A switch-on near-infrared fluorescence-ready probe for Cu(I): live cell imaging

<div><p>A reaction-based strategy exploiting metal ion mediated oxidative C–O bond cleavage affords selective ‘switch-on’ near-infrared (NIR) emitting cyanine probe for Cu⁺ in aqueous media and live cells. Near-infrared fluorescence-ready probe <b>TPACy</b> readily reacts with Cu⁺ to release the quinone embedded heptamethine cyanine (<b>Cy-quinone</b>) with extended π-electron conjugation responsible for the switch-on NIR fluorescence in aqueous buffer solution (50 mM HEPES, pH 7.2). This probe can be conveniently used for monitoring Cu⁺ without the interference from pH dependent effects of physiological media. Utility of the probe has been demonstrated by its application in the detection of unbound copper species (Cu⁺) in live cells.</p></div

Oligo(<i>p</i>‑phenyleneethynylene)-Derived Porous Luminescent Nanoscale Coordination Polymer of Gd^III: Bimodal Imaging and Nitroaromatic Sensing

Self-assembled highly luminescent nanoscale coordination polymer of {[Gd­(OPE)­(NO₃)­(H₂O)₂]·H₂O} (<b>NCP-1</b>), (oligo-(<i>p</i>-phenyleneethynylene)­dicarboxylate) was synthesized by coordination-driven self-assembly of oligo-(<i>p</i>-phenyleneethynylene)­dicarboxylic acid and Gd^III in polar solvent under refluxing conditions. This nanostructure has been characterized by FESEM, TEM, powder X-ray diffraction, and adsorption study. Interdigitation between 1D coordination polymers through alkyl chains results in a porous supramolecular 3D extended structure. <b>NCP-1</b> shows permanent microporosity as revealed by type-I CO₂ uptake profile. FESEM and TEM studies of <b>NCP-1</b> reveal nanorod-like morphology with square-type cross section having dimensions of 50–100 nm diameter and 0.5–0.8 μm length. High-magnification TEM images show long-range structural ordering present in <b>NCP-1</b> with uniform dark lines having an interspacing distance of 0.9–1.1 nm. Physiological stability and strong luminescence features of <b>NCP-1</b> have been exploited for bioimaging based on internalization into mammalian cultured cell lines HEK 293T and H1299. Magnetic resonance imaging studies suggest that <b>NCP-1</b> could act as a potential negative (T2) contrast agent. Furthermore, this porous luminescent <b>NCP-1</b> shows efficient nitroaromatic sensing as realized by the fluorescence quenching in solution as well as in vapor phase of the analyte like 2,4-dinitrotoluene (2,4-DNT). These results demonstrate that hybridization of a paramagnetic metal center and luminescent linker in a nanoscale porous coordination polymer culminates in a functional hybrid material with potential bimodal imaging and sensing applications

The 12^th base contacts R41 in p65.

<p>The 12^th base in the DNA equivalent to the position of rs28416813 in the IL28B promoter contacts R41 of p65 in the co-crystal structures (PDB ID 2O61). The figure generated by UCSF CHIMERA (available on the World Wide Web) shows that arginine 41 (red) of the p65 protein (in cyan, whereas p50 is in yellow) is in close contact (<3 angstroms) distance from the nucleotide at position equivalent (12^th position) to the SNP rs28416813 (magenta). The DNA strands are colored green and orange. A consensus oligonucleotide is used in the co-crystal structures.</p

The non-protective C allele at rs28416813-rev reduces transcription of reporter gene likely by decreasing NF-κB binding at the non-consensus site.

<p>A) A schematic of the ΔNF construct made with the p1.4IL28B construct with G allele at rs28416813-rev. TSS-transcription start site. The grey rectangle represents the non-consensus NF-κB-binding site between +115 to +124. The vertical line next to the grey rectangle denotes the SNP rs28416813-rev. The dashed line denotes the deletion made between +115 and +163 to generate ΔNF construct. The sequences of the other constructs used are shown in the right with the non-cosensus NF-κB site underlined and the SNP rs28416813-rev in italics. B) A representative plot (of at least two independent experiments, error bars show SD of triplicates) of luciferase activities of p1.4IL28B constructs with G or C alleles at rs28416813-rev and with different mutations introduced in to the non-consensus NF-κB site at +115 position to the plasmid with G allele background.</p

HCV RdRp can stimulate transcription from <i>IL28B</i> promoter.

<p>The gene encoding genotype 2a HCV RdRp (NS5B) was co-transfected at 50 ng/well along with the respective p1.4IL28B constructs at 10 ng/well and either RIG-I(WT) (A) RIG-I(K861E) (B) or MDA5 (C) at 0.5 ng/well <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075495#pone.0075495-RanjithKumar1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075495#pone.0075495-SubbaReddy1" target="_blank">[31]</a> along with pRLTK and luciferase assays were carried out after 36 hr. The experiment was repeated independently at least two times with similar results and the error bars show SD of triplicates from one experiment.</p

The alleles at rs28416813-rev and rs71356849-rev affect NFκB binding in EMSA.

