Notes: DNA was isolated from a variety of environmental samples including surface soil, drinking water biofilm, sludge from an anaerobic digester, bioreactor samples, ground water, peat soil and glacial deposit soil. The 16S rRNA gene was amplified from the DNA. PCR amplifications were run on agarose gels, and bands of the predicted sizes excised and purified using the Wizard® SV Gel and PCR Clean-Up System before pooling for pyrosequencing. (4554)

Notes: Sputum samples were collected from cystic fibrosis patients and 16S rRNA sequences amplified by PCR. These products were cloned into a T-vector, transformed into competent cells and the resulting colonies grown in 2ml LB broth in 96-deep-well plate for 20 hours. Of this culture, 1.9ml was pelleted and the clones isolated using the Wizard® SV Plasmid Purification System. The purified plasmid DNA was subjected to agarose gel electrophoresis and sequenced. (4133)

Notes: These authors investigated the ability of Phyllanthus plant extracts to affect the metastatic activity of human lung (A549) and breast (MCF-7) cancer cell lines. They initially used CellTiter 96® AQ_ueous Non-Radioactive Cell Proliferation Assay and the GloMax®-Multi Detection System absorbance module to determine cytotoxicity of various Phyllanthus extracts. After determining the effective dose, the authors investigated the ability of these plant compounds to inhibit/reduce metastatic activity. They then evaluated the mechanism of cell death in treated cells using the Caspase-Glo® 3/7 Assay and the Glomax® Multi Detection System luminescence module to measure caspase activity, and the CytoTox-ONE™ Homogeneous Membrane Integrity Assay to measure LDH activity. (4196)

Notes: The authors of this study sought to correlate promoter methylation with gene expression. Gene expression data was generated by RNA-seq of Jurkat cells. Amplified cDNA was prepared from total RNA, the cDNA was treated with S1 nuclease to remove single stranded nucleic acids and used as template for the sequencing libraries. (4536)

Notes: These authors used the Maxwell^® 16 Blood DNA Purification Kit to isolate genomic DNA from leukocytes. (4210)

Notes: Here the authors describe development of an HTS cell viability assay protocol for use with cultured cyropreserved human primary hepatocytes. Cryopreserved hepatocytes for culturing were prepared as suspensions and dispensed at 2,000 or 4,000 cells/5µl/well in collagen I-coated 1,536-well plates. Cells were allowed to attach and then 23nl of each test compound was added in a dilution series from 2.8nM to 92µM, and cells incubated for 24 or 40 hours. Five microliters of CellTiter-Glo^® Reagent was added and cells were incubated 30 minutes before reading the luminescent output. IC₅₀ values for 12 compounds were determined; a summary of the protocol is provided in Table 1 of the paper. Cultured cryopreserved hepatocytes were assayed for function using the P450-Glo^® CYP3A4 assay with the Luciferin-IPA substrate. (4182)

Notes: This proof-of-principle study investigated whether attenuated Salmonella typhimurium could be used as a vehicle for delivering shRNA-expressing plasmid DNA into cancer cells in mice. The authors delivered S. typhimurium bearing plasmids encoding anti-GFP shRNA orally to mice harboring tumors that expressed GFP. They were able to show that the bacteria accumulated and persisted for 40 days within the tumors, and that GFP expression in infected tumors was reduced. The AccessQuick™ RT-PCR System was used to analyze GFP expression levels in cultured cells and tumors. (4347)

Notes: The authors constructed red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) for ratiometric imaging of protein-protein interactions (PPIs) in cell culture and deep tissues of small animals. These BRET systems consist of Renilla reniformis luciferase (RLuc) variants, RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. They used the EnduRen™ Live Cell Substrate in their BRET systems to produce a red-shift emission maxima optimal for deep-tissue imaging. To demonstrate this capability, the authors imaged HT-1080 cells accumulating in the lungs of nude mice. The cells expressed BRET fusion proteins in the context of rapamycin-induced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. Mice were injected with luciferase substrate at 1.5 hours after cell injection to produce a bioluminescence image. Their data suggest that the BRET systems could be used for drug screening and target validation applications. (4130)

