Notes: In this study the authors looked for molecular mechanisms underlying the role of kainite receptors in ischemic stroke. In their studies, the researchers examined binding of Src kinase to GluK2, and the site of this interaction. A GST pulldown assay confirmed a direct interaction between GluK2 and Src in vitro. Then a bioluminescence resonance energy transfer (BRET) assay was used to examine this GluK2-Src interaction in live HEK293 cells, using NanoLuc® Luciferase as the energy donor, and HaloTag-labeled GluK2 as the energy acceptor. As reported, the co-expression of NLuc and HaloTag® fusions resulted in a significant NanoBRET ratio. The authors added untagged GluK2 as a competitor of the GluK2-HaloTag and Src-NLuc interaction, which resulted in reduction of the NanoBRET ratio. These results demonstrated that GluK2 interacts directly with Src in living cells. The GloMax® Discover Detection System was used to measure the output of these assays.

Their source of NanoLuc® Luciferase and HaloTag® tags was the NanoBRET™ PPI Starter System (Cat.# N1811, N1821). (4696)

Notes: Hepcidin is a small peptide secreted by the liver that plays a key role in iron homeostasis by binding and internalizing the iron efflux transporter ferroportin (Fpn). The authors of this paper used NanoLuc® luciferase-tagged and GFP-tagged Hepcidin fusion proteins to measure the internalization of Fpn in HEK293T cells. Once the NanoLuc®-tagged Fpn was internalized, luminescence was significantly decreased. The authors coexpressed both NanoLuc®-tagged Fpn and GFP-tagged Fpn using a doxycycline-inducible bidirectional promoter and were able to measure hepcidin-induced Fpn internalization qualitatively (GFP fluorescence) and quantitatively (bioluminescence).  (4436)

Notes: These authors developed a sensitive receptor-binding assay for detection of interactions between the peptide hormone Insulin-like peptide-3 (INSL3) and the relaxin family peptide receptor RXFP2.  Recombinant INSL3 was tagged with NanoLuc® Luciferase by chemical modification of INSL3 to include an active disufide bond, and engineering of a 6× His-Cys-NanoLuc with an exposed N-terminal cysteine. The NanoLuc®-conjugated INSL3 was used to monitor the receptor-binding of a variety of ligands. (4438)

Notes: These authors generated luciferase biosensors and used them to screen for potential inhibitors of Middle East respiratory syndrome coronavirus (MERS-CoV) replication. The biosensors were developed using the pGloSensor™-30F plasmid backbone and were engineered to include cleavage sites for the viral papain-like protease (PLpro) or 3-chymotrypsin-like protease (3CLpro). Lytic, endpoint assays and live-cell assays were performed to detect protease activity. The authors identified a small-molecule inhibitor that blocked the activity of MERS-CoV 3CLpro. These biosensor-based assays allowed rapid evaluation of viral protease activity and screening for protease inhibitors. (4526)

Notes: To understand the mechanism of Panicle blast 1 (Pb1) gene-mediated resistance to rice blast, a rice fungal disease, researchers investigated Pb1 interacted with a transcription factor involved in resistance, WRKY45 that is regulated by the ubiquitin system. To study how these proteins interacted, inner rice leaf sheaths were bombarded with gold particles coated with 0.5 µg of effector plasmid, 0.3 µg of NanoLuc® luciferase reporter and 0.1 µg of reference Renilla luciferase. After incubating overnight at 28°C, samples were ground in liquid nitrogen and reporter activities assayed using the Dual-Glo® Luciferase Reporter Assay System and Nano-Glo® Luciferase Assay System. The Renilla luciferase gene was also split into an N-terminal construct and C-terminal construct, expressed in rice protoplasts and assayed for reconstituted Renilla luciferase activity. Expression was normalized to firefly luciferase. (4510)

Notes: The authors sought to determine whether different residues had specific roles in binding and activation of the k-opioid receptor (KOR) by agonist molecules with distinct chemotypes. For function assays they introduced point mutations into human KOR using site-directed mutagenesis. All mutations were verified by Sanger automated sequencing. HEK293T cells were cotransfected with pGloSensor™-22F cAMP Plasmid plus the different KOR variants. Cells were plated, and after 24 hours, medium replaced with drug buffer and the different drug treatments. cAMP production was detected by treatment with isoproterenol in GloSensor™ Reagent. (4519)

