Notes: Biochemical oxygen demand (BOD) is useful for determining water biodegradability, and the application of this measure allows researchers to analyze the efficiency of a given water treatment process in reducing the biodegradable natural organic matter (NOM). Such analyses allow researchers to determine the amount of oxygen required for the biochemical degradation of organic material over time. BOD protocols are well established for freshwater and wastewater; however, no satisfactory standard protocols have been developed for seawater and saltwater. The authors of this study set out to develop a protocol for a reliable Mediterranean seawater analysis using an appropriate seeding method. Raw seawater was collected from the Mediterranean Sea at the desalination plant at El Prat de Llobregat (Spain). The BacTiter-Glo™ Microbial Cell Viability Assay, which uses ATP indicator as the presence of viable cells, was used to monitor inoculum activity. Assay results were measured using the GloMax® 20/20 Luminometer. The authors determined a minimum seed quantity required for reliable BOD determination. (4260)

Notes: The authors used the Beta-Glo® Assay System to determine the transfection efficiency of electroporation. Control of transfection efficiency was done by using a rous sarcoma virus-β-galactosidase expression plasmid at 0.5μg/electroporation. The Plasminogen activator inhibitor-1 (PAI-1) was expressed with a luciferase reporter in a variety of cell lines to show that the effect of insulin to increase PAI-1 is increased by expression of Forkhead-related transcription factors (Fox). To identify which Fox mediated the effects of insulin on PAI expression, small interfering RNAs specific for each Fox were tested along with chromatin cross-linking and ChIP experiments. The authors were able to demonstrate that insulin acts through FoxO3a to increase PAI-1 gene expression, a major regulator of fibrinolysis. (4128)

Notes: These authors investigated the relationship between VEGF and oxidative stress related to preeclampsia. They showed that VEGF activates Nrf2 in an ERK1/2-dependent manner, protecting against oxidative stress. They first used a dual-luciferase reporter assay and a pGL3-ARE vector construct to show that VEGF activates ARE in the cytotrophic cell line BeWo. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase® reporter assay system and the GloMax®-96 microplate luminometer. The authors then showed that inactivation of the transcription factor Nrf2 by shRNA abolished this VEGF-dependent ARE activation. To determine whether Nrf2 protected BeWo cells from oxidative stress, cells were pretreated with VEGF and then exposed to H₂O₂ before monitoring cell viability and cytotoxicity. Cytotoxicity assays were performed using the CytoTox-Glo™ Assay. (4199)

Notes: These authors studied KLF6 expression during human trophoblast cell differentiation, and its role in the regulation of genes associated with placental development and pregnancy maintenance. They used immunofluorescence microscopy, RT-qPCR and luciferase reporter assays to investigate cellular localization, mRNA expression, and transcriptional activation. Reporter assays were performed using various luciferase reporter constructs, the Dual-Luciferase^® Assay, and the GloMax^®-Multi Detection System. KLF6 was shown to play a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG. (4197)

Notes: The authors used the Dual-Glo™ Luciferase Assay system to detect induction of NF-κB activity in transfected HEK-TLR4/MD2 cells expressing human CD14, toll-like receptor 4 (TLR4) and MD2. An NF-κB-driven luciferase reporter gene and a thymidine kinase (TK) promoter-driven Renilla luciferase reporter gene were measured 24 hours after the cells were exposed to long palate, lung, and nasal epithelium clone 1 protein (LPLUNC1) 1 hour prior to stimulation with lipopolysaccharide (LPS) from either E. coli or V. cholera. Figure 4 shows how LPLUNC1 was able to inhibit the TLR4 response to LPS from V. cholera. It was hypothesized that LPLUNC1 serves to reduce inflammation to enteric pathogens. Transfection details: Cells were seeded in 96-well plates at 30,000 cells/well and transfected with a total of 0.3µg of DNA per well. The transfected DNA contained 80ng of NF-κB-firefly luciferase plasmid and 20ng HSV-TK promoter-driven Renilla luciferase plasmid, along with human CD14 construct cloned into pCDNA3 at 10ng/well. (4177)

