Notes: The ability to detect trace protease activity is important for assessing protein purification methods during process development and for confirming the absence of protease in final purified proteins. The authors describe a general protease assay using five peptide-conjugated luciferase substrates and compare it to fluorophore-conjugated general protease assays. (4156)

Notes: This paper describes the evaluation of the HDAC-Glo™ I/II and SIRT-Glo™ Assays. For all enzymes assayed (HDAC-I, HDAC-3, HDAC-6 and SIRT-1), the K_m values obtained using the luminogenic assays were comparable to those reported in the literature. Furthermore, the assays tolerated well the concentrations of DMSO used to deliver known HDAC inhibitors. The authors used the assays to generate dose-response curves and IC₅₀ values for standard inhibitors of several HDAC isoforms. They indicate that all data sets were of high quality and that the IC₅₀ values obtained were comparable to those reported in the literature. The authors screened four HDAC isoforms (HDAC-1, HDAC-3, HDAC-6 and SIRT-1) in duplicate against 640 FDA-approved drugs, and they screened HDAC-6 and SIRT-1 against the Hypha Discovery MycoDiverse natural products library. For both screens, “hits” were classified as compounds that showed >50% inhibition at their screening concentration. For the 640-drug screen, the confirmation of hits was >80%, and the false-positive hit rate was <20%. The Z´-factor values were 0.65 for the HDAC-6 screen and >0.85 for the SIRT-1 in the Hypha Discovery MycoDiverse natural products library screen. A Z´ factor of 0.5 or greater is indicative of an assay with low data variability but good dynamic range. The authors conclude that the HDAC-Glo™ I/II and SIRT-Glo™ Assays met the criteria of sensitivity, signal stability, low background, DMSO tolerance, data variability and dynamic range, and scalability required for a suitable drug-screening assay. (4137)

Notes: The authors of this paper investigated mutations affecting the hinge loop of protein kinases that appear to confer resistance to both Type I and Type II inhibitors. They introduced individual amino acid substitutions into the hinge region of six distantly related protein kinases and determined the inhibitor sensitivity of these kinases. The ADP-Glo™ Kinase Assay was used to asses the activity of the Haspin and c-Src kinases and the engineered mutants in this study. (4144)

Notes: The authors of this study evaluated the ADP-Glo™ Assay technology for use in high-throughput screening applications for inhibitors of all four known mammalian PI 4-kinases. They found that K_m values, IC₅₀ values of known inhibitors, and dose-response curves were comparable to values reported in the literature or those obtained using the standard isotopic assay. Z´-factor values for the assay in a low-volume, 384-well format were 0.72 and 0.74, indicating that the assay would be suitable for screening activities in 384- or 1536-well formats. (4129)

Notes: This paper describes the use of the ADP-Glo™ Assay technology to screen for small-molecule inhibitors of PI 4-kinases (phatidylinositol 4-kinases). The authors characterized E. coli-expressed proteins using the ADP-Glo™ Kinase Assay and saw strong signal-to-background ratios, no signal from their negative controls or the D1899A protein, and no signal in the absence of micelles. Next they compared data from the ADP-Glo™ Assay with results obtained from the standard isotopic method or results available in the literature. They were able to obtain K_m values for PI4KA and PI4KB that corresponded to published values. Finally, the authors evaluated the potential of the ADP-Glo™ Assay as a high-throughput screening tool. To assess the assay, they used Z´ factor as their statistical measure, which is a description of the dynamic range and the variability of the assay. In this test, the Z´ factor values were 0.72 and 0.74. From these results, the authors conclude that the assay would work well for screening in a 384-well system and could likely be optimized for screening in a 1536-well format. (4135)

Notes: Human Genomic DNA used as the starting material in the NGS workflow.  GoTaq® Flexi Colorless Buffer and GoTaq® Flexi Polymerase were used in amplification of template in step added to the beginning of library preparation. (4531)

Notes: DNA templates are often amplified by PCR during library generation prior to next-generation sequencing, but amplification can introduce biases and duplications that are not easily corrected. In this paper, the authors developed a simple method to count the number of input template molecules to reduce these PCR-related problems: The ligation of a degenerate base region to all fragments during library creation. To evaluate their approach to correct for biases and duplications, the authors created a library using Human Genomic DNA, amplified the library by inverse PCR using the GoTaq^® Hot Start Polymerase and 1X Colorless GoTaq^® Flexi Buffer, sequenced the resulting DNA fragments and assessed the quality of the next-generation sequencing data. (4160)

Notes: To confirm the role of a mutation in the miR-96 microRNA (miRNA) associated with an autosomal dominant hearing lost, HeLa cells (250,000 cells per well in six-well plates) were transfected with 4µg of plasmid carrying wild type or mutant miR-96 miRNA using FuGENE® HD Transfection Reagent. After 24 hours, the cells were washed and total RNA extracted. After quantitation, the RNA used in RT-PCR analysis. The entire 3´UTRs of eight putative target genes were amplified by PCR from genomic DNA and cloned into the psiCHECK™-2 Vector. HeLa cells were transiently transfected with 2µg of the 3´ UTR psiCHECK™-2 constructs and 0.2µg of a wild-type, single or double mutant miR-96 plasmid using FuGENE® HD Transfection Reagent. Forty-eight hours after transfection, the Dual-Luciferase® Reporter Assay System was used to quantify the firefly and Renilla luciferase in cell lysates. (4251)

Notes: This study characterized interactions between the proton-pumping membrane complex V-ATPase, the Arf nucleotide binding site opener ARNO, and aldolase. The authors used a combination of protein-protein interaction techniques to identify downstream effectors of ARNO and V-ATPase signaling, and identified aldolase as a specific interaction partner of ARNO that could be involved in intracellular trafficking and cytoskeletal modulation. As part of the study, the FluoroTect™ Green_Lys in vitro Translation Labeling System was used to fluorescently label recombinant proteins during in vitro translation reactions. The labeled proteins were used in pull-down experiments. (4250)

Notes: The transcription factor Nanog is required for the maintenance of embryonic stem (ES) cell pluripotency. These authors showed that the Nanog N-terminal domain is regulated by post-transcriptional modification, and that alternative splicing generates Nanog variants with different capacities for maintaining an undifferentiated cell state. As part of their study, the authors used GoScript® Reverse Transcriptase to generate cDNA from RNA extracted from cell lines expressing different Nanog variants. The cDNA was used in RT-qPCR to quantify relative expression levels. (4184)

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)