Notes: Heregulin is a growth factor that binds to the HER/ErbB family of receptors, which includes four HER members. HER1 and HER2 overexpression in breast cancer cells is associated with higher malignancy and poorer prognosis. However, HER4 expression is associated with longer survival and more positive prognosis. The authors of this study investigated HER4-dependent growth inhibition that is mediated by heregulin. The authors determined the absolute levels of HER4 mRNA in several human breast cancer and mouse cancer cell lines. For this determination, relative cell numbers were determined using a CellTiter® AQ_ueous MTS assay. Apoptosis in heregulin-treated or untreated cells was also determined using the DeadEnd™ Colorimetric TUNEL System. The authors concluded that heregulin decreases growth of HER4-positive breast cancer cells, and that this growth inhibition is dependent on BRCA1. (3596)

Notes: Because of concerns about the consumption of histamine in wine (which makes histamine more potent), the quantity of lactic acid bacteria (LAB), which can produce histamine during winemaking, was determined in 264 samples of red wine from 116 wineries. DNA was isolated from LAB strains grown in De Man Rogosa Sharpe (MRS) broth using the Wizard^® Genomic DNA Purification Kit. Glass beads were used to disrupt the cells, and a modified Wizard^® Genomic DNA Purification protocol that included PVP treatment was performed. The purified DNA was used in qPCR analysis. (3741)

Notes: The authors compared two DNA extraction methods: the International Commission on Missing Persons silica method and the standard phenol:chloroform method to determine the preferred method for extraction of DNA from skeletal remains. The efficacy of DNA extraction was measured by real-time PCR to quantify DNA and to check for the presence of PCR inhibitors, and by amplification with the PowerPlex^® 16 System. DNA was extracted from processed bone powder, and 10µl of the final extract was amplified using the PowerPlex^® 16 System and GeneAmp^® PCR System 9700 according to the manufacturer's recommendations, except that the extension time was doubled from 30 seconds to 60 seconds for the first 10 cycles and from 45 seconds to 90 seconds for the next 22 cycles. Amplified products were detected using the ABI PRISM^® 3100 Genetic Analyzer. The authors concluded that the silica-based method gave better results in autosomal STR typing than the organic extraction method. (3818)

Notes: Heterogeneous culture maturation over several days provided an opportunity to influence several different steps in the differentiation process from ES to mature, post-mitotic cells. The authors procured a mouse genomic committee rearrayed IRAV library of approximately 8,000 full-length mouse cDNAs in an expression vector under the CMV promoter. For an easy and efficient gene delivery method, they used liposome-based transfection and were able to reliably achieve greater than 80% transfection efficiency of monolayer cultures as detected by flow cytometry for CMV-eGFP expression. This high rate of transfection suggests that differentiating cells were accessible over the course of at least 4 days to exogenously expressed genes from plasmid DNA. DNA isolation was accomplished using PureYield™ Midiprep System (Cat. A2492). (3921)

Notes: These authors developed a strategy for screening large numbers of genes that influence the pluripotency and differentiation of embryonic stem cells to specific fates. A plasmid expression library was grown in deep-well plates, 32 clones were pooled and the plasmid isolated using the PureYield™ Plasmid Midiprep System. The purified plasmid DNA was used to transfect E14 ES cells and measure expression of a specific cell fate reporter construct. (3583)

Notes: The Fanconi anemia (FA) pathway is inactivated in a variety of human tumors. Identifying novel compounds that affect FA-pathway deficient cells could provide further information on the FA pathway as well as new therapeutic possibilities. In this study, RKO cells, a human cancer cell line stably expressing firefly luciferase under the control of the p53 consensus DNA-binding site, were treated with 10 and 20µmol/L 80136342, a new compound that enhances cancer cell survival, or 50µmol/L etoposide, a known activator of p53, for 24 hours. The reporter activity was assessed using the Steady-Glo^® Luciferase Assay System and compared to that seen in untreated controls. (3600)

