Notes: The authors were interested in examined the relationship between pregnane X receptor (PXR) and protein arginine methyltransferase 1 (PRMT1). Cells were transiently transfected for 60 hours including chemical treatment, and the Luciferase Assay System was used to analyze reporter activity. For the Checkmate™ Mammalian Two-Hybrid Assay, CV-1 cells were transfected with the pBIND Vector containing PXR, pACT Vector containing PRMT1 and pG5luc Vector. After 12 hours, the cells were treated with rifampicin and 48 hours later, luciferase activity was measured. Full-length PRMT1 protein was synthesized using the TNT^® SP6 Coupled Reticulocyte Lysate System and used in a GST pulldown assay. (4027)

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)

Notes: In this study, the authors wanted to examine the ability to trace the origin of tomato goods from fresh to processed. They tested several DNA extraction procedures for fresh tomato, tomato sauce, tomato puree, tomato pulp, whole peeled S. Marzano PDO (Protected Designation of Origin) tomato, whole peeled tomato, tomato concentrate and ‘‘Arrabbiata sauce”. Homogenized material (200mg) was extracted in three replicates using seven different methods including the Wizard^® DNA Clean-Up System. The DNA extracted was then analyzed by agarose gel electrophoresis, quantified and tested in PCR using SSR loci. The authors concluded that the Wizard^® DNA Clean-Up System was the most effective of the DNA extraction methods tested and yielded the greatest number of successful amplification reactions with lowest investment of personnel time and money. (4003)

Notes: This study examined the effects of folic acid supplementation on the offspring of pregnant rats fed a protein-restricted diet. Genomic DNA was extracted from rat adipose tissue using the Wizard^® SV Genomic DNA Purification System. The purified DNA was then incubated with methylation-sensitive restriction enzymes and then used in real-time PCR. (4066)

Notes: The lymphocytic choriomeningitis virus (LCMV) was used as a model to create a trisegmented recombinant arenavirus in which viral genes were replaced by a gene of interest. One such engineered virus, r3LCMV CAT/FLuc, was used in a pilot screen to identify anti-arenaviral compounds. Firefly luciferase (FLuc) activity was measured using the ONE-Glo™ Luciferase Assay System. (3957)

Notes: The authors were interested on confirming the identity of the exhumed skeletal remains thought to be Nicolaus Copernicus. DNA isolation from teeth was performed by pulverizing the tooth in liquid nitrogen, soaking the powdered sample in 0.5M EDTA, 5% SDS and 3 mg proteinase K at 37 °C and extracting genomic DNA using the Wizard^® Genomic DNA Purification Kit. Bone and other tooth samples were extracted using another method and the Wizard^® Genomic DNA Purification Kit used for a salting-out method. Purified DNA was subjected to mitochondrial, autosomal and Y chromosome STR amplification and analysis. (4063)

Notes: In 1991, the remains of murdered Emperor Nicholas II, Empress Alexandra and three of their five children were discovered. Until recently, the remains of the two other children were never found. In July of 2007 human bone fragments were discovered at a second grave site in the Ural region of Russia. The authors performed DNA typing to determine if these remains were those of the two missing children. Bone fragments and teeth were subjected to mitochondrial and nuclear DNA typing. DNA was quantitated using the Plexor® HY System. Nuclear DNA analysis was performed, in part, using the PowerPlex® S5 System. A comparison of mitochondrial DNA sequences from remains in the first and second graves and from maternal reference samples confirmed that the remains constituted a family with a "Queen Victoria" mitotype (Empress Alexandra was the granddaughter of Queen Victoria). Y-STR analysis of both sets of remains was performed, and the results confirmed that the Y-STR haplotypes of the two sets of male remains matched, and this haplotype matched that of several descendants from an unbroken paternal lineage of Nicholas I, father of Nicholas II. The mitochondrial and Y-STR haplotypes and autosomal STR profile also matched those obtained from a bloodstained shirt that Nicholas II was wearing during an assassination attempt. (3967)