Notes: The authors were interested in quantitating the presence as well as the prevalence of Lactobacillus gasseri K7 in humans that did not consume the probiotic bacteria. Fecal samples from 45 healthy adults were collected, frozen, diluted, centrifuged and digested with proteases. After sonication, DNA was extracted using the Maxwell® 16 Tissue DNA Purification kit on the Maxwell® 16 instrument. This same kit and instrument also were used to isolate bacterial DNA from 1ml bacterial cultures. The purified DNA was PCR amplified using primers for gassericin K7 A and K7 B (bacteriocin) genes and GoTaq® Flexi DNA Polymerase in Green GoTaq® Flexi Buffer for 30 cycles. PCR products were analyzed by 1.8 % agarose gel electrophoresis. (4522)

Notes:   (5200)

Notes: GoTaq^® Green Master Mix was used to amplify Tribolium castaneum  satellite DNA that had been bisulfite treated to detect methylated cytosines. The bisulfite-treated satellite DNA was amplified using methyl-specific primers in a total volume of 30µl using 2X GoTaq® Green Master Mix, 2mM mix of the primer sets, and 1µl of the bisulfite modified DNA.

  (4356)

Notes: These authors identified a previously unknown interaction between the transcription factor HIF1A and the cyclin-dependent kinase CDK8 (a component of the Mediator complex) in the regulation of genes associated with cellular survival under low-oxygen conditions. As part of the study, HaloTag technology was used to identify proteins interacting with CDK8 in a colorectal cancer cell line. Specifically, cells were transfected with CDK8 and CDK19 HaloTag fusion constructs obtained from Kazusa Institute. The cell lysates were then used in pulldown assays to capture interacting proteins. The results showed that CDK8 and CDK19 are present in mutually exclusive Mediator complexes. Details of the transfection are as follows: HCT116 cells were plated in 150 mm dishes and grown to 70%–80% confluence before transfection with 30 μg of plasmid DNA using FuGENE HD Transfection Reagent. (4355)

Notes: In vivo imaging using bioluminescent reporters is a powerful tool for real-time detection of viral load and spread in an animal over time. However, construction of influenza reporter viruses is complicated because the small viral genome does not tolerate large insertions and all the viral genes are critical in vivo, making it impossible to replace any gene with a reporter. These authors describe construction of a replication-competent influenza reporter virus containing the small (19kDa), bright NanoLuc® luciferase gene. NanoLuc® luciferase activity was then used to monitor viral infection in real time in an animal model.  Bioluminescent imaging of the reporter virus allowed serial observations of viral load and dissemination in infected animals, even following clearance of a sublethal challenge. (4435)

Notes: This article describes how a miniaturized high-throughput biochemical assay for Yes1 kinase was developed and used for screening inhibitors in small molecule libraries. Kinase activity of Yes1 was assessed by dispensing 2µl of Yes1 enzyme in a 1,536-well white multiwell plate and mixing with compounds and substrate for a total kinase reaction volume of 25µl. The amount of ADP was quantitated using the ADP-Glo™ Kinase Assay and normalized to vehicle and minus-enzyme controls. Some of the inhibitors identified in the in vitro assay were tested in a cell-based assay. Two rhabdomyosarcoma cell lines, RD and RH30, were seeded at 4,000 cells/well in a 96-well plate with 100µl of culture medium. After an overnight incubation, 100µl of medium with 0, 0.1, 1, 5, 10, 15 or 20μM of inhibitor (final concentration) was added. After 48 hours, the number of cells was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. (4354)

Notes: These authors demonstrated use of NanoLuc® Luciferase bioluminescence for imaging applications in mice. They showed that bioluminescence could be imaged in superficial and deep tissues, and were able to monitor changes in bioluminescence over time to quantify tumor growth. Secreted NanoLuc® Luciferase was also detectable in small volumes of serum. The paper also details use of both NanoLuc® and firefly luciferase reporters in a dual assay to quantify two steps in TGFβ signaling both in intact cells and in living mice. (4439)

