Highly Sensitive and Selective Rhodamine-Based “Off–On” Reversible Chemosensor for Tin (Sn⁴⁺) and Imaging in Living Cells

A structurally characterized new oxo-chromene functionalized rhodamine derivative <b>L1</b> exhibits high selectivity toward Sn⁴⁺ by forming a 1:1 complex, among other biologically important metal ions, as studied by fluorescence, absorption, and HRMS spectroscopy. Complexing with Sn⁴⁺ triggers the formation of a highly fluorescent ring-open form which is pink in color. The sensor shows extremely high fluorescence enhancement upon complexation with Sn⁴⁺, and it can be used as a “naked-eye” sensor. DFT computational studies carried out in mimicking the formation of a 1:1 complex between <b>L1</b> and Sn⁴⁺ resulted in a nearly planar pentacoordinate Sn­(IV) complex. Studies reveal that the <i>in situ</i> prepared <b>L1</b>–Sn complex is selectively and fully reversible in presence of sulfide anions. Further, confocal microscopic studies confirmed that the receptor shows <i>in vitro</i> detection of Sn⁴⁺ ions in RAW cells

Transcriptional activity of MCMV.

<p>(A) Whole genome visualization using IGV viewer of RNA-Seq reads mapping to the MCMV genome showing different data ranges. Row 1, range of 20–50,000 reads; Row 2, range of 20–5000 reads; Row 3, range 20–500 reads; Row 4, annotation from NC_004065. (B to D) Quantitation of transcript abundance varies with annotation. The most expressed MCMV genes (RPKM>10 000) relative to NCBI NC_004065 and (B) and GU305914.1 (C). (D) Percentage of reads mapping to coding (exon) or intergenic regions using NC_004065.1 (NC) or GU305914.1 (GU). (E) Example of a transcriptionally active region between M85 and M87.</p

Top 20 host genes¹ downregulated in infection.

1<p>p<0.05 identified using SAMMate with EdgeR.</p><p>Genes associated with genetic networks identified by IPA are shown in bold.</p>2<p>antisense transcripts.</p>3<p>recently withdrawn from Mouse Genome Informatics (MGI) database.</p>4<p>uncharacterized RNA.</p>5<p>lincRNA.</p>6<p>microRNA record discontinued.</p

Dual Analysis of the Murine Cytomegalovirus and Host Cell Transcriptomes Reveal New Aspects of the Virus-Host Cell Interface

<div><p>Major gaps in our knowledge of pathogen genes and how these gene products interact with host gene products to cause disease represent a major obstacle to progress in vaccine and antiviral drug development for the herpesviruses. To begin to bridge these gaps, we conducted a dual analysis of Murine Cytomegalovirus (MCMV) and host cell transcriptomes during lytic infection. We analyzed the MCMV transcriptome during lytic infection using both classical cDNA cloning and sequencing of viral transcripts and next generation sequencing of transcripts (RNA-Seq). We also investigated the host transcriptome using RNA-Seq combined with differential gene expression analysis, biological pathway analysis, and gene ontology analysis.</p><p>We identify numerous novel spliced and unspliced transcripts of MCMV. Unexpectedly, the most abundantly transcribed viral genes are of unknown function. We found that the most abundant viral transcript, recently identified as a noncoding RNA regulating cellular microRNAs, also codes for a novel protein. To our knowledge, this is the first viral transcript that functions both as a noncoding RNA and an mRNA. We also report that lytic infection elicits a profound cellular response in fibroblasts. Highly upregulated and induced host genes included those involved in inflammation and immunity, but also many unexpected transcription factors and host genes related to development and differentiation. Many top downregulated and repressed genes are associated with functions whose roles in infection are obscure, including host long intergenic noncoding RNAs, antisense RNAs or small nucleolar RNAs. Correspondingly, many differentially expressed genes cluster in biological pathways that may shed new light on cytomegalovirus pathogenesis. Together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus-associated diseases.</p></div

Analysis of the novel most abundant MCMV transcript and protein.

<p>(A) Comparison of RNA-Seq data and the longest MAT cDNA clone (E125) with current annotation (GU305914). The predicted exons are shown in white boxes. (B) Predicted amino acid sequence of the MAT protein. The first 127 residues match a truncated m169 translation and the C-terminal 20 residues highlighted in gray are derived from exon 2, mapping to the m168 gene. (C) Northern analysis of MAT RNA in MEF cells infected with various deletion mutants. Note that the single gene mutants are partial gene deletions and thus truncated transcripts accumulate. (D) Immunoblot analysis of MEF cell lysates probed with monoclonal antibody generated to the predicted m169 ORF or monoclonal antibody to actin (45 kDa band). (E) Immunoblot analysis of the time course of MAT protein accumulation in infected cells and (F) quantitation. (G) Immunoblot analysis of MAT protein from cells exposed to wild virus isolates. (H) MAT protein accumulation in WT and m168mut virus infected Balb/c MEF. Mutation of the binding site for miR27-b in MAT 3′UTR did not alter regulation of MAT protein expression.</p

Comparison of cDNA cloning and RNA-Seq data in relation to current genome annotation.

<p>Comparison of poly(A) cDNA library (green arrows) and RNA-Seq analysis of murine cytomegalovirus (gray histograms). The longest clone from each group of clones in the cDNA library is shown. ELAND alignments of RNA-Seq reads were loaded in Integrative Genomics Viewer and compared to NC_004065.1, (red arrows) and GU305914.1 (blue arrows). The data range for RNA-Seq data was set to 20–5000. Data is shown in 30 kb ranges with 1 kb overlap. Data is shown for the first 120 kb of the MCMV genome and the figure legend is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003611#ppat-1003611-g002" target="_blank">Figure 2</a>.</p

Gene enrichment analysis of differentially regulated mouse genes in MCMV infection.

<p>Differentially expressed genes were identified by SAMMate and analyzed with IPA Core Analysis with fold change ratio cutoff of 2. Shown are top diseases and disorders, molecular and cellular functions, and physiological system development and functions (A) and top canonical pathways (B) of DE genes.</