Notes: The authors of this study describe a novel bioluminescent HTS screen using the BacTiter-Glo^™ Assay to identify inhibitors of biofilm formation. They compare the BacTiter-Glo^™ Assay to conventional crystal violet staining and show that it produces higher Z´-factor values, is less messy and provides results over a greater range of bacterial OD. They used the BacTiter-Glo^™ Assay to screen more than 60,000 compounds in 384-well plates, and identified 30 compounds representing six structural classes that inhibited biofilm formation. (3735)

Notes: In this study, the Renilla luciferase gene from the phRL-CMV Vector was incorporated into polioviral RNA in place of a structural protein-coding region. Renilla luciferase activity was then monitored as a convenient indicator of viral replication. Specifically, these authors tested whether the guanine nucleotide exchange factors GBF1 and BIG2 could rescue brefeldin A (BFA)-induced inhibition of polioviral replication in HeLa cells. Cells were transfected with plasmids encoding either GBF1 or BIG2, together with the firefly-luciferase-encoding pGL4.13 Vector, which was used as a control to monitor transfection efficiency. Eighteen hours post-transfection, the cells were transfected with polioviral replicon RNA containing the Renilla luciferase gene. One hour later, normal growth medium containing EnduRen^® Live Cell Substrate and either BFA or solvent alone, was added and Renilla luciferase activity was monitored at hourly intervals for 7 hours. Cells were then lysed and firefly luciferase activity was measured to assess transfection efficiency using the Dual-Glo^® Luciferase Assay System System. In the presence of BFA, GBF1 was able to partially rescue viral replication. In the presence of BIG2 and BFA, no viral replication occurred. (3562)

Notes: The authors examined whether genetic differences at the HLA class I locus affect development of Epstein Barr Virus-associated diseases. Peripheral blood mononuclear cells were isolated from asymptomatic EBV-seropositive and seronegative individuals and patients with acute infectious mononucleosis. DNA was isolated, and genotypes at two HLA class I loci and one HLA class III locus, as a control, were determined by PCR. The 10µl PCRs contained 25ng of DNA, 1X GoTaq^® Flexi Reaction Buffer, 2.5mM MgCl₂, 200µM dNTP, 0.5 units of GoTaq^® Flexi DNA Polymerase and 25µM of forward and reverse primer, one of which was labeled with 6-FAM fluorescent dye. The results show that HLA class I polymorphisms might predispose people to develop infectious mononucleosis upon EBV infection. (3712)

Notes: In this study, the Wizard® Genomic DNA Purification Kit was used to extract DNA from diluted fecal samples. The extracted DNA was used in PCR with specific primers to detect viral sequences. PCR fragments were gel-purified using the SV Gel and PCR Clean-Up System prior to sequencing to confirm the Bocavirus DNA identity. (4221)

Notes: The authors cloned the novel equine aldo-keto reductase AKR1C23 and characterized its expression patterns in the preovulatory follicle. The AKR1C23 cDNA was amplified from equine ovarian RNA using the Access RT-PCR System and primers designed by sequence alignments of known AKR sequences, then cloned into the pGEM^®-T Easy Vector. Levels of AKR1C23 and ribosomal protein L17a mRNAs in various equine tissues were quantified using the Access RT-PCR System and 21 cycles and 18 cycles, respectively, followed by agarose gel electrophoresis, transfer to nylon membranes, and hybridization to radiolabeled probes synthesized using the Prime-a-Gene^® Labeling System. (3791)

Notes: The authors used biochemical and mutational analysis to characterize human lysosomal DNaseIIα. Native DNaseIIα and site-directed mutants were expressed as Flag-His₆-DNaseIIα and HA-tagged DNaseIIα using expression constructs created with the pCI Vector. Protein was expressed by transiently transfecting HEK 293-T cells using the TransFast™ Transfection Reagent. Flag-His₆-DNaseIIα was purified using the MagneHis™ Ni-Particles, and this purified protein was used in nuclease assays to monitor catalytic activity and in gel filtration experiments and coimmunoprecipitation assays with HA-DNaseIIα to determine whether the active enzyme is monomeric. (3786)

