Notes: To determine the usefulness of multilocus sequence typing (MLST) in differentiating Candida species during epidemiological studies, the authors investigated the population structure of C. dubliniensis by amplifying the same 10 MLST loci found to be useful in differentiating isolates of C. albicans, a closely related species. PCRs were performed using 1.25 units of GoTaq^® Flexi DNA Polymerase and 1ng of DNA template in a 50µl reaction. (3880)

Notes: In this study, FuGENE® 6 was used to perform transient transfection of K562 cells using 0.2 or 0.3µg of DNA in a 3:1 ratio of reagent to DNA. The transfections were performed in 24-well plates using 1 × 10⁵ cells/well. FuGENE® HD was used to transiently transfect HepG2 cells using 2µg of DNA in a 3:1 ratio of reagent to DNA using 2× 10⁵ cells  seeded onto coverslips 1 day prior to transfection. (4418)

Notes: The authors of this study investigated the relationship of somatic mutations that deregulate the RAS-RAF-MEK-ERK pathway and Acute Lymphoblastic Leukemia (ALL) and its progression to relapse in some patients. They show that such mutations are common in ALL and its recurrence. Furthermore, lymphoblasts from patients with mutations that were associated with upregulation of ERK showed increased cytotoxicity over wild-type controls when treated with U0126, suggesting that specific ERK inhibitors may eventually yield useful therapeutics. Following drug exposure, cytotoxic effects were assessed using the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay. (3905)

Notes: To study how mutations in the quinolone resistance determining region (QRDR) of Staphylococcus epidermidis may have a role in fluoroquinolone resistance, 138 samples of S. epidermidis were swabbed from the conjunctival sacs of 129 patients. These samples were cultured overnight in tryptic soy broth, and genomic DNA isolated using the Wizard^® SV 96 Genomic DNA Purification System. One microliter of the isolated DNA was used in PCR for the QRDR genes (gyrA, gyrB, parC and parE). (3939)

Notes: This article showed that expression of several immune response genes could be induced in lung adenocarcinoma H460 cells by treatment with farnesol and that this induction proceeds through an NF-κB pathway. To determine which MAPKs were involved in the activation of these genes, the authors used the MEK inhibitor U0126 and showed that it inhibited the expression of several of the immune response genes and specifically inhibited the phosphorylation of p65 on Ser²⁷⁶. (3904)

Notes: The authors were interested in testing platinum (Pt) anticancer drugs that do not bind to DNA to see how they worked in cisplatin- and carboplatin-resistant cells. The human ovarian cancer cells, A2780 and A2780/C30, were seeded in T75 cm² flasks with 1.0 × 10⁷ cells. After 24 hours, the cells were treated with 0, 10, 20, 30 and 50µM Pt compounds for 24 hours. Medium was removed, the cells washed, trypsinized and centrifuged. Genomic DNA was isolated using the Wizard^® SV Genomic DNA Purification System and quantitated using absorbance at 260nm. This DNA was using in DNA-Pt binding assessment. (4019)

Notes: Nuclear transport of hypoxia-inducible factors (HIF) allows these factors to activate transcription of genes including epo,vegf an glut1 to maintain oxygen homeostais in cells. In this study the authors synthesized HIF-1α,β and HIF-2α in vitro, and used the expressed proteins in binding studies to determine the nature of HIF binding to nuclear pore complex proteins. The HIF proteins were transcribed and translated in vitro in the presence of ³⁵S-methionine using the TNT^® Coupled Reticulocyte Lysate System according to the protocol. After incubation, 10µl of the reaction batch was allowed to bind to the immobilized fusion-proteins importin alpha3, alpha5, and alpha7. The direct interaction of HIF-1α with alpha importins was dependent on functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS was not effective. In a typical experiment 100µl GST beads were pre-equilibrated in IP buffer (20mM Hepes pH 7.5, 100mM KOAC, 0.5mM EGTA, 5mM MgOAc, 250mM sucrose, 4°C), mixed with 15µg GST-fusion proteins and His-tagged importin beta and incubated at 4° C for 1 h. (3947)

Notes: To examine radiation-bystander responses in neonatal mouse cerebellum, heterozygous radiosensitive Patched-1 (Ptch1) mice were exposed to either whole body (WB) x-rays or shielded head/rest of body (SH) irradiation. Genomic DNA was isolated from tumors and normal tissue using the Wizard^® SV Genomic DNA Purification System. Loss of heterozygosity was tested using PCR and sequencing of exon 23 of the Ptch1 gene. (3940)

