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)

Notes: In this paper, the researchers describe and demonstrate a new method for creating precise genome modifications in Saccharomyces cerevisiae. The mutagenic inverted repeat assisted genome engineering (MIRAGE) was tested in S. cerevisiae W303a by deleting gal7 as well as point and frameshift mutations. Genomic DNA was isolated using the Wizard^® Genomic DNA Purification Kit, amplified and modifications verified by gel analysis or DNA sequencing. (4014)

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)

Notes: Human boule (BOLL) gene was subcloned into the HaloTag^® pHT2 Vector and transformed into JM109 and HeLa cells. (4060)

Notes: To examine the prevalence of Campylobacter species in asymptomatic 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)

Notes: In this paper, the role of prostaglandin E2 (PGE2) stimulation of integrin-linked kinase (ILK) in human lung carcinoma was explored. Mutations of Sp1 and NF-κB cis-acting elements in an ILK promoter-pGL3-Basic Vector construct were created using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The mutations were confirmed via sequencing. Human non–small cell lung carcinoma (NSCLC) cells were plated at a density of 5 × 10⁵ cells per well in six-well plates and transfected with 2µg of ILK promoter reporter vectors with or without 0.2µg of the phRL-TK Renilla Luciferase Reporter Vector. After 24 hours, the transfected cells were exposed to PGE2 and the cells lysed for assessment using the Dual-Luciferase^® Reporter Assay System. NSCLC cells were transfected with inactive (ILK-S343A) and superactive ILK (ILK-S343D) cDNA, incubated for 24 hours, treated with or without exogenous PGE2 or with an Sp1 inhibitor for 2 hours. The numbers of viable cells were measured using the CellTiter-Glo^® Luminescent Cell Viability Assay. (4026)

Notes: These authors compared 6 different detection strategies for protein-protein interactions on protein arrays. They expressed HaloTag^® labeled bait proteins in a cell-free expression system, and captured these bait proteins onto coated glass slides using the HaloLink™ Array System. They then compared detection strategies using prey proteins labeled as follows: 1)³⁵S methionine, 2) fluorescence (BODIPY-FL) and 3) biotin labeling of lysine residues using modified Lys tRNA, 4) chemical labeling after expression, 5) HaloTag^® fusion, and 6) N-terminal FLAG tag. The authors evaluated signal:background ratios, adaptability to high-throughput screening, and ease of use. (3999)

Notes: The authors used the Halotag^® Labeling Technology in pulse-chase experiments. They pulse labeled proteins in cultured mammalian cells. Using the HaloTag^® Technology, they were able to monitor the degradation of the labeled protein, Smad1, that was induced by coexpression of Smurf1. They conclude that the HaloTag^® Technology could be used to monitor the regulation of SMAD1 degradation. (4055)

Notes: In this paper, the role of the KAE1/osgep/ygjD gene family, a universally conserved gene set without an assigned function, was investigated in yeast and Caenorhabditis elegans. Genomic DNA was isolated from Saccharomyces cerevisiae using the Wizard^® SV Genomic DNA Purification System. This purified DNA was then used in PCR. (4065)

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)

Notes: The association of thiopurines (anticancer drugs) with acute skin sensitivity to ultraviolet A (UVA) radiation and a high risk of skin cancer was tested using six human fibroblast and lymphoid cell lines. The thiopurine 6-thioguanine (6-TG) was added at 0.8 or 0.6mM to each of six cell lines and incubated for 48 hours to ensure incorporation. DNA and RNA were extracted and 40µg of nucleic acid were digested to nucleosides, separated by HPLC, and the 6-TG 20-deoxy and ribonucleosides quantified by absorbance at 342 nm. The same DNA isolation and digestion method was used when the cells were treated with 1µM of 6-TG and then irradiated with 5 kJ/m² UVA after 48 hours. The Wizard^® Genomic DNA Purification Kit was used for DNA extraction. (4013)

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 AQ_ueous 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)

Notes: These authors expressed recombinant dynactin and dynein in Saccharomyces cerevisiae and investigated their interactions in motility assays. They created a c-terminal Halotag-Dynactin fusion, and were able to site-specifically label the fusion protein with the fluorescent dye tetramethylrhodamine (TMR). They studied the effect of the purified dynactin fusion protein on the motility of dynein complexes using total internal reflection fluorescence microscopy. Dynactin alone did not interact with microtubules. However, when coincubated with recombinant dynein, the TMR-labeled dynactin moved processively along microtubules. The authors then used truncation mutants of dynactin to identify the region of the dynactin molecule required for localization and enhanced processivity of dynein. (3960)

Notes: Mutations in the extracellular matrix glycoprotein cartilage oligomeric matrix protein (COMP) cause skeletal dysplasia. This study evaluated the efficacy of a ribozyme (a short catalytic RNA oligonucleotide) to knock down COMP expression. As part of the study, COS7 cells were transfected with plasmids that expressed mutant or wild-type COMP, as well as with a ribozyme targeting COMP. Transfection conditions were as follows: Cells were grown to 80%–90% confluency in 6-well plates, then transfected with plasmid DNA using a 3:2 ratio of FuGENE 6 reagent:DNA. Ribozyme treatment reduced expression of COMP mRNA in the transfected cells. (4357)

Notes: To try to understand the mechanism by which certain strains of Shiga-toxigenic E.coli adhere to host intestinal epithelium, the authors characterized the novel autotransporter protein Sab. Expression levels and protein localization were examined by Western blot analysis and enzyme-linked immunosorbent assay. The mouse anti-Sab antibody used in these studies was raised against an N-terminal His₆-Sab fusion protein purified using the HisLink™ Resin. (4102)

Notes: In this study, FuGENE® 6 reagent was used to transiently transfect Jurkat T lymphocyte cells and HEK 293 adenovirus-transformed  HEK 293 cells with 0.25µg DNA at a 6:1 ratio of FuGENE:DNA. The Jurkat cells were plated at 1 × 10e6 cells/plate and the HEK cells were plated at 5 × 10e5 cells/plate. (4375)