Notes: The authors examined the interaction between various proteins involved in mRNA deadenylation and degradation using GST pull-down assays. Cytoplasmic poly(A)-binding protein (PABPC1) was expressed in E. coli as a GST fusion and immobilized using the MagneGST™ Glutathione Particles. This bait protein was incubated with various [³⁵S]Methionine-labeled prey proteins expressed in the TNT^® Coupled Reticulocyte Lysate System. Bait:prey complexes were washed, eluted with 1X SDS loading buffer, then analyzed by SDS-PAGE to determine which proteins interacted. (3782)

Notes: The authors characterized a juvenile hormone response element in Drosophila melanogaster (DmJHRE1) and identified two proteins that bound to a DmJHRE1 affinity column. Proteins eluted from the column were digested with Sequencing Grade Modified Trypsin, subjected to liquid chromatography-tandem mass spectrometry and identified as FKBP39 and Chd64. DmJHRE1 transcription regulatory activity was confirmed using reporter constructs with DmJHRE1 sequences regulating expression of firefly luciferase in Drosophila L57 and S2 cells. A vector with Renilla luciferase and the Autographa californica multicapsid nucleopolyhedrovirus IE1 promoter was used for normalization. Luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. Potential interactions between FKBP39, Chd64 and several candidates proteins for the JH receptor were examined using the MagneGST™ Pull-Down System. Each bait protein was expressed as a GST-fusion protein in E. coli and immobilized using MagneGST™ Glutathione Particles. [³⁵S]Methionine-labeled prey proteins were expressed using the TNT^® T7 Quick Coupled Transcription/Translation System. (3784)

Notes: The authors analyzed duplicate buccal swabs and observed eight disconcordant phenotypes with SGM Plus™ (2 at TH01, 4 at vWA and 2 at D18S51) and one disconcordant phenotype (at D18S51) with PowerPlex^® 16 out of a total of 1,377 individuals. In each case, the discrepancy was due to allele dropout of the second allele in a heterozygous genotype. DNA sequencing using primers that encompass the locus-specific STR primers was performed to characterize the mutations responsible for allele dropout for each locus. (3769)

Notes: The STAT proteins are involved in the transcriptional response to interferon (IFN), which includes induction of CXCL11 and ISG15 genes. The authors used quantitative real-time PCR to examine CXCL11 and ISG15 expression levels in IFN-sensitive and IFN-resistant cells after IFN treatment. Expression levels of the IFN-responsive genes were normalized to that of β-actin. Total RNA was isolated from untreated and IFN-treated cells, and qPCR was performed on a Bio-Rad iCycler^® instrument using the AccessQuick™ RT-PCR System and SYBR^® Green I. Reverse transcription was performed at 48°C for 45 minutes, followed by 35 cycles of PCR. Prior to qRT-PCR, PCR product size was confirmed by agarose gel electrophoresis, and PCR specificity was checked by analyzing melting curves. (3767)

Notes: The authors used H₂¹⁸O to increase the mass shift of recombinant antibody fragments upon asparagine deamidation and aspartic acid isomerization during a month-long incubation at 50°C, as detected by HPLC-mass spectrometry. The increase shift in mass makes it easier to detect these amino acid modifications. The levels of isoaspartate residues in the antibody fragments were quantified using the IsoQuant^® Isoaspartate Detection Kit and HPLC. (3973)

Notes: NVP-TAE684 was identified as an inhibitor of the constitutive anaplastic lymphoma kinase (ALK) activity associated with the NPM-ALK fusion. NPM-ALK fusion protein is created by translocation event characteristic of anaplastic large-cell lymophomas. NVP-TAE684 was screened against a panel of 35 Ba/F3 cell lines expressing a variety of tyrosine kinases constitutively activated by fusion to TEL. The CellTiter-Glo^® Luminescent Cell Viability Assay was used to detect any decrease in viability as a result of treatment with NVP-TAE684. The inhibitory effect of NVP-TAE684 was specific for ALK-associated cell proliferation. In a secondary screen to determine potency, TAE684 was screened against two human anaplastic large-cell lymphoma cell lines. Inhibition of proliferation correlated with dosage and was assessed using the Bright-Glo^® Luciferase Assay System. (3738)

