Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

Notes: When infected with Borrelia burgdorferi, humans respond by producing borreliacidal antibodies against outer surface protein C (OspC). The authors examined the immune response in infected canines to determine if the response was similar. Infected dogs produced borreliacidal antibodies, only some of which were against OspC. To examine the borreliacidal activity of these other antibodies, sera from infected canines were depleted of OspC antibodies, and the borreliacidal activities of the depleted sera were measured. Sera were depleted by passage over an OspC column, which was created by immobilizing 0.5mg of biotinylated OspC protein with 1ml of TetraLink™ Tetrameric Avidin Resin. (3780)

Notes: To explore the role of C/EBPb isoforms in regulating p53 expression during the cell cycle, the 1.7 kb murine p53 promoter was cloned into the pGL3-Basic Vector. Using TransFast™ Reagent, Swiss3T3 and 6629 (C/EBPb-null) cells were transfected with 0.1–0.75 µg of pGL3-1.7-kb p53 promoter construct with or without co-transfection of 0.25 µg of C/EBPb-2, and with 50 ng of pRL-TK Vector as an internal control. Twenty-four hours post-transfection, the cells were harvested and assayed for luciferase activity, normalizing reporter activity to Renilla luciferase activity. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to either mutate or delete the –972/–953 cis-acting element carrying the C/EBPb-binding site within the p53 promoter, and 0.1–0.75 µg of the mutant constructs were then tested in the same reporter assay. The C/EBPb-1, -2, and -3 cDNAs were cloned into an expression vector and translated using 0.5µg of plasmid in the TNT^® T7 Quick Coupled transcription/translation system either in the presence of unlabeled or [³⁵S]methionine. The synthesized proteins were analyzed on 12% SDS-polyacrylamide gel. (3675)

Notes: The authors wanted to examine how the Camelpox virus (CMLV) gene 176R (v-slfn) might affect orthopoxvirus virulence since it shares sequence similarity to murine schlafen (m-slfn) proteins that inhibit T cell and fibroblast growth. HeLa cells were transiently transfected with either pCI Mammalian Expression Vector alone or pCI Mammalian Expression Vector expressing v-slfn with or without a HA tag. Cellular localization of the protein was visualized using either α-v-slfn antiserum or α-HA mAb. (3757)

Notes: The authors tested their hypothesis that Na⁺-H⁺ exchangers (NHE) are involved in the formation of multicullar dome structures in confluent Madin-Darby canine kidney (MDCK) cells and that the Stat3 pathway is involved in regulation of NHEs. The authors performed semi-quantitative RT-PCR to monitor NHE3 mRNA levels in MDCK cells expressing a constitutive Stat3 mutant or a dominant-negative Stat3 mutant. The reverse transcription step was performed using Promega M-MLV Reverse Transcriptase. RAlso, Stat3 activities in low-density cultures and high-density cultures were compared using a reporter gene assay. Four copies of the Stat3-binding site were cloned upstream of a firefly luciferase reporter gene, and the resulting vector, along with the pRL-TK Vector for normalization, were transfected into MDCK cells. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System. (3910)

Notes: The role of V₀ subunit isoform and its cellular environment in the disassociation of vacuolar (H⁺)-ATPases (V-ATPases) in yeast was explored in this paper. An EcoRI-BamHI fragment of HA-Stv1p, a hemagglutanin-tagged Golgi-targeting subunit of V-ATPase, had two different mutations introduced (R795K and R795Q) using the Altered Sites^® II in vitro Mutagenesis System. The mutant vectors were then transformed into yeast strain MM112 and used in studies with vacuolar protein sorting (vps) mutants. (3693)

Notes: During casework analysis using the PowerPlex^® 16 System, the authors identified an off-ladder allele at the Penta D locus as the microvariant allele 9.2. DNA from individuals with the 9.2 allele was amplified using a single primer pair specific for Penta D, and the amplification products were purified and sequenced to characterize the microvariant allele. PCR products were purified using the Wizard^® PCR Preps DNA Purification System. Sequence analysis revealed that the 9.2 allele has 10 STR repeats and a TAA deletion in the 3´ flanking region. (3771)