Notes: The authors determined that PERP, a tetraspan membrane protein, is required for enamel formation during tooth development in mice. Using microarray analysis, they then identified genes that were differentially expressed in wildtype and Perp-null mice and might be involved in enamel formation. Differential expression of these genes was confirmed by qPCR using the GoTaq^® qPCR Master Mix. The authors also identified an upstream regulator of Perp, P63, by transfecting cells derived from embryonic mouse teeth with a Perp-luciferase reporter construct that contained a P63 response element or mutated P63 response element. A Renilla luciferase vector was used for normalization of transfection efficiency, and luciferase assays were performed using the Dual-Luciferase^® Reporter Assay System. (4168)

Notes: Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind specific RNA segments and protect the adjacent RNA by serving as a barrier to exoribonucleases. This study showed that protection by PPR or PPR-like proteins is the predominant mechanism for defining the positions of processed 5′ and intercistronic mRNA termini in land plant chloroplasts. The authors used RNasin^® Ribonuclease Inhibitor in binding reactions between labeled RNA and PPR proteins prior to Gel mobility shift assays. They also used the pGEM^®-T Vector to clone various 3´ RNA terminal sequences amplified by RT-PCR. (4185)

Notes: The authors used stably transfected HEK cells expressing the human β2 adrenergic receptor, (β2AR) where the C-terminal tail was replaced with the C-terminal tail of the V2 vasopressin receptor (to increase signal-to-noise ratio) followed by a Tobacco Etch Virus (TEV) protease cleavage site and a tetracycline-controlled transcription factor (tTA). A second construct encoding β-arrestin 2 fused to TEV protease was also transfected into the same cell line. To follow the recruitment of β–arrestin upon ligand stimulation of the β2AR, the authors detected the cleavage of tTA and its translocation to the nucleus where it transcribed a stably expressing luciferase reporter gene. The Bright-Glo™ Luciferase Assay Reagent was used to detect luciferase activity and confirm positive recruitment of β-arrestin. The luminescent GloSensor™ cAMP Assay was used to assess ligand stimulation of the β2AR signaling through G proteins, resulting in an increase in cAMP. The authors used both of these assays to quantify ligand bias and identify weakly biased compounds of the β2AR and angiotensin II type 1A receptors. A number of known compounds were assessed to show the value of this strategy in helping to decipher complex signaling pathways. This approach may be useful in the development of novel biased ligands for therapeutic use. (4146)

Notes: The full-length 3´ UTR of mouse RNA-binding protein HuR was amplified and cloned into psiCHECK™-1 Vector to create pEM429 plasmid. To generate radiolabeled RNA substrates for use in cleavage assays, RNA was synthesized from 1µg of linearized plasmid using 50µCi of [α-³²P]UTP, 0.8mM Ribo m⁷G Cap Analog and T7 RNA Polymerase by incubating for 1.5 hours at 37°C. The RNAs were then treated with 1 unit of RQ1 RNase-Free DNase per 1µg of template DNA for 15 minutes at 37°C before extracting with phenol:chloroform, precipitating with ethanol and resuspending in DEPC-treated water. The cleavage assay used 60fmol of ³²P-labeled substrate RNA with 10U Recombinant RNAsin Ribonuclease Inhibitor in a reaction incubated for 2.5 hours at 30°C. RNA probes were labeled with biotin using T7 or T3 RNA Polymerases with a biotin-UTP labeling NTP mixture and incubated for 2 hours at 37°C. The biotinylation reaction was then treated with RQ1 RNase-Free DNase following the same protocol used for radiolabeled RNA. To form HuR/RNA complexes, 2µg of biotinylated RNA was mixed with 100µg nuclear extract and 40 units Recombinant RNAsin^® Ribonuclease Inhibitor in a total volume of 20µl, and incubated for 30 minutes at room temperature. For CstF-64/RNA complexes, 1µg of biotinylated RNA was used and the complexes were stabilized by UV crosslinking using 10U Recombinant RNAsin Ribonuclease Inhibitor during the UV treatment. NIH 3T3 cells were UV crosslinked and either total or nuclear RNA used for immunoprecipitation. After extraction the RNAs were treated with RQ1 RNase-Free DNase for 15 minutes at 37°C before RT-PCR using HuR-specific primers. Total RNA purified from cultured cells were incubated with 50U/ml RQ1 RNase-Free DNase at 37°C for 30 minutes before use in RT-qPCR. (4187)