Notes: The authors describe the modification of the Promega MAO-Glo™ Assay to create a robust high-throughput screen for semicarbazide-sensitive amine oxidases (SSAOs). These proteins are implicated in inflammatory diseases and are believed to play a role in immune cell trafficking and cell activation. Their modified MAO-Glo™ Assay was tested with enzyme from recombinant and cell sources, and was able to identify specific inhibitors. (4155)

Notes: The authors amplified and sequenced the KRAS and BRAF genes in 803 patients with metastatic colorectal cancer to determine the frequency of mutation. They also examined the level of microsatellite instability (MSI) in BRAF-mutated cancer samples using the MSI Analysis System, Version 1.1. Of the 34 BRAF-mutated tumors, 8 had microsatellite instability. (4108)

Notes: Cell death was detected using TUNEL techniques in honey bee larvae that had been treated with different pesticides. (5158)

Notes: The authors determined the degree of polymorphism and population diversity of STR loci in 1,156 Russian individuals using the PowerPlex^® 16 System. They examined populations of six cities and 11 ethnic groups in the Russian Federation and reported the levels of intra- and interpopulation genetic differentiation and genetic relations between populations. There were significant differences between Russian populations and the U.S. reference database that was used in forensic medical examinations in Russia. The authors created a database of allele frequencies for 15 STR loci in Russian populations for DNA identification and forensic medical examination. (4150)

Notes: Briefly, BHK-21 cells were plated the day before transfection at a density of 5 × 10⁵ cells into a 100-mm cell culture dish. A total of 19 μg of each plasmid was mixed with Fugene HD reagent and incubated for 10 min at room temperature. Each mixture was added to confluent monolayers of BHK-21 cells and incubated at 37°C in a 5% CO₂ incubator. At 24 hours posttransfection, the cells were lysed. (4427)

Notes: This paper describes a rapid dual-luciferase-based assay for L1 retrotransposition that is amenable to high-throughput screening. A firefly luciferase vector in which the luciferase gene was disrupted by an antisense intron was constructed by introducing a 900-bp fragment of the human γ-globin intron into pGL4.13. This Fluc gene, interrupted by an antisense intron, gives only minimal luciferase expression unless the luciferase gene is restored by a retrotransposition event. The authors also tested a similar retrotransposition reporter using the pGL4.73 Renilla luciferase vector, but found that the firefly construct gave much higher signals. They therefore used the firefly luciferase retrotransposition reporter, a Renilla luciferase normalization control and the Dual-Luciferase^® Assay to characterize profiles of retrotransposition by various human and mouse L1 elements, and to measure the kinetics of L1 retrotransposition in cultured cells. The GloMax^® Multi Luminometer was used to quantify luciferase activity. (4205)

Notes: In this paper, FuGENE® 6 reagent was used to transfect PC3 human prostate cancer cells at a 4:1 FuGENE®:DNA ratio. (4370)

Notes: These authors compared the performance of four DNA purification methods for recovery of bacterial and archaeal DNA from fecal material. One of the methods tested was the Wizard® Genomic DNA Purification Kit, which uses a solution-based, enzymatic method for extraction. The Wizard® Genomic method was rated highly on extraction speed, and gave the highest DNA yields. A second method involving mechanical disruption (repeated bead beating) was rated more highly on extraction efficiency from archaea and some bacterial species. The criteria for performance comparison are described fully in the paper. (4219)

Notes: The authors developed a loop-mediated isothermal amplification (LAMP) assay to distinguish between virulent and nonvirulent strains of Vibrio vulnificus by targeting the virulence-correlated gene (vcg). The authors performed PCR using vcg-specific primers and GoTaq^® Hot Start Polymerase in parallel to confirm the LAMP results. (4163)