Notes: The let-7 microRNA family are thought to act as tumor suppressors. Let-7 activity is downregulated in several cancers, and overexpression of let-7 inhibits cancer growth in some mouse models. The authors of this paper describe a sensitive luciferase-based reporter assay for detecting let-7 miRNA activity in cells. The reporter construct was based on the pmirGLO Vector, which contains firefly luciferase as the reporter gene and Renilla luciferase as an internal control. The authors inserted let-7 miRNA target sites at the 3′ end of the firefly luciferase gene. Interaction of let-7 miRNA with these target sequences resulted in reduced luciferase activity. The authors used the reporter construct to screen a kinase inhibitor library for compounds that repress let-7 activity in ovarian cancer cells, and identified GSK-3β as a potential target for therapeutics. (4406)

Notes: These authors analyzed the performance of the following reporter enzymes used to measure biological pathway modulation by small molecules: firefly luciferase, Renilla reniformis luciferase, β-lactamase, mutated forms of Renilla luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and NanoLuc® luciferase. They screened a collection of more than 42,000 PubChem compounds using purified enzyme preparations to determine hit rates and then examined structure:activity relationships. The study evaluated hit rates and inhibitor overlap between reporters. Based on these results, the authors suggest strategies to improve the construction and interpretation of assays using these reporter enzymes. (4437)

Notes: FuGENE^® HD Transfection Reagent was used to transiently transfect HeLa cells seeded on 12mm coverslips in 24-well plates. Cells were seeded at a concentration of 5 × 10⁴ cells/well and transfected after 24 hours using a 2:1 ratio of FuGENE^® HD reagent to DNA (4µl of reagent, 2µg of DNA). Transiently transfected cells were used for confocal microscopy. For luciferase assays, HEK293 cells were seeded into 48-well plates at 40% confluency, and 24 hours later, FuGENE^® HD reagent was used to transfect the cells with one of a variety of constructs. (4411)

Notes: HepG2 cells were plated, 5 × 106 cells in 15cm plates. Transfections were performed 24 h later using Fugene HD. The authors used 15 μg of DNA per transfection and a Fugene:DNA ratio of 7:2. (4425)

Notes: The authors transiently transfected 5 × 10⁶ HEK293 cells per 150cm dish using the FuGENE® HD Transfection Reagent, 15µg of DNA and a reagent:DNA ratio of 7:2. (4432)

Notes: This paper describes a bioluminescence-based method for the detection of DNA methyltransferase activity based on methylation-resistant cleavage and protein expression. The authors used Dam methylase as a model enzyme, MboI as the methylation-resistant endonuclease, and luciferase reporter DNA (LR-DNA) as the target. LR-DNA was amplified by PCR and then used as substrate in a Dam methylation reaction. If the target sites in the LR-DNA were fully methylated, they were resistant to subsequent MboI cleavage and expressed luciferase upon in vitro transcription/translation. Incomplete methylation or the absence of methylation resulted in DNA digestion and diminished/absent luciferase activity. TNT® T7 Quick for PCR DNA was used for in vitro translation of the LR-DNA, and the Luciferase Assay System and GloMax® 20/20 Luminometer were used to measure luciferase activity. (4191)

Notes: In this report, the original investigators who found XMRV and pMLV (polytropic murine leukemia virus) in blood of subjects with Chronic Fatigue Syndrome (CFS) report that this association is not confirmed in a blinded analysis of samples from rigorously characterized subjects.

The CDC performed nucleic acid testing assays. Plasma was centrifuged and RNA isolated from the pellet. Quantitative real-time RT-PCR assays (qRT-PCR) for generic pMLV/XMRV pro (protease) and gag detection were performed on RNA extracts, using the AccessQuick™ RT-PCR System and an AgPath one-step RT-PCR kit.