Notes: These authors investigated whether HaloTag® technology could be used to specifically target and degrade intracellular proteins. They developed a method to degrade specific proteins of interest using a small molecule by tagging the HaloTag® protein with an adamantyl moiety. The hypothesis was that appending a hydrophobic residue to a protein surface domain would mimic partial denaturation and induce proteasomal degradation. They first used a HaloTag®-luciferase fusion protein to determine the biological activity of various small molecules that enhanced hydrophobicity. After selecting the small molecule that reduced luciferase activity in HEK293 cells, they went on to test their hypothesis in various in vitro and in vivo models. They were able to demonstrate degradation of various HaloTag®-linked transmembrane proteins expressed in HEK293 cells, to show degradations of target proteins in Zebrafish, and to show degradation of the HRAS oncogene product both in NIH3T3 cells and in a mouse tumor model. (4125)

Notes: The authors used the Dual-Luciferase® Reporter Assay System to measure NF-κB firefly luciferase activity normalized to Renilla luciferase (pRL-TK) in transfected HEK293T and EL4 cells. Coumermycin-treated HEK293 and EL4 cells transiently transfected with B-cell CLL/lymphoma 10 (BCL10) led to transcriptional NF-κB activation in a dose dependent manner. Dimerization of BCL10 by coumermycin was used to mimic physiological stimulation through T cell receptor cross-linking, which initiates BCL10-mediated activation of the NF-κB signaling pathway. Overexpression experiments showed that E3 Ubiquitin Ligase Mind Bomb-2 (MIB2) controlled BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ, a kinase responsible for phosphorylating IκBα protein. The authors identified that the C-terminal RING finger domain of MIB2 was critical for protein ubiquitination in NF-κB activation, which was confirmed by the NF-κB luciferase reporter response to various MIB2 mutants. (4180)

Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag^® CMV Flexi^® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag^® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag^® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase^® Reporter Assay System. (4123)

Notes: Proteasome activity was determined in cells expressing the β2 proteasome subunit using the Proteasome-Glo™ Trypsin-Like Cell-Based Assay. The authors determined the proteasome activity in the cells after exposure to the proteasome inhibitor MG132. Luminescence indicative of proteasome activity was measured using a GloMax^®-Multi Detection System Luminometer. (4195)

Notes: The authors of this study set out to describe the mechanism of cell death through which TNF-like weak inducer of apoptosis (TWEAK) exerts its apoptotic effect on certain cancer cells. The used the CellTiter-Glo® Cell Viability Assay and the Caspase-Glo® 3/7 Assay to investigate cell viability and mechanism of cell death in HSC3 cells treated with TWEAK. They looked at caspase-8 activity in cells treated with TWEAK in the presence or absence of a caspase-8 inhibitor using the Caspase-Glo® 8 Assay. They showed that TWEAK induces caspase-dependent apoptosis in these cells. (4170)

Notes: pGL4.35  and pGL4.36 reporter vectors were used. (4264)

