Notes: The authors of this study investigated the energetic effects of direct fed microbials on 1-day-old broiler chicks. They looked at body weight, feed consumption, whole-body energy expenditure, organ mass, tissue respiration rates and peripheral blood mononuclear cell (PBMC) ATP levels to explore effects on energy metabolism in control-diet chicks versus chicks receiving direct fed microbials. An ATP assay was used because previous attempts to monitor O₂ use by PMBCs were inconclusive. PBMC ATP levels were measured using the CellTiter-Glo™ Luminescent Cell Viability Assay in 96-well plates using 10 × 10⁵ cells/well. The authors also looked for differences in ATP depletion in control and experimental chick populations by using an ATP synthase inhibitor and a proton ionophore. In this study, the authors concluded that the PMBC isolated from the direct microbial fed animals consumed more ATP/cell than the controls. (4202)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Bacillus amyloliquefaciens. (4312)

Notes: These authors describe the engineering of an enzyme and substrate to create a novel highly efficient bioluminescence system. The paper introduces NanoLuc™ Luciferase, a small luciferase subunit (19kDa) from the deep-sea shrimp Oplophorus gracilirostris, which provides ∼2.5 millionfold improved luminescence activity in mammalian cells by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The glow-type luminescence (signal half-life >2 hours) produced by the new luciferase has a specific activity ∼150-fold greater than either firefly or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc™ Luciferase shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55°C or in culture medium for >15 hours at 37°C.
The authors discuss utility of NanoLuc™ Luciferase as a genetic reporter configured for high sensitivity or for response dynamics through addition of a degradation sequence to reduce intracellular accumulation. Data shows the effect of adding a signal sequence to allow export of NanoLuc™ Luciferase to the culture medium, allowing measurement of enzyme activity without cell lysis. (4295)

Notes: The authors examined cellular adaptation to increased salinity in Broussonetia papyrifera by measuring protein and mRNA levels of vacuolar H⁺-ATPase (V-H⁺-ATPase) subunits and the activities of V-H⁺-ATPase and vacuolar H⁺-pyrophosphatase. Relative expression levels of V-H⁺-ATPase subunits A, B, E and c in salt-stressed and control plants were determined by RT-PCR using actin as a normalization control gene. cDNA was synthesized using the GoScript™ Reverse Transcription System as described by the manufacturer’s protocol, then amplified by PCR for 25 cycles. The amplification products were analyzed and quantified by agarose gel electrophoresis and ethidium bromide staining. (4258)

Notes: The authors used microarray analysis, bisulfite sequencing and pyrosequencing to examine a possible link between aberrant DNA methylation and abnormal human spermatogenesis and male infertility. They identified almost 600 genes that were differentially methylated in testis tissue of men with secretory male infertility. Genomic DNA used in the microarray analysis was extracted from testicular biopsies using the Wizard® Genomic DNA Purification Kit. For the bisulfite sequencing experiments, genomic DNA was bisulfite-modified, amplified, cloned using the pGEM®-T Easy Vector System, then sequenced. (4257)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Escherichia coli. (4332)

Notes: In this study, MCF7 human breast adenocarcinoma cells were transfected with plasmid constructs using FuGENE^® 6 transfection reagent. Cells were grown to 50% confluence and transfected with 2µg of DNA at a 3:2 ratio of FuGENE^® 6 reagent:DNA. (4358)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Bacteroides stercoris. (4313)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Acinetobacter baumannii. (4296)

Notes: To better understand what roles FAM123A may play in signaling, cell behavior and human disease, HT1080 sarcoma cells were plated on MatTek dishes coated with 5mg/ml fibronectin before transfection with Venus (a yellow fluorescent protein)-tagged FAM123A or Venus-WTX, another member of the FAM123 gene family, using FuGENE® HD Transfection Reagent. The cells were imaged the next day for low-resolution analysis. For a higher magnification, dynamic analysis, HT1080 cells were plated onto Delta T dishes coated with 5mg/ml fibronectin and transfected with EGFP-FAM123A and FuGENE® HD Transfection Reagent. The next day, cell images were captured every 10 seconds. Examining the effect of silencing FAM123A, a reporter assay used the pGL4.34[luc2P/SRFRE/Hygro] Vector cotransfected with a CMV-Renilla coreporter and other effector plasmids or siRNA, and luciferase levels assessed using the Dual-Glo® Luciferase Assay System. (4246)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Flavobacterium psychrophilum. (4334)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Clostridium tunisiense TJ. (4291)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Dickeya zeae. (4327)

