Differential expression of these genes was confirmed by qPCR using the GoTaq® qPCR Master Mix. The authors also identified an upstream regulator of Perp, P63, by transfecting cells derived from embryonic mouse teeth with a Perp-luciferase reporter construct that contained a P63 response element or mutated P63 response element. A Renilla luciferase vector was used for normalization of transfection efficiency, and luciferase assays were performed using the Dual-Luciferase® Reporter Assay System. (4168)

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Nucl. Acids Res. Dec 8, Epub ahead of print. Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts. 2011

Zhelyazkhova, P., Hammani, K., Rojas, M., Voelker, R., Vargas-Suárez, M., Börner, T., and Barkan, A.

Notes: Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind specific RNA segments and protect the adjacent RNA by serving as a barrier to exoribonucleases. This study showed that protection by PPR or PPR-like proteins is the predominant mechanism for defining the positions of processed 5′ and intercistronic mRNA termini in land plant chloroplasts. The authors used RNasin® Ribonuclease Inhibitor in binding reactions between labeled RNA and PPR proteins prior to Gel mobility shift assays. They also used the pGEM®-T Vector to clone various 3´ RNA terminal sequences amplified by RT-PCR. (4185)

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Nucl. Acids Res. [Epub ahead of print]. RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage. 2011

Dai, W., Zhang, G. and Makeyev, E.V.

Notes: The full-length 3´ UTR of mouse RNA-binding protein HuR was amplified and cloned into psiCHECK™-1 Vector to create pEM429 plasmid. To generate radiolabeled RNA substrates for use in cleavage assays, RNA was synthesized from 1µg of linearized plasmid using 50µCi of [α-32P]UTP, 0.8mM Ribo m7G Cap Analog and T7 RNA Polymerase by incubating for 1.5 hours at 37°C. The RNAs were then treated with 1 unit of RQ1 RNase-Free DNase per 1µg of template DNA for 15 minutes at 37°C before extracting with phenol:chloroform, precipitating with ethanol and resuspending in DEPC-treated water. The cleavage assay used 60fmol of 32P-labeled substrate RNA with 10U Recombinant RNAsin Ribonuclease Inhibitor in a reaction incubated for 2.5 hours at 30°C. RNA probes were labeled with biotin using T7 or T3 RNA Polymerases with a biotin-UTP labeling NTP mixture and incubated for 2 hours at 37°C. The biotinylation reaction was then treated with RQ1 RNase-Free DNase following the same protocol used for radiolabeled RNA. To form HuR/RNA complexes, 2µg of biotinylated RNA was mixed with 100µg nuclear extract and 40 units Recombinant RNAsin® Ribonuclease Inhibitor in a total volume of 20µl, and incubated for 30 minutes at room temperature. For CstF-64/RNA complexes, 1µg of biotinylated RNA was used and the complexes were stabilized by UV crosslinking using 10U Recombinant RNAsin Ribonuclease Inhibitor during the UV treatment. NIH 3T3 cells were UV crosslinked and either total or nuclear RNA used for immunoprecipitation. After extraction the RNAs were treated with RQ1 RNase-Free DNase for 15 minutes at 37°C before RT-PCR using HuR-specific primers. Total RNA purified from cultured cells were incubated with 50U/ml RQ1 RNase-Free DNase at 37°C for 30 minutes before use in RT-qPCR. (4187)

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J. Exp. Bot. 62, 5217–31. The barley amo1 locus is tightly linked to the starch synthase IIIa gene and negatively regulates expression of granule-bound starch synthetic genes. 2011

Li, Z., Li, D., Du, X., Wang, H., Larroque, O., Jenkins, C.L., Jobling, S.A. and Morell, M.K.

Notes: The authors investigated starch synthesis in barley (Hordeum vulgare) by examining mutations in class I, class II and class III starch synthases (ssI, ssII and ssIII, respectively). Mutations of ssIIa and ssIIIa were detected by PCR using the GoTaq® Hot Start Polymerase. (4162)

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Biochem. J. 436, 387–397. The novel Nrf2-interacting factor KAP1 regulates susceptibility to oxidative stress by promoting the Nrf2-mediated cytoprotective response. 2011

Maruyama, A., Nishikawa, K., Kawatani, Y., Mimura, J., Hosoya, T., Harada, N., Yamamato, M. and Itoh, K.

Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag® CMV Flexi® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase® Reporter Assay System. (4123)

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Med. Vet. Entomol. 25, 58–63. The potential of house flies to act as a vector of avian influenza subtype H5N1 under experimental conditions. 2011

Wanaratana, S., Panyim, S. and Pakpinyo, S.

Notes: To investigate if house flies could act as a vector for avian influenza virus H1N5, flies exposed to the virus were homogenated and the homogenate used to inoculate 10-day-old embryonated chicken eggs (ECEs). Allantoic fluids were collected from the eggs, RNA purified from the samples and the presence of H1N5 assessed using M-specific primers and the AccessQuick™ RT-PCR System and looking for the 276bp amplimer. (4339)

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J. Biol. Chem. 286, 19478–19488. Thrombomodulin is silenced in malignant mesothelioma by a poly(ADP-ribose) polymerase-1-mediated epigenetic mechanism. 2011

Nocchi, L., Tomasetti, M., Amati, M., Neuzil, J., Santarelli, L. and Saccucci, F.

Notes: Thrombomodulin (TM) expression was examined by isolating genomic DNA from biopsies of human malignant mesothelioma and normal mesothelial tissue, and cultured cell lines with or without PARP1 silencing treated with 5-aza-2´-deoxycytidine and trichostatin alone or in combination and then subjected to biosulfide modification. To analyze methylation of TM, a CpG island in the promoter, 5´ UTR and an exon region containing 44 CpG dinucleotides were PCR amplified, cloned into the pGEM®-T Easy Vector, transformed and positive clones selected using IPTG/X-Gal and analyzed by PCR. Colonies were cultured, the plasmids isolated using the Wizard® Plus SV Minipreps DNA Purification System then 10 clones
from each sample type were sequenced. (4132)

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Genome Res. 20, 1590-604.
Next-generation sequencing identifies the natural killer cell microRNA transcriptome

T. A. Fehniger, T. Wylie, E. Germino, et al.

Notes: RNasin was used in the small RNA library preparation step before sequencing on an Illumina platform.


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Nucl. Acids Res. 38, 522–33. An integrated pipeline for next-generation sequencing and annotation of mitochondrial genomes 2010

Jex, A.R., Hall, R.S., Littlewood, D.T. and Gasser, R.B.

Notes: Wizard® SV Gel and PCR Clean-Up System was used to clean up genomic DNA isolated from parasitic nematodes isolated from a variety of animals. Species identification of each nematode specimen was determined via PCR amplification of specific nuclear DNA followed by purification of the amplified product using Wizard® PCR Preps DNA Purification System before sequencing using BigDye chemistry. Wizard® SV Gel and PCR Clean-Up System was also used to prepare amplicons generated by long-PCR of mt genomes from the nematodes before NGS sequencing. Results from NGS were confirmed using PCR-based sequencing of short mt DNA tracts. Short mtDNA regions were amplified by conventional PCR. Amplicons were purified using Wizard® PCR Preps DNA Purification System before sequencing using BigDye chemistry. (4533)

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Appl. Environ. Microbiol. 76, 3863–8. Comparison of normalization methods for construction or large multiplex amplicon pools for next-generation sequencing 2010

Harris, J.K., Sahl, J.W., Castoe, T.A., Wagner, B.D., Pollack, D.D. and Spear, J.R.

Notes: GoTaq® Master Mix was used for PCR of bacterial ribosomal RNA genes prior to pyrosequencing. (4529)

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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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J. Am. Soc. Hort. Sci. 135, 291–302. Transcriptional profiling of rapidly growing cucumber fruit by 454-pyrosequencing analysis 2010

Ando, K. and Grumet, R.

Notes: The Wizard® SV Gel and PCR Clean-Up System was used to purify PCR products prior to pyrosequencing. (4546)

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