Notes: The authors knocked down expression of the hydin gene, which encodes a flagellar protein, to determine the function and location of hydin in Chlamydomonas reinhardtii. Amplification steps in the creation of the RNAi expression vector to target hydin expression were performed using GoTaq^® Flexi DNA Polymerase. (3723)

Notes: Taxol-induced peripheral neuropathy is a common side-effect of treatment that has been associated with disturbed intracellular calcium homeostasis in neuronal cells. These authors investigated whether prolonged exposure to Taxol caused alterations in calcium signaling in human neuroblastoma and rat dorsal root ganglia. They found that expression of the inositol 1,4,5-triphosphate receptor modulator NCS-1 was reduced in Taxol-treated neuronal cells. The authors also found that Taxol treatment activated calpain, which degrades NCS-1, and that the calpain inhibitor AK295 prevented Taxol-mediated suppression of calcium release. These findings suggest that calcium-mediated activation of calpain is responsible for the observed degradation of NCS-1. (3681)

Notes: The authors identified an adherence factor of enterohemorrhagic E. coli that is involved in colonization of cultured epithelial cells. This factor, named E. coli common pilus (ECP), is encoded by the ecpA gene, which is present 96% of E. coli strains tested, as determined by PCR. The remaining 4% of the strains were found to be deficient in the ECP operon, as determined by multiplex PCR amplification of ecpR, ecpA, epcB and ecpC sequences. PCR were performed using GoTaq® Green Master Mix. An ecpA deletion mutant exhibited impaired adherence compared to the wildtype E. coli strain. Complementation of the mutant strain with the plasmid pMR13, the pGEM®-T Vector containing the ecpA gene, restored the strain's ability to adhere to epithelial cells. (3719)

Notes: The authors analyzed 1,308 samples for concordance between the Identifiler^® kit, AmpFlSTR^® Minifiler™ kit and PowerPlex^® 16 System. DNA was isolated from liquid blood using the manual DNA IQ™ System protocol, and STR amplifications were performed as per the manufacturer's recommendations except that reaction volumes were decreased by half. Amplified products were analyzed using an Applied Biosystems 3130xl and POP™-4 or POP™-6 polymer. Twenty seven disconcordant phenotypes were identified between the Minifiler™ and Identifiler^® kits, 14 between Minifiler™ and PowerPlex^® 16 kits, and 4 between PowerPlex^® 16 and Identifiler^® kits. (3770)

Notes: The authors studied the defective prophage KplE1 in E. coli K12 to map the binding sites of proteins required for recombination. Prior to in vivo excision assays in two E. coli K12 strains, the presence of three DNA sequences required for recombination was confirmed by PCR using GoTaq^® DNA Polymerase. In vitro excision assays were also performed using linear and supercoiled DNA substrates that were purified using the Wizard^® PCR Clean-Up System. Finally the phage-encoded integraseS (IntS) mRNA was quantitated by real-time RT-PCR. The RNA template was purified from E. coli K12 using the PureYield™ RNA Midiprep System. (3722)

Notes: This study looked at how OX40, a member of the TNFR superfamily, affected the differentiation of CD8 T cells with an OX40 agonist. Recombinant lentiviruses were generated by cotransfection of a vector plasmid, virus-packaging plasmid, envelope plasmid and the helper pAdVAntage™ Vector into HEK 293 cells. The supernatant was collected and used to infect tumor cells which were injected into mice. (3753)

Notes: The authors studied hsp70 mRNA degradation in Drosophila Schneider cells. mRNA deadenylation and decay were monitored by Northern blot. Two of the Northern blot probes used to visualize the mRNA decay products were synthesized by transcription of linearized plasmids using T7 RNA Polymerase and [α-³²P] UTP. A population of deadenylated mRNA was created by hybridizing mRNA with oligo(dT) and treating with RNase H. CCR4•NOT was identified as the main deadenylase involved in mRNA decay, and the PAN2:PAN3 deadenylase was a minor contributor. RNA interference was used to knock down expression of PAN2 and CAF1, a subunit of CCR4•NOT, to assess the effect on mRNA decay. Reduced expression levels of PAN2 and CAF1 were confirmed by semi-quantitative RT-PCR and Western blotting, respectively. RT-PCR was performed using 1.5µg total RNA and 150 units of MMLV Reverse Transcriptase in a 25µl reaction. One microliter of the RT reaction was used as a template in an 80µl PCR using 0.5 units of GoTaq^® DNA Polymerase, 1.5mM MgCl₂ and 1X Green GoTaq^® Flexi Reaction Buffer. (3707)