Notes: The authors developed a high-throughput assay in a 1536-well format using the Kinase-Glo^® Luminescent Kinase Assay to test for small-molecule ceramide kinase (CERK) inhibitors. The assay was performed using a final compound concentration of 10µM, and an ATP concentration of 5µM in a total volume of 5µl. The assay was robust with Z´-factors of 0.54. (3590)

Notes: The authors of this study sought to identify inhibitors of myelin-reactive T-cell proliferation. They used spleen cells isolated from transgenic mice expressing T-cell receptors that specifically recognize myelin proteolipid protein residues 139-151 (PLP_139-151). Spleen cell suspensions were prepared from the transgenic mice and exposed to PLP in the presence of test compounds. Proliferation was assessed relative to positive and negative controls using the CellTiter-Glo® Luminescent Cell Viability Assay. Of the 41,184 compounds screened, 302 hits were obtained. These 302 compounds were next evaluated for nonspecific cytotoxicity using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay, and compounds causing nonspecific toxicity were eliminated from further consideration. Z´-factor values for the primary screen were robust (> 0.5). (3733)

Notes: These authors used the coelenterazine analog ViviRen^® as a substrate for Renilla luciferase in Bioluminescence Resonance Energy Transfer (BRET) experiments. Using this technique, they were able to demonstrate protein interactions in single mammalian cells. (3631)

Notes: The authors used RT-PCR to quantitate the level of rnpB expression after complementing the Bacillus subtilis rnpB mutant strain SSB318 with wildtype or mutant rnpB genes from S. aureus or B. subtilis. Preliminary experiments with decreasing amounts of RNA template were performed to show that rnpB RT-PCR product yields were linearly dependent on RNA template amounts after 12 PCR cycles. RT-PCR was performed using the Access RT-PCR System. (3794)

Notes: The brome mosaic virus (BMV) contains four RNAs (B1, B2, B3 and B4), each of which includes a tRNA-like structure (TLS) that is required for packaging in vitro. To confirm the necessity of the TLS for packaging in vivo, the authors created TLS-less B1, B2 and B3 RNAs; TLS-less B1 and B2 RNAs were packaged, while the TLS-less B3 RNA was not. Co-infiltration of Nicotiana benthamiana leaves with wildtype B1 and B2 and TLS-less B3 RNA resulted in restoration of the B3 TLS. To determine whether the resulting TLS was regained by homologous or heterologous recombination with B1 or B2 RNA, the 3´ end of the B3 RNA was amplified from leaf total RNA by RT-PCR using the AccessQuick™ RT-PCR System, then sequenced. (3768)

Notes: The authors of this paper describe the generation of induced pluripotent stem (iPS) cells from human dermal fibroblasts. STR analysis using the PowerPlex^® 16 System showed that patterns of 16 STRs in the clones matched the parent cell line. Luciferase assays to assess activity of the OCT3/4 and Rex1 promoters were performed using the Dual-Luciferase^® Assay System. (3951)

Notes: The authors studied the rat VP V1b receptor (V1bR) gene including the 826 base 5´ UTR, which has five ORFs upstream (uORF) of the V1bR protein start codon, to examine the effect of the upstream peptides on V1bR translation. The V1bR gene was cloned into the pALTER^®-MAX Vector, and substitution mutations were created in the uORF translation initiation codons with the Altered Sites^® II in vitro Mutagenesis System. One microgram of the linearized pALTER^®- V1bR construct was transcribed using the Riboprobe^® System and translated using Wheat Germ Extract with or without ³⁵S-methionine. The unlabeled protein was analyzed by Western blot. The radiolabeled protein was spun through a 3% sucrose cushion, precipitated and separated by SDS-PAGE. For a possible membrane-targeted protein, the uORF1 was transcribed in vitro and then translated using Rabbit Reticulocyte Lysate, Canine Pancreatic Microsomal Membanes and ³⁵S-methionine. The proteins were spun through a 3% sucrose cushion and then run on a 15% SDS-PAGE gel to determine membrane presence. (3749)