Notes: The authors performed ChIP and quantitated immunoprecipitated DNA with the QuantiFluor® dsDNA System prior to NGS on an Illumina Instrument. (4918)

Notes: The authors studied the effect of butyrate treatment on histone acetylation and characterized how H3 acetylation affects DNA sequence binding specificity. To examine sequence specificity, they performed chromatin immunoprecipitation with butyrate-treated and untreated bovine kidney epithelial cells, quantified the recovered DNA, then sequenced the DNA by Illumina next-generation sequencing. DNA was quantitifed using the QuantiFluor® dsDNA System. (4240)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Halanaerobacter jeridensis. (4337)

Notes: This paper provides an overview of the many applications of HaloTag® Technology. The authors describe the development of the technology, focusing on it's multifunctional utility for protein imaging, protein isolation and display, and in the study of protein complexes and interactions. They also discuss it's potential to facilitate proteomics research studies across complex biological systems at the biochemical, cell-based and whole animal level. (4325)

Notes: GoTaq® Green Master Mix was used in the PCR amplification of genomic DNA template for pyrosequencing. (4541)

Notes: The authors transiently transfected 2 × 10⁴ HEK293 cells per well of a 96-well plate using the FuGene® HD Transfection Reagent, 0.1µg of DNA and a reagent:DNA ratio of 3:1. (4430)

Notes: Caspase-Glo® 3/7 and Caspase-Glo® 8 Assays were used to assess activation of apoptosis pathways in MC3T3-E1 cells (clonal mouse), U-2 OS human osteoscarcoma cell line and CCL-51 cells (mouse mammary gland tumor cells). Although ibandronate reduced cell proliferation in all cell lines, its effect on activation of caspases was different in neoplastic versus non-neoplastic cells. Caspase-8 and caspase-3/7 activities were reduced in MC3T3-E1 cells after 72 hours treatment with ibandronate. In two tumor cell lines assayed, opposite results were seen: caspase-8 and caspase-3/7 activities increased in U-2 OS cells and in CCL-51 cells. Luminescence was detected using a GloMax® 96 Microplate Luminometer. (Figure 1 in the paper)

To analyze FAS promoter methylation levels, fragments of the targeted promoter regions were generated by digestion of genomic DNA using CpG methylation insensitive restriction enzymes MboII and PstI from cells cultured in the presence of ibandronate for varying lengths of time. The authors demonstrated that FAS promoter methylation is altered in tumor cells in the presence of ibandronate. (4253)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Campylobacter coli. (4316)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Bartonella. (4314)

Notes: Specific neuronal connectivity is thought to be based on the expression of the protocadherins (Pcdh)--a family of membrane adhesion proteins. Each neuron expresses only a specific subset of the Pcdh genes. The authors of this paper used a bioinformatics approach to identify conserved, gene-specific regions upstream of the Pcdh genes. They showed that this specific sequence element (SSE) is involved in transcription regulation together with a conserved sequence element (CSE), and identified a potential interacting protein partner. To demonstrate promoter activity, the SSE-CSE region was cloned upstream of the luciferase gene in the pGL3 Basic Vector and its effect on luciferase expression was evaluated. The authors then isolated protein complexes that bound the SSE-CSE region and characterized the interacting proteins by mass spectrometry. The CCTC binding-factor (CTCF) was identified as a key molecule that binds and activates Pcdh promoters. As part of the study, human CTCF  and the CTCF-binding domain were expressed in the TNT® T7 Quick Coupled Transcription/Translation System. The in vitro expressed proteins were fluorescently labeled using the FluoroTect™ Green_Lys System and were used in EMSA to confirm interaction with the SSE-CSE region. (4249)

Notes: The authors used the Roche 454 sequencing platform to perform a differential transcriptome analysis and identify genes that are differentially expressed in human buccal cancer and normal tissue. The quantity of first-strand and second-strand cDNA products was estimated using the QuantiFluor™-ST Fluorometer as well as the High Sensitivity DNA Chip kit and Agilent Bioanalyzer 2100. (4238)

Notes: Oropouche fever is a arboviral infection in Brazil, surpassed in frequency only by dengue. Oropouche virus (OROV) causes large outbreaks of acute febrile illness in areas along the Amazon and Central-Plateau regions. RNA was extracted from CSF and underwent reverse transcription-polymerase chain reaction and sequencing to identify OROV. Reverse transcription was performed with 5ml of the random primers, using the AccessQuick™ RT-PCR System. (4320)