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    Binding of nuclear factor kappa B to noncanonical consensus sites reveals its multimodal role during the early inflammatory response
    (2016) Kolovos, Petros; Georgomanolis, Theodore; Koeferle, Anna; Larkin, Joshua D.; Brant, Lilija; Nikolic, Milos; Gusmao, Eduardo G.; Zirkel, Anne; Knoch, Tobias A.; van Ijcken, Wilfred F.; Cook, Peter R.; Costa, Ivan G.; Grosveld, Frank G.; Papantonis, Argyris
    Mammalian cells have developed intricate mechanisms to interpret, integrate, and respond to extracellular stimuli. For example, tumor necrosis factor (TNF) rapidly activates proinflammatory genes, but our understanding of how this occurs against the ongoing transcriptional program of the cell is far from complete. Here, we monitor the early phase of this cascade at high spatiotemporal resolution in TNF-stimulated human endothelial cells. NF-kappa B, the transcription factor complex driving the response, interferes with the regulatory machinery by binding active enhancers already in interaction with gene promoters. Notably, >50% of these enhancers do not encode canonical NF-kappa B binding motifs. Using a combination of genomics tools, we find that binding site selection plays a key role in NF-kappa B-mediated transcriptional activation and repression. We demonstrate the latter by describing the synergy between NF-kappa B and the corepressor JDP2. Finally, detailed analysis of a 2.8-Mbp locus using sub-kbp-resolution targeted chromatin conformation capture and genome editing uncovers how NF-kappa B that has just entered the nucleus exploits pre-existing chromatin looping to exert its multimodal role. This work highlights the involvement of topology in cis-regulatory element function during acute transcriptional responses, where primary DNA sequence and its higher-order structure constitute a regulatory context leading to either gene activation or repression.
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    Super-resolution measurement of distance between transcription sites using RNA FISH with intronic probes
    (2016) Larkin, Joshua D.; Cook, Peter R.
    Nascent transcripts being copied from specific human genes can be detected using RNA FISH (fluorescence in situ hybridization) with intronic probes, and the distance between two different nascent transcripts is often measured when studying structure-function relationships. Such distance measurements are limited by the resolution of the light microscope. Here we describe methods for measuring these distances in cultured cells with a precision of a few tens of nanometers, using equipment found in most laboratories (i.e., a wide-field fluorescence microscope equipped with a charged-coupled-device camera). Using images of pairs of transcripts that are often co-transcribed, we discuss how selection of cell type, design of FISH probes, image acquisition, and image processing affect the precision that can be achieved. (C) 2015 The Authors. Published by Elsevier Inc.
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    Preparation of photoluminescence tunable Cu-doped AgInS2 and AgInS2/ZnS nanocrystals and their application as cellular imaging probes
    (2016) Chen, Siqi; Demillo, Violeta G.; Lu, Minggen; Zhu, Xiaoshan
    In this work, high-quality Cu doped AIS and AIS/ZnS NCs have been first synthesized via a surface doping approach. By varying the Cu doping concentrations, Cu doped AIS NCs exhibit a photoluminescence red-shift from around 600 nm to 660 nm with a decrease of quantum yield from around 30% to 20%. After ZnS coating or zinc etching on the Cu doped AIS NCs, Cu doped AIS/ZnS NCs present photoluminescence peaks from around 570 nm to 610 nm and high quantum yields in the range of 50-60%. Moreover, it is found that Cu doping can prolong the photoluminescence lifetime of NCs, and the average photoluminescence lifetime of Cu doped AIS and AIS/ZnS NCs is in the range of 300-500 ns. The resultant Cu doped AIS/ZnS NCs were further encapsulated with amphiphilic polymers and used as biocompatible photoluminescence probes in cellular imaging. The cellular imaging study shows that peptide-conjugated probes can specifically target U-87 brain tumor cells and thus they can be applied to the detection of endogenous targets expressed on brain tumor cells.
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    BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models
    (2016) Bilgin, Cemal C.; Fonteney, Gerald; Cheng, Qingsu; Chang, Hang; Han, Ju; Parvin, Bahram
    BioSig3D is a computational platform for high-content screening of three-dimensional (3D) cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i) morphogenesis of a panel of human mammary epithelial cell lines (HMEC), and (ii) heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation.
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    Quality control of subgrade soil using intelligent compaction
    (2016) Barman, Manik; Nazari, Moeen; Imran, Syed A.; Commuri, Sesh; Zaman, Musharraf; Beainy, Fares; Singh, Dharamveer
    Intelligent Compaction (IC) of subgrade soil has been proposed to continuously monitor the stiffness of subgrade during its compaction. Modern IC rollers are vibratory compactors equipped with (1) an onboard measuring system capable of estimating the stiffness of the pavement material being compacted, (2) Global Positioning System (GPS) sensor to precisely locate the roller, and (3) an integrated mapping and reporting system. Using IC, the roller operator is able to evaluate the entire subgrade and address deficiencies encountered during compaction. Continuous monitoring of quality during construction can help build better quality and long-lasting pavements. However, most of the commercially available IC rollers report stiffness in terms of Original Equipment Manufacturer (OEM) specified indicator, known as Intelligent Compaction Measurement Value (ICMV). Although useful, additional tests are required to establish the correlation between these ICMV values and the resilient modulus of subgrade (M-r). Since the mechanistic design of the pavement is performed using M-r, it is important to know if the design M-r is achieved on the entire subgrade during compaction. This paper presents a systematic procedure for monitoring the level of compaction of subgrade in real time using intelligent compaction (IC). Specifically, the Intelligent Compaction Analyzer (ICA) developed at the University of Oklahoma was used for estimating the modulus of the subgrade. Results from two demonstration studies show that the ICA is able to estimate subgrade modulus with an accuracy that is acceptable for quality control activities during the construction of pavements.