Notes: To learn about the ideal target binding sequence for SATB1, the T-lineage-enriched chromatin organizer and transcription factor, random oligonucleotides underwent SELEX and five rounds of selection by EMSA. The enriched library of oligos was cloned into the pGEM®-T Easy Vector, transformed and sequenced. Several variants of SATB1-binding consensus sequences were annealed, ligated into the pGL3-Promoter Vector and cotransfected into HEK 293 cells with a plasmid that either contained SATB1 or was empty. After 48 hours, the cells were harvested and luciferase activity measured. The CheckMate™ Mammalian Two-Hybrid System was used to assess how the N-terminal PDZ domain of SATB1 interacted with the Cut and homeodomain in the C-terminus. (3982)

Notes: To examine the role of NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) during greening, the PORA gene was mutated by changing each of the four conserved Cys to Ala using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The resulting genes were subcloned into an expression vector and transformed into XL1-Blue cells. The proteins were expressed either in E. coli cells after IPTG induction or in a wheat germ extract system and tested for interaction with Pchlide molecules and NADPH. (3994)

Notes: A plastic support suitable for use in microfluidic systems for highly sensitive DNA microarray hybridizations was developed and tested. Human DNA from Hsap-11 cells was isolated using the MagneSil^® KF, Genomic System on a KingFisher ML instrument. Ten nanograms of the isolated DNA was used in RT-PCR. (4021)

Notes: The authors generated population data for unrelated Caucasian-Mestizos from Columbia using the PowerPlex^® 2.1 System and the GammaSTR^® Multiplex. DNA was isolated from whole blood using the Wizard^® Genomic DNA Purification Kit or ReadyAmp™ Genomic DNA Purification System, then amplified per the manufacturer's recommendations. Amplified fragments were detected using a Hitachi FMBIO^® II fluorescence imaging system. (3856)

Notes: This article examines the interaction between CAR, a member of the nuclear steroid/thyroid hormone receptor family, which translocates from the cytoplasm to the nucleus when cells are exposed to phenobaritol, and the membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A), which is a novel CAR-binding protein. The R16A protein, it was expressed using the TnT^® Coupled Reticulocyte Lysate System and labeled with ³⁵S. This protein was incubated with GST-hCAR-fusion protein attached to a glutathione resin; the resin was washed, and the bound protein was separated by PAGE and detected by autoradiography. Affinity-tagged R16A was expressed in HepG2 cells, purified and separated by SDS-PAGE. The two major bands were excised, digested with Sequencing Grade Modified Trypsin, lyophilized, resuspended in acetonitrile and subjected to mass spectrometric analyses. The CheckMate™ Mammalian Two-Hybrid System was used to examine the interaction between wildtype and mutated R16A. Forty-eight hours posttransfection into HepG2 cells or 16 hours after DNA injection into mice and liver homogenization, the luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. (3752)

Notes: The authors generated population data for 100 unrelated individuals from three main ethnic groups in Bosnia and Herzegovina. Autosomal STR analysis was performed for 100 male and female individuals, and Y-STR analysis was performed with 100 male individuals. DNA was collected as buccal swabs or blood samples, isolated and amplified, and amplification products were detected using an ABI PRISM^® 377 DNA Sequencer. (3877)

Notes: These authors prepared a complete collection of Vibrio cholerae ORF clones using an automated amplification and cloning procedure. They then tested this set of clones for protein expression and capture using a nucleic acid programmable protein array method. To do this, they used the TNT^® T7 Coupled Reticulocyte Lysate System to perform in situ transcription/translation of cDNA clones containing a GST fusion tag. Proteins were captured using an anti-GST antibody and subjected to further analysis. (3879)

Notes: The authors used a microfabricated capillary electrophoresis instrument to rapidly assess the genetic relationship between same-sex twins and their parents and siblings. STR typing was performed to determine if the twins were monozyotic or dizygotic and to confirm familial relationships. The authors used the PowerPlex^® 16 System to examine 15 STR loci in this study. (4045)

Notes: These authors describe use of a red recombinase mediated method for generation of reporter constructs in Salmonella enterica setrovar typhimurium. Among the reporter constructs created was a HaloTag^® reporter using the HaloTag^® coding region from the pHT2 promoter. (3924)

Notes: The authors of this paper describe the development of a cell-free assay system derived from Agrobacterium tumefaciens NTL4(pCF218) (pCF372), which expresses β-galactosidase under the control of a tra1 promoter. The tra1 promoter is responsive to quorum-sensing signals. The assay was developed to address time-consuming cell conditioning and incubation times of the standard whole-cell assays used to detect quorum-sensing signals in natural systems. Replacing the X-Gal substrate with the luminescent Beta-Glo^® substrate increased the assay sensitivity by tenfold. (3936)