Notes: The authors used Dual-Glo^® Luciferase Assay to measure a pISG54 promoter-driven firefly luciferase gene (0.5µg) and pRL-TK plasmid constitutively expressing Renilla luciferase (0.1µg), and either a plasmid (0.5µg) expressing the corresponding variants of Ebola virus (EBOV) structural protein VP24 construct (phCMV-EBOV-VP24) or empty phCMV in HEK 293T and GPC-16 transfected cells. A total of 1.1µg of DNA was transfected. Cells were stimulated with interferon (IFN) 24 hours post-transfection, harvested 16 hours later, and assayed for dual-luciferase activity. Data indicated that mutations in the V24 protein were associated with EBOV virulence but that this virulence was not linked to the IFN-antagonist function of V24 protein. (4178)

Notes: The authors determined that a recombinant catalytic domain of rat matriptase (His₆t-S-CD) caused detachment of small-intestinal epithelial cells (IEC-6 cells) from laminin-coated plates. His₆t-S-CD was expressed in the yeast P. pastoris and purified by ammonium sulfate precipitation, gel filtration through a PD-10 column, then Ni²⁺-chelating chromatography using HisLink™ Resin. The authors also treated IEC-6 cells with purified His₆t-S-CD to determine if this domain induced apoptosis by monitoring annexin-V staining, DNA fragmentation and caspase-3 activity. For the DNA fragmentation analysis, IEC-6 cells were treated with His₆t-S-CD, then harvested, and genomic DNA was purified using the Wizard® SV Gel and PCR Clean-Up System. DNA fragmentation was assessed by agarose gel electrophoresis and ethidium bromide staining. (4100)

Notes: The authors created two new osteosarcoma models expressing the firefly luciferase enzyme, using a modified pGL3 plasmid to insert luciferase into lentiviral particles for transfection. Luciferase activity was detected using Steady-Glo® Luciferase Assay System with varying numbers of osteosarcoma cells in 96-well microplates. The luciferase-expressing osteosarcomas showed conserved osteolytic and osteogenic activities in mice, and could be imaged in vivo. The authors developed protocols to administer small interfering RNA to its osteosarcoma target (luciferase), and demonstrated its efficiency in vivo to suppress luciferase expression as a model for siRNA treatment of cancer. (4127)

Notes: The authors identified a single nucleotide polymorphism (SNP) in the 3´ untranslated region (3´ UTR) of MDM4, which promotes tumorigenesis by decreasing p53 tumor suppressor function, in ovarian cancer cells. This A to C transversion creates a putative target site for the hsa-miR-191 microRNA in the MDM4-C allele, but not the wildtype MDM4-A allele. To determine if this SNP affects MDM4 translation efficiency or mRNA stability, the authors cloned a 224-bp fragment of the MDM4 3´UTRs into the psiCHECK™-2 Vector and transfected the ovarian cancer cell line A2780 with the 224A or 224C variants of the MDM4 3´ UTR. The authors used the luciferase-based psiCHECK™-2 Vector and Dual-Glo^® Luciferase Assay System to show that the C variant dramatically reduces translation efficiency and/or mRNA stability. They also assessed MDM4 expression levels in A/A, A/C and C/C ovarian cancer cells and tissues using RT-qPCR. qPCR primers for MDM4 were designed using the Plexor^® Primer Design Software, and assays were performed using the Plexor^® qPCR System. (4157)

Notes: The authors explore barley (Hordeum vulgare) as a means of expressing recombinant human Flt3 ligand, which is a growth factor involved in proliferation and differentiation of stem cells and development of various immune cells. As part of their quality control, they performed in-gel proteolytic digestion and mass spectrometry. The recombinant Flt3 ligand was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Coomassie® blue staining, excision of the protein band, destaining, drying of the gel slice and digestion with Sequencing Grade Modified Trypsin at 30°C overnight prior to mass spectrometry. To test biological activity, the authors treated human acute myeloid leukemia cells with Flt3 expressed in barley or a commercial source of Flt3 then measured cell proliferation using the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay. (4352)