Notes: The authors note that while many protein fusion tags are available to assist in purifying functional, recombinant proteins in soluble form and adequate amounts, none of the available tags are ideal when applied to the diversity of proteins studied. Available tags are often best-suited to specific aspects of the overall process, be it expression, solubilization, protein capture, etc. HaloTag was designed to address multiple features. The authors demonstrated that HaloTag provided enhanced expression and solubility in E. coli, with efficient protein purification and labeling for screening and quantitation. Enhanced solubility coupled with covalent capture gives HaloTag advantages over conventional affinity fusion tags, demonstrated by the successful purification of a wide variety of proteins, including low expressers, with higher yield and purity compared to the other tested tags. Covalent capture coupled with efficient tag removal to release the target protein, provides higher purity by eliminating tag contaminants. Using full-length cDNAs encoding 23 human proteins of varying size and function, the authors compared the efficacy of HaloTag as an expression and purification tag to the frequently used solubility enhancers MBP and GST. As reported previously, the three larger tags (HaloTag, MBP and GST) provided higher total and soluble expression compared to the smaller His6tag. In comparing soluble protein expression levels for the three larger tags, it was found that HaloTag solubilized 74% of the 23 human proteins examined, compared to 52% for MBP and 39% for GST. G2681 was one of the T7, bacterial promoter-based Flexi vectors used.
(4005)

Notes: These authors used proteolysis and mass spectrometry to identify the specific protein domains involved in copper-dependent phosphorylation. They expressed wildtype and mutant ATP7B in COS1 cells. The band of interest was excised from a gel, cut into small pieces and subjected to tryptic digestion in the presence of ProteaseMax Surfactant. (4086)

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)

Notes: The authors examined whether HMGB1 and HMGB2 proteins could affect promoter activity of the topoisomerase IIα gene. Portions of the topoisomerase IIα gene promoter were cloned into the pGL3 Basic Vector, and Saos-2 cells were cotransfected with the resulting constructs, an HMGB1- or HMGB2-expressing plasmid and the pRL-tk Vector as a control for normalization. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay. To determine whether HMGB1 and HMGB2 promoted binding of the transcription factor nuclear factor-Y (NF-Y) to the topoisomerase IIα promoter, the authors used a chromatin immunoprecipitation (ChIP) assay. Two populations of Saos-2 cells, one of which expressed HMGB1 or HMGB2 and one that had expression inhibited, were fixed with formaldehyde, then treated to shear chromatin. Immunoprecipitation was performed using an anti-NF-Y antibody, and the amount of DNA bound to the NF-Y was quantified by semi-quantitative PCR using GoTaq^® Hot Start DNA Polymerase and Green GoTaq^® Flexi Reaction Buffer. (4037)

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)

Notes: The authors explored the role of a DNA replication factor, flap endonuclease I (FEN1), in regulating telomerase activity in mammalian cells. PCR was used to add a myc tag to the N terminus of FEN1 cDNA. The amplimer was gel purified, digested with NheI and SmaI, and cloned into the same sites in the pCI-neo Mammalian Expression Vector. The insert was confirmed by sequencing. (4030)

Notes: The researchers examined if a CD20 mutation would affect resistance to rituximab, an adjunct cancer therapy drug used for CD20-positive B-cell lymphoma. CD20 PCR products amplified from genomic DNA were cloned into the pTARGET™ Mammalian Expression Vector. These CD20 mutant constructs were stably introduced into K562 chronic myelogenous leukemia cells by electroporation and selected using G-418. One microgram of CD20 mutant construct DNA was transcribed and translated using an in vitro translation kit from Promega. (4032)

Notes: In this study, small molecule enhancers of cAMP response element binding (CREB) were studied using quantitative high-throughput screening. After an initial screen of 73,000 compounds, 1,800 compounds were classified as potentiators of CREB activity. A second screening to confirm the compound potential was performed using the GloResponse™ CRE-luc2P HEK293 Cell Line. Five microliters of cells in assay medium were seeded in 1,536-well plates at a density of 2,500 cells/well. The next day, 23 nl of compound in DMSO or DMSO alone was dispensed into each well, then 1 μl of NKH477 (final concentration, 200 nM) or media alone was added to the assay plates. After incubating the cells for 4 hours at 37 °C, 6 μl of Bright-Glo™ Luciferase Assay Reagent was added to each well, incubated at room temperature for 10 minutes and the luminescence measured. (4004)