<p>A) The distal region nucleotide sequence of the <i>IL28B</i> promoter is shown along with the two SNP positions (italics, underlined) and the non-consensus NFκB-binding site (underlined) identified by Osterlund et al. (2007) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075495#pone.0075495-Osterlund1" target="_blank">[21]</a>. The start codon is underlined and is in italics. The numbering is with respect to TSS. Note that the naming is on the opposite DNA strand to that used for naming SNPs in dbSNP, hence the SNPs have a “rev” notation. B) The oligonucleotides used for EMSA and pull-down experiment. The consensus NF-κB binding sequence is underlined in the competitor oligonucleotide. C) Autoradiograph of EMSA carried out from nuclear extracts prepared from HeLa cells after stimulation for NF-κB signaling and overexpression. Increasing concentrations of the extracts (0, 2, 4 and 6 µl respectively in lanes 1–4 and 5–8) were used at a constant concentration of the probe. The arrow indicates a likely NF-κB dimer. The fold-change in binding at the band positions (arrow) for the two probes G-T and C-C are shown below each lane. D) EMSA with recombinant p50. Arrow indicates the shifted probe position due to binding with p50. C-oligo- competitor oligo. E) Pull-down assay and western blot. The streptavidin-bound biotin labeled probes were incubated with nuclear extracts from HEK293T cells overexpressed with activated NF-κB. The bound proteins were probed with antibodies against p65 (Abcam).</p

The distal fragment of the <i>IL28B</i> promoter is responsive to NF-κB.

<p>A) A schematic of the 1.4 kb fragment of the <i>IL28B</i> promoter fragment cloned in to pGL3basic vector. The rectangles show the different transcription factor binding sites identified by Osterlund et al. (2007) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075495#pone.0075495-Osterlund1" target="_blank">[21]</a>. The vertical lines next to the non-consensus NF-κB site indicate the position of two SNPs rs28416813 and rs71356849 respectively. ISRE, Interferon-stimulated response element, PRDI, positive-regulatory domain I. The TSS is shown by the arrow before the non-consensus NF-κB-binding site (at position +115 with respect to TSS). B) Luciferase assays carried out with HEK293T cells in 6-well plates. The transfection of the p1.4IL28BC-T construct was done either in presence or absence of p50+p65 and activity compared with the empty pGL3basic vector. The error bars show SD from triplicates.</p

TNF-α and 5′ppp-dsRNA stimulate transcription from <i>IL28B</i> promoter.

<p>A) p1.4IL28B constructs (10 ng/well) with either G or C alleles at rs28416813-rev along with pRLTK were transfected in to 96-well plates and after 24 hr recombinant human TNF-α was added in to the media at indicated concentrations and plates were incubated for a further 8 hr before subjecting to luciferase assays. The experiment was done at least twice with similar results and the error bars show SD from triplicates. B) and C) p1.4IL28B constructs (10 ng/well) with either G or C alleles at rs28416813-rev along with pRLTK and either pUNO (B) or pUNO-RIG-I (C) were transfected in to 96-well plates and after 24 hr a 19-mer 5′ppp-dsRNA was transfected at 250 nM concentration. Luciferase assays were done after 12 hr. The error bars show SD of 6 replicates from two experiments.</p

The C allele at rs28416813-rev in <i>IL28B</i> promoter decreases luciferase expression.

<p>A) Luciferase activity with p1.4IL28B constructs shows that the C allele at rs28416813-rev when present along with the T allele at rs71356849-rev decreases reporter gene expression. The ratio of firefly to <i>Renilla</i> luciferase values for the other three constructs was normalized to that of p1.4IL28BG-T construct as % activity and plotted. The data is representative of at least two independent experiments with the error bars showing SD of triplicates. B) A representative chromatogram from a small cohort of HCV infected patients (n = 25) who were sequenced at the two SNP positions (shown in bold) and only rs28416813-rev was dimorphic (C/G) with a minor allele frequency (MAF) of 0.3 whereas none of the samples showed any variation at rs71356849-rev (T). C) The C allele at rs28416813-rev consistently shows a reduction in luciferase activity by 20–30%. Results show the mean (76.5) of four independent experiments where samples were processed in triplicate and were controlled for transfection efficiency and were normalized to the G allele. Error bars show SE.</p

rs28416813 is in LD with rs12979860.

<p>A) EtBr-stained gel of the PCR products that were amplified from the <i>IL28A</i> and <i>IL28B</i> promoters that included the SNP rs28416813 by the same set of primers because of high homology between the two genes at their 5′ ends <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075495#pone.0075495-Smith1" target="_blank">[35]</a>. The ∼2 kb fragment specific to <i>IL28B</i> was excised from gels purified and subject to DNA sequencing to genotype the SNP rs28416813. 1 and 2 are samples from two different patients. Ma-DNA Mol. Wt. marker. B) Pairwise LD plots for the three SNPs genotyped from a cohort of patients with chronic HCV infections in India (n = 20) (top) and LD plots for the SNPs from genotype data of a CEU population available from the 1000 genomes project database (bottom). The plots were generated using LDHeatmap <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075495#pone.0075495-Shin1" target="_blank">[20]</a>.</p