Notes: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) tends to cause apoptosis in tumor cells over normal cells, and so the TRAIL ligand and signaling pathway is an attractive pathway for developing cancer therapeutics. Bortezomib is a proteasome inhibitor that is used for therapy in many cancers. Studies indicate that it can sensitize tumor cells to the apoptotic effects of TRAIL. In this study, the authors investigated the ability of bortezomib on renal cell carcinoma (RCC). Growth inhibition of RCC in response to treatment with bortezomib followed by TRAIL treatment in the presence or absence of caspase inhibitors was assessed using the CellTiter® 96 AQ_ueous Non-Radioactive Cell Proliferation Assay. They assessed caspase activity in bortezomib/TRAI- treated RCC using the Caspase-Glo® 8 Assay. The effect of bortezomib on the proteasome of the RCCs was investigated using the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay. Their studies suggest that bortezomib can sensitize some RCC to TRAIL signaling. (4169)

Notes: The authors investigated genetic diversity in a Florida population of Illicium parviflorum, an endangered evergreen shrub, by amplifying intersimple sequence repeats (ISSRs). Amplifications were performed using GoTaq^® Hot Start Polymerase. (4161)

Notes: These authors investigated the role of the cardiac lineage protein 1 (CLP1/HEXIM1) in skeletal myogenesis. They showed that CLP1 knockout C2C12 cells were unable to differentiate, and then investigated the hypothesis that CLP1 associates with MyoD and HDAC proteins to downregulate cell cycle genes, such as cyclin D1, and allow expression of differentiation-specific genes. RNasin® Ribonuclease inhibitor was used in coimmunoprecipitation assays investigating the interaction between CLP1 and HDAC in C2C12 cells under differentiation and non-differentiation culture conditions. RNasin® was included during cell lysate preparation prior to coimmunoprecipitation assays with antibodies directed against various HDAC proteins. The authors also performed  a luciferase reporter assay using the Dual-Luciferase® Assay System to investigate the regulation of cyclin D1 expression by CLP1, MyoD and HDAC. A luciferase reporter-cyclin D1 construct was co-transfected with MyoD, HDAC and CLP1 constructs and the effect on luciferase expression examined under differentiation and non-differentiation conditions. In differentiation medium MyoD, CLP1 and HDAC5 acted synergistically to reduce cyclin D1 expression. (4225)

Notes: The authors used a yeast two-hybrid system to identify proteins that interact with the autoimmune regulator (AIRE) protein. DAXX, a multifunctional protein involved in apoptosis and transcription regulation, interacts with AIRE, as shown through coimmunoprecipitation and colocalization studies. Colocalization of AIRE and DAXX in HeLa cells was demonstrated by confocal microscopy using a Monster Green^® Fluorescent Protein-AIRE fusion protein and endogenous DAXX, which was detected using an anti-DAXX primary antibody and an anti-rabbit secondary antibody conjugated with Texas Red fluorophore. However, AIRE and DAXX did not interact in vitro in a GST pull-down assay using a GST-AIRE construct, radiolabeled DAXX protein expressed in a TNT^® system, and MagneGST™ Glutathione Particles, leading the authors to speculate that the interaction is weak or there are scaffold proteins required for protein interaction. To examine the effect of DAXX on AIRE transcriptional activity, the authors transfected COS-1 and HeLa cells with AIRE and DAXX expression constructs and a luciferase reporter plasmid with the human insulin promoter, then performed Dual Luciferase^® Reporter Assays. AIRE induced transcription of the insulin promoter, but coexpression of DAXX suppressed this transcriptional activation. (4153)

Notes: This study describes an enrichment method for cysteinyl adducts of human serum albumin. Electrospray ionization mass spectrometry was used to detect mercaptalbumin and HSA-Cys34 modifications before and after enrichment. After enrichment, mercaptalbumin was no longer observed in mass spectra. Trypsin and ProteaseMax Surfactant were used to prepare samples for mass spectrometry analysis. (4083)