Notes: This paper describes analysis of DNA samples at a mock crime scene using automated DNA purification followed by STR analysis on a portable microchip system. The mock crime scene was created using "victim" and "suspect" blood stains prepared by spotting 3µl of liquid blood onto paper towels and a cloth shirt. The samples were allowed to dry overnight before placement at the crime scene. Samples were processed at the scene using the Maxwell^® 16 Instrument and DNA IQ™ Casework Sample Kit for DNA extraction, and a microchip analyzer to perform amplification and STR analysis. The 9-plex autosomal STR typing system used in the microchip system included primer sequences from the PowerPlex^® 16 System (D3S1358, THO1, D21S11, D5S818, D13S317, D7S820, vWA and D8S1179) and amelogenin for sex identification. DNA purification from suspect samples was completed in 2 hours, and subsequent STR analysis and generation of a "suspect" DNA profile took a further 3 hours. The entire process from sample collection to generation of a CODIS "hit" took 6 hours. (3922)

Notes: The authors of this study used the gene module map method to identify genotype-specific therapies for human cancers. They studied breast cancer cell lines that expressed the "mitochondria" and "wound signatures". These cell lines, which are associated with tumors that have poor prognosis, were also shown to have a "proteasome signature" and were sensitive to the proteasome inhibitor bortezomib. The authors used the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay to show that genotype-specific killing of "wound signature" cells was not due to differential degrees of proteasome inhibition, since 1 and 1µmol/L of bortezomib completely inhibited the ability of the wound signature and control cells to cleave the luminogenic substrate. More likely, the wound signature genotype conferred increased susceptibility to death from proteasome inhibition. (3870)

Notes: The authors examined the role of sxy expression in hypercompetence of Haemophilus influenza. The 1.8kb sxy gene was subcloned into the pALTER®-1 Vector and point mutations made using the Altered Sites® II in vitro Mutagenesis System. The mutated gene was sequenced and subcloned back into the original plasmid. The wildtype and mutant sxy genes were transcribed, dephosphorylated and labeled with 32P ATP. The end-labeled RNAs were then subjected to S1 nuclease mapping. The E. coli S30 Extract System for Linear Templates was used with the wildtype and mutant sxy constructs and the expression levels of the expressed protein measured by spot blotting. (3995)

Notes: In this study, A549 human lung carcinoma cells and adenovirus-transformed human embryonic kidney cells (HEK293) were transfected using FuGENE® 6 Transfection Reagent. Cells were transfected with 1.2µg of DNA at a 3:1 FuGENE:DNA ratio. (4364)

Notes: The authors determined the role of various domains of the hepatocyte growth factor activator inhibitor type 1 (HAI-1) in the inhibition of the protease matriptase. HAI-1 mutants lacking one or more domains were expressed as His-tagged fusion proteins in CHO-K1 or COS-1 cells, and proteins were purified using the HisLink™ Protein Purification Resin. Purified proteins were then used in protease assays with recombinant matriptase. (3788)

Notes: The authors developed an in vitro selection system that is based on ribosome display and favors identification of 5´-untranslated regions (UTRs) with high translation efficiencies. A 5´-UTR random library was created in which the 5´-UTRs were upstream of a polyhistidine-tag/Renilla luciferase-coding region. In vitro transcripts from this library were translated in vitro using the Flexi^® Rabbit Reticulocyte Lysate System. The authors preferentially selected mRNAs with high translational efficiencies by shortening the translation time and capturing ternary complexes of mRNA, ribosome and nascent proteins. These complexes were captured using MagneHis™ Ni Particles. RNA was extracted from these complexes and used as a template in RT-PCR for the next round of selection. Before and after each round of selection, 9µl of RNA was translated in vitro, and 20µl of translated product was removed every 5 minutes to measure Renilla luciferase activity and monitor translation efficiency. Renilla luciferase was measured using the Renilla Luciferase Assay System. After two rounds of selection, RT-PCR products were cloned into a pUC18 vector, the sequences of the resulting plasmids were confirmed, and 0.5µg of plasmid was translated in vitro using the TNT^® T7 Coupled Rabbit Reticulocyte Lysate System to further evaluate translation efficiency. (3963)

Notes: The authors show that SepN, a selenoprotein of unknown function, and ryanodine receptor (RyR) intracellular calcium release channel are both required for normal muscle development in zebrafish. Furthermore SepN and RyR interact, and SepN is required for full activity of the RyR channel. As part of their study, the authors expressed SepN as a polyhistidine-tagged (8X His) protein in TNT^® Coupled Reticulocyte Lysate System and purified it using the MagZ™ Protein Purification System. The TNT^® reaction was modified to optimize selenocysteine incorporation efficiency; the reaction contained 80% rabbit reticulocyte lysate (RRL), 1mM methionine, 0.4mM spermidine, 0.01µg/ml SepN-8X-His DNA and 300nM of the C-terminus of Secis Binding Protein 2 , which is required for efficient incorporation of selenocysteine in RRL-based reactions. (3898)