Notes: Here the authors studied various non-synonymous SNPs of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) that are essential for detecting viral RNA and triggering antiviral responses. Various point mutations were introduced into RIG-1 and MDA5 using the GeneEditor™ in vitro Site-Directed Mutagenesis System with pEF-FLAG clones. Mouse embryonic fibroblasts (MEFs) and L929 cells were cotransfected with RIG-I mutants or MDA5mutants and pRL-TK Vector, and stimulated with RNA or viral infection. Reporter activity was measured using the Dual-Luciferase^® Reporter Assay System. (4024)

Notes: The authors of this study describe a proof-of-concept screen to use mammary epithelial cells that have been induced to undergo an epithelial to mesenchymal transition (EMT) as model cells to identify agents that may be selectively toxic against "epithelial cancer stem cells" (CSCs). They induced the transformed breast cancer cell line HMLER to undergo a mesenchymal transition using shRNA directed against the E-cadherin gene. They characterized the responsiveness of these transitioned cells to common cytotoxic agents using the CellTiter® 96 AQueous Cytotoxicity Assay and compared the response to that of HMLER cells containing a control shRNA. They showed that the HMLER cells induced to undergo EMT behaved more like CSCs. The researchers then performed a proof-of-concept high-throughput screen to identify compounds that targeted the HMLER cells induced to undergo EMT, using the CellTiter-Glo® Assay to assess cell viability. (4006)

Notes: The authors review the use of in vitro cytotoxicity testing in drug discovery to characterize the toxic potential of new chemical entities (nce) at the earliest stages of profiling. DOI: 10.2174/1875397300903010033 (4000)

Notes: Traditionally light microscopy resolution has been limited by the diffraction of light. However several new technologies have emerged that partially overcome that limitation. One of these stimulated emission depletion (STED) microscopy is now commercially available and has been integrated into confocal microscope platforms. Because STED depends on fluorescent markers that fulfill specific spectroscopic needs, its uses have been limited. The authors of this paper demonstrate successful high resolution of β1-integrin-Halotag®-fusion protein distribution using STED microscopy. Use of the HaloTag® technology allows researchers to create a reporter that can be labeled with STED-optimized fluorescent tags. (3955)

Notes: In this study, the researchers wanted to determine the role of kelch-like ECH associated protein 1 knockdown (Keap1-kd) mice protein products, which are thought to protect against oxidative and electrophilic stress, and compare the hepatic phenotype with that of transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2)-null and wild-type mice. Microsomal suspensions from liver homogenates were prepared, and bile was collected from wild-type, Nrf2-null, and Keap1-kd mice. Reduced GSH was quantified using the GSH-Glo™ Glutathione Assay. (4012)

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

Notes: To better understand tumor progression in mice carrying the Min allele of Adenomatous polyposis coli (Apc), a longer lived cross was generated and studied. Intestinal tumors and adjacent normal tissue were microdissected, frozen in liquid nitrogen and genomic DNA isolated using the Magnesil^® Genomic, Fixed Tissue System. The purified DNA was then used for microsatellite instability (MSI) analysis. (4067)

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)

Notes: The authors describe a multiplexed in vitro assay to detect nephrotoxicity and gain information about mechanism of cell death in HK-2 (human kidney-2) cells. The multiplexed assay involved an LDH assay to detect necrosis, a caspase-3/7 assay to detect apoptosis, a reazurin assay to assess metabolic state, and a DNA dye staining assay to monitor nuclear morphology. (4002)

Notes: The authors examined the role of thymidine kinase (TK) in establishing a herpesvirus infection via the upper respiratory tract. DNA was purified from ex vivo organs of female BALB/c mice infected with a murid herpesvirus-4 (MuHV-4) TK knockout using the Wizard^® Genomic DNA Purification Kit. Real-time PCR was used with 50–80ng of purified DNA to determine viral load of the animals. (4015)