Notes: These authors used extensive computational analysis of isolates from the 2009 H1N1 outbreak to identify conserved H1N1 sequence signatures that could potentially be used in diagnostic assays to track the spread of specific strains during viral outbreaks. They identified target sequences in the hemagglutinin and neuraminidase genes that were highly conserved among 2009 H1N1 isolates. They then designed primers to amplify those sequences and used them in Taqman® and SYBR® Green-based qPCR assays to create a discriminatory 2009 H1N1 detection assay. They used the AccessQuick™ System in conventional RT-PCR to first establish whether their chosen primers were specific for the 2009 H1N1 isolates. In that assay they amplified representative H1N1 strains spanning from 1930 to the 2009 H1N1, and showed that only the 2009 isolates generated product. (4284)

Notes: The authors sought to determine the function of ceramide and ceramide synthase (CerS6) in mitochondria during post-natal brain development. They derived oligodendrocytes (OLs) from cultured, dissociated rat neonatal cortices. To determine if CerS6 plays in cell death, OLs were treated with glutamate to induce excitotoxicity, and cell death was assessed using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. To determine if the glutamate-induced excitotoxicity is dependent on ceramide or CerS6, OLs were treated with glumate in the presence or absence of the CerS6 inhibitor, fumonisin B1. To assess the mechanism of cell death resulting from ceramide-dependent glutamate toxicity in OLs, caspase activation during glutamate treatment was measured using the Apo-ONE^® Homogeneous Caspase-3/7 Assay. (4171)

Notes: Serum-starved ARPE cells were treated with the oxidant, t-butylhydroperoxide (tBH), in increasing concentrations for 30 minutes, 1, 2 and 24 hours, and caspase-3/7 activity was measured using the Apo-ONE^® Homogeneous Caspase 3/7 Assay to determine the mechanism of cell death. A dose-dependent increase in caspase-3/7 activity was observed. (4172)

Notes: The authors of this paper were investigating the role of mycobacterial membrane proteins in pathogenesis of Mycobacterium avium subsp. paratuberculosis, causative agent of Bovine Johne’s disease. Membrane enriched protein fractions, either mucosa-derived membranes (MDM) or culture-derived (CDM) of M. avium paratuberculosis from three cows with clinical disease were examined. Initial 2D DIGE and MALDI-TOF-MS analysis was unsatisfactory, so the researchers subjected the membrane preparations to tube-gel trypsin digestion supplemented with the ProteaseMax™ Surfactant, Trypsin Enhancer. They analyzed the proteins using nanoflow-liquid-chromatography-coupled tandem MS. A total of 130 proteins were detected in both MDM and CDM, with 48 predicted membrane proteins; four were not detected in the CDM, implying differential expression in the host. (4214)

Notes: This study analysed the roles of Dcp2 and Nudt16 in nonsense-mediated mRNA decay miRNA-mediated silencing. The authors used various luciferase reporter constructs to evaluate the significance of Dcp2 and Nudt16 in miRNA- and siRNA-mediated gene silencing in wildtype and knockout MEF cells. Various Renilla luciferase constructs containing or lacking miRNA target sites were cotransfected along with a control plasmid encoding firefly luciferase (for normalization purposes). Renilla and firefly luciferase luminescence were measured using the Dual-Luciferase^® Reporter Assay and the GloMax^®-Multi Luminometer. (4204)

Notes: This paper explored potential compounds as agonists of dopamine D2 receptor (D2R) with a bias toward β-arrestin signaling. Based on the aripiprazole scaffold, compounds were synthesized and tested in a D2-mediated Gi-coupled isoproterenol-stimulated cAMP production assay using HEK293T cells expressing D2R transfected with pGloSensor™-22F cAMP Plasmid. Assessing β-arrestin recruitment to agonist-stimulated receptors was determined using HTLA cells stably expressing β-arrestin-TEV protease and a tetracycline transactivator-driven luciferase exposed to agonist or D2 test ligand with or without reference agonist. After 18 hours, medium was removed from the cells, 1X Bright-Glo™ Reagent added and luminescence measured. (4518)

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 bla_OXA-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 bla_OXA-58 gene. (4344)

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 G_s-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 (E_max) after isoproterenol pretreatment. (4138)