Notes: The authors of this paper describe the validation of a bioluminescent assay using the CellTiter-Glo^® Reagent to identify novel compounds that inhibit the cytopathic effect of SARS CoV. In this study, three cell viability assay reagents were evaluated: MTS, neutral red, and CellTiter-Glo^® Reagent. The CellTiter-Glo^®-based assay was chosen because it did not require washing or medium removal; it involved minimal pipetting steps and had a short incubation time, which reduced time in the BSL3 containment facility. The assay was used to screen 100,000 compounds and identified several compounds that inhibited CPE while having a minimal effect on cell viability. (3736)

Notes: To examine the effects of hormones on Gas6, a plasma vitamin K-dependent protein that may play a role in cardiovascular disease, the authors developed an ELISA test for Gas6, which they tested on blood from male and female volunteers. A recombinant Gas6 control was developed by reverse transcribing the full-length human Gas6 mRNA from human umbilical vein endothelial cells, amplifying the cDNA using nested PCR and after restriction digestion, ligating the insert into the EcoRI and XbaI sites of the pCI-neo Mammalian Expression Vector. The full-length construct was confirmed by sequencing and then tested in the Gas6 ELISA. (3688)

Notes: The authors examined transcriptional regulation of the vitamin D receptor (VDR) by p53 and p63, a member of the p53 family, under stressed and unstressed conditions. Reporter constructs with the full-length and minimal VDR promoters controlling expression of firefly luciferase were cotransfected with p53 or p63 expression constructs, and transcriptional activation of the VDR promoter was monitored using the Dual-Luciferase^® Reporter 100 Assay System. Results were normalized to Renilla luciferase activity. Interaction between p73, another member of the p53 family, and the VDR promoter was examined using chromatin immunoprecipitation. The imunnopreciptated chromatin was reverse crosslinked, DNA was eluted and VDR and p21 sequences were detected by PCR using GoTaq^® Green Master Mix. (3715)

Notes: To study how chemotherapy affects hair follicles (HF), the researchers microdissected and cultured human anagen VI scalp follicles and treated the cells with 4-Hydroperoxycyclophosphamide (4-HC) at similar concentrations to those received during therapy. After incubation for 24 hours with 30µmol/L of 4-HC, the HFs were homogenized and genomic DNA was purified using the Wizard^® SV Genomic DNA Purification System. To determine if the treatment induced the mitochondrial common DNA deletion, the isolated DNA was subjected to real-time PCR. Further examination of gene expression was performed by isolating total RNA from two HF samples, reverse transcribing 3µg of the RNA using 15U of AMV Reverse Transcriptase and 0.025 µg/µl random primers, and amplifying seven human genes in a TaqMan^® Assay. (3746)

Notes: The authors characterized mutations in the acetyl-coenzyme A carboxylase (ACCase) gene that confer resistance to the herbicide clethodim in the grass weed Lolium rigidum. The ACCase gene was amplified from clethidem-resistant and susceptible plants, then sequenced to identify previously unknown mutations. Amplifications of ACCase were performed using 300ng of genomic DNA and GoTaq^® Green Master Mix. The Wizard^® SV Gel and PCR Clean-Up System was used to purify PCR products directly or from agarose gels. (3721)

Notes: The authors used the PowerPlex^® 16 System to perform DNA typing of 27 sets of World War II skeletal remains found in two mass graves in Slovenia. Each bone sample was sanded to remove potential contaminants from exterior surfaces, then washed and air-dried. The same procedure was used to process teeth, except that the teeth were not sanded. DNA was isolated from the bone powder using organic extraction, then quantified. DNA was amplified using the PowerPlex^® 16 System as per the manufacturer's recommendations; for samples with small amounts of DNA, the number of cycles was increased to 32 and the elongation time extended to 90 seconds. Fifteen sets of remains yielded full profiles, and 12 sets yielded partial profiles, with the least successful profile including 13 loci. DNA was also extracted and amplified from 69 reference buccal swab samples from potential relatives. (3817)

