Notes: In this study, caspase-3/7 activity in HUVECs was measured using the Caspase-Glo^® 3/7 Assay. Luminescence was measured with the GloMax^®-96 Microplate Luminometer. (4207)

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: Mutations in the KRAS gene occur in 30–50% of colorectal cancers. The authors amplified and sequenced the KRAS gene in 198 colorectal cancer samples to detect KRAS mutations. They then determined the frequency of microsatellite instability (MSI) in both KRAS-mutated and KRAS wildtype cancers to show that KRAS-mutated samples had a lower frequency of MSI. MSI analysis of paired normal and cancer tissues were performed using the MSI Analysis System. (4107)

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: In this paper, FuGENE® HD reagent was used to transiently transfect U937 cells. Cells were plated in 6-well plates and transfected with a total of 2.155µg plasmid DNA.
(4416)

Notes: The authors of this study used GFP to create a fluorescent reporter that can report the functional state of proteins that are activated by a post-translational event such as enzymatic cleavage or other modification. They created a caspase-activatable GFP (CA-GFP) and analyzed the mechanism of action of the reporter. They subjected purified CA-GFP and GFP to trypsin digestion in the presence of ProteaseMax™ Surfactant, Trypsin Enhancer for matrix-assisted-laser desorption Time-of-Flight (MALDI-TOF) and subsequent MS/MS. They were able to show that the absorbance spectra of CA-GFP does not have the characteristic signature of GFP suggesting that the GFP chromaphore is not mature and in the dark state (Figure 6 in the paper). (4216)

Notes: These authors performed metagenomic analysis to investigate any changes in composition of the microbial flora of the abomasa (ruminant 4th stomach compartment) in immune cattle after reinfection with the nematode Ostertagia ostertagi. DNA was extracted from abomasal contents using a QIAamp stool kit and the integrity verified using an Agilent Bioanalyzer 2000. PCR was then performed using primers targeting hypervariable regions of 16S rRNA genes. The amplified material was gel-purified and sequenced using the Roche 454 pyrosequencing system. DNA concentration was measured before and after PCR using the QuantiFluor™ Fluorometer.

Notes: This study characterized the strains of hand, foot and mouth disease (HFMD) that circulated in Peninsular Malaysia and Sabah during the last ten years. Viral RNA was extracted from vesicle, throat and rectal swabs, and 5µl of purified RNA was amplified in a 50µl reaction using the AccessQuick™ RT-PCT System with primers for VP4 or VP1. The amplified products were then sequenced and compared to known HFMD sequences. (4341)

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect C2C12 cells using a 4:2 FuGENE® reagent:DNA ratio. (4421)

Notes: These authors evaluated the effect of knockdown of Parp1 on survival of tumor cells after in vivo delivery of anti-Parp siRNA in a lipid-based nanoparticle format. Inhibition of Parp1 expression in mouse tumors was confirmed by qPCR analysis. Total RNA was extracted from the tumors using TRIzol® reagent (Life Technologies) and reverse transcribed using the ImProm-II™ Reverse Transcription System prior to qPCR using SYBR Green PCR Master Mix (Applied Biosystems) . (4222)

Notes: The authors of this study investigated the role of lipid raft microdomains that are present in detergent-resistant membranes (DRMs) during Plasmodium ookinete invasion of A. gambiae midguts. The invasion of the mosquito midgut is a critical step in the Plasmodium life cycle and therefore a promising target of transmission-blocking vaccines. The authors analyzed midgut DRMs by tandem MS and also the glycosylphosphotidyl inositol-anchored subproteome of A. gambiae midgut brush-border microvilli as well. To prepare the GI-anchored proteins for analysis, an in-gel protein digestion protocol was followed using trypsin in the presence of ProteaseMAX™ Surfactant, Trypsin Enhancer. Nearly 97% of the GI-anchored proteins identified were also present in the DRMs, perhaps providing additional targets for transmission-blocking vaccines. (4218)

Notes: To confirm the  Mycoplasma contamination in HIV-1 infected samples detected by high-throughput sequencing, a PCR-based Mycoplasma test was performed. Products were separated on an agarose gel and purified using the Wizard® SV Gel and PCR Clean-Up System before confirmatory sequencing. (4537)

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: In this study, HEK293 cells were grown to 50-60% confluence in 100mm dishes and then transfected with plasmid constructs using FuGENE® 6 transfection reagent at a 3:1 FuGENE:DNA ratio. (4363)

Notes: Wizard® SV Gel and PCR Clean-Up System was used to purify products after barcodes were attached by PCR prior to sequencing on an Illumina Genome Analyzer GA2. (4553)

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: In this study, FuGENE® 6 Reagent was used to transiently transfect the S2R+ drosophila Schneider cell line with 0.6µg DNA at a 8.3:1 FuGENE:DNA ratio. 0.6 x 10e6 cells were plated 1 day prior to transfection in 12-well plates. (4369)

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: These authors compared the performance of automated DNA purification using the Maxwell® 16 Instrument with manual methods. DNA yield and quality from milk, blood (buffy coat), and semen samples from cattle were evaluated. The Maxwell® 16 System performed best, consistently generating high quantity and quality DNA across the sample range tested. (4213)

Notes: This study explored whether the Agr-like quorum-sensing system regulates sporulation and production of the Clostridium perfringens toxins CPE and beta2 toxin. An agrB null mutant was inhibited for production of beta2 toxin during vegetative growth and in sporulating culture, had reduced production of alpha-toxin and perfringolysin O during vegetative growth, and did not form spores efficiently. The AccessQuick™ System was used in RT-PCR analysis to confirm the presence or absence of agrB transcripts in the wild type, mutant, and complemented strains used in the study. (4546)