ArrayScript RT and AmpliTaq Gold DNA polymerase were used for cDNA synthesis and amplification in the pro and gag qRT-PCR assays, respectively. A third PCR was done using the primers XPOLOF and XPOLOR, followed by a nested PCR with the primers XPOLIF and XPOLIR for the generic detection of MLV/XMRV 216-bp pol sequences. For this reaction, cDNA synthesis and amplification of RNA was done using Promega AMV Reverse Transcriptase and a RobustI RT-PCR kit. Each PCR experiment included 20 water-only reactions to control for contamination. (4300)

Notes: These authors developed a protein-aggregation reporter that uses huntingtin exon 1 containing 72 glutamines fused to the N-terminal end of firefly luciferase (httQ72-Luc). This construct did not aggregate unless seeded by a non-luciferase-containing polyglutamine (polyQ) protein. Upon co-aggregation, httQ72-luc becomes insoluble and loses its enzymatic activity.  httQ72-Luc and a polyQ protein were used to screen a library for compounds that prevent polyQ aggregation. Leflunomide and its active metabolite teriflunomide inhibited protein aggregation independently of their known role in pyrimidine biosynthesis. This study demonstrated the usefulness of luciferase-based protein aggregate reporters for high-throughput screening applications. Luciferase activity was measured using the Luciferase Assay System and the GloMax®-Multi Detection System. (4188)

Notes: RAW 264.7 mouse macrophage cells were plated in 12-well plates at 1.5 × 10⁵ cells/well for transient transfection with 0.5μg of DNA and 1.5μl of FuGENE® 6 Transfection Reagent in 50µl of Opti-MEM I medium. After 24 hours, the cells were treated with IgG IC for 5 hours and luciferase activity analyzed using the Dual-Luciferase® Reporter Assay System. (4402)

Notes: Polyglutamine diseases are neurodegenerative disorders associated with the presence of proteins containing polyglutamine repeats. These authors studied the mechanism of polygluatmine toxicity. Mutant RNAs carrying an expanded CAG repeat were shown to activate the nucleolar stress pathway and induce apoptosis. Expanded CAG RNAs were shown to interact with nucleolin, preventing it from binding to an upstream control element of the rRNA promoter and causing decreased rRNA transcription, which in turn induced apoptosis. Perturbations in rRNA transcription were identified by real-time PCR, and fluorescence in situ hybridization was used to determine that expanded CAG RNAs localized to the nucleolus. Hybridization solutions were supplemented with RNasin® Ribonuclease Inhibitor. ImProm-II™ Reverse Transcriptase was used in RT-qPCR assays. (4228)

Notes: Sleep deprivation increases susceptibility to diseases such as diabetes and cancer. Because chronic inflammation is a feature of these diseases, the authors of this paper investigated whether the circadian oscillator component Cryptochrome (CRY) has a role in regulating the immune response. They found constitutive NF-κB and protein kinase A (PKA) activation in Cry1(-/-);Cry2(-/-) knockout mice, and high basal levels of cAMP. As part of the study, the effect of CRY overexpression on intracellular cAMP levels was evaluated in 293T cells using the bioluminescence-based GloSensor™ cAMP Assay. Overexpression of CRY1 reduced the cAMP production induced by forskolin, prostaglandin E2 or isoproterenol. Immunoprecipitation analysis with Flag-tagged CRY1 showed that CRY1 binds to adenylyl cyclase and limits cAMP production. The authors suggest that reduced CRY expression in chronic sleep deprivation may result in elevated cAMP levels, leading to increased PKA and NF-κB activation and offering a potential link between sleep deprivation, circadian rhythm disruption and chronic inflammation. (4224)

Notes: The authors used the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) as a third assay (in addition to ³H thymidine and ³H leucine assays) to assess the response of cells to CoArgIPEG and demonstrated not only a decline in cell survival with treatment but also a decline in proliferation and protein synthesis in most cases.

The CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was also used to evaluate the effect of recovery in cancer cells. The authors determined that prolonged treatment resulted in cytotoxicity; however, assessment at 24 hours showed that some cells survived in the short term. Because the authors knew proliferation/protein synthesis inhibition was occurring, evaluation with a cell survival assay like the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was required for a more complete understanding of cellular health and response. (4511)