Notes: The authors explored the possibility of a domain-based level of gene expression regulation for chromosomes by integrating the green fluorescent protein (GFP) reporter in 90 different locations in cultured human cells. This integration was accomplished by infecting human embryonic kidney cells (HEK293) with a lentiviral construct carrying the GFP gene under the control of the ubiquitously expressed human phosphoglycerate kinase (PGK), sorting GFP-positive cells by FACS and selecting clones for expansion. Genomic DNA was isolated from the various clones using the Wizard^® SV Genomic DNA Purification System and analyzed by PCR or restriction digested for Southern blotting. (3743)

Notes: Telomeres shorten by 50–100 bases with each cell division, making the telomere a "mitotic counter" that can limit cellular lifespan. Telomerase is a two-component protein consisting of a reverse transcriptase (hTERT) bound to its own RNA template that can act to maintain telomere length in dividing cells. Telomerase is highly active in dividing cells such as germ cells, stem cells and many cancers. This paper investigated the role of methylation of the hTERT promoter and the transcription factor CTCF in regulation of telomerase activity. LacZ reporter plasmids driven by the hTERT minimal promoter were transiently transfected into HeLa cells, and reporter assays were performed on lysate generated using Passive Lysis Buffer. The hTERT minimal promoter did not show activity if all of the CpG sites were methylated. The promoter and first exon of hTERT were amplified using PCR Master Mix from sodium bisulfite-treated genomic DNA isolated from telomerase-positive cell lines and tissues. The resulting fragments were cloned using the pGEM^®-T Vector System II. For the methylation cassette assay, methylated and unmethylated fragments were cloned into a methylated or unmethylated vector using the LigaFast™ Rapid DNA Ligation System. The authors conclude that methylation plays a dual role in regulating hTERT expression. CTCF will bind to the first exon of hTERT when the hTERT CpG island is not methylated, resulting in downregulation of hTERT expression. Although CTCF cannot bind the hTERT promoter when the DNA is completely methylated, the methylation itself completely represses transcription. In situations where there is partial methylation of the promoter, such as in tumor cells, CTCF cannot bind to the promoter, but the partial methylation is not enough to repress transcription, and hTERT is expressed. (3641)

Notes: The authors examined the deacylation activity of acyloxyacyl hydrolase (AOAH) against a protein:lipooligosaccharides (LOS) complex. A soluble, truncated form of endotoxin-binding protein CD14 (sCD14) was expressed with a hexapolyhistidine tag from a baculovirus vector in High Five insect cells and used to form [³H]LOS:protein complexes. These complexes were incubated with AOAH for 4 hours, then purified using the HisLink™ Protein Purification Resin. The degree of deacylation of the LOS was determined using liquid scintillation spectroscopy. No imidazole was used in the binding buffer or rinses. The buffer did contain 0.1% bovine serum albumin. (3698)

Notes: This paper explored the process by which the Eph receptor EphA2 regulates repulsive response in prostatic carcinoma cells via the ephrinA1 ligand. Focal adhesion kinase (FAK) non-related kinase (FRNK; a target in the ephrinA1 ligand cascade) was subcloned into the pTargeT™ Mammalian Expression Vector. This mutant FAK was transfected into PC-3 cells and subjected to a GST-pulldown assay to examine RhoA activation. In addition, PC-3 cells transfected with several targets in the EphA2/ephrinA1 activation cascade including FRNK were subjected to an in vitro three-dimensional migration assay. (3690)

Notes: These authors compared standard culture conditions, DNA isolation and analysis (e.g, sequencing) with a liquid culture, DNA isolation and a homogeneous fluorescent detection system for identifying mycobacterial species. The standard DNA extraction began with a loopful (3–mm³ sphere) of bacterial colony grown on Ogawa slants that used glass beads to mechanically disrupt the cells. The resulting lysate was extracted using phenol/chloroform, and DNA purified from the aqueous phase using a robotic liquid handler AGE-96 (Biotec) and the MagneSil^® Blood Genomic, Max Yield System. The DNA extractions were used in PCR and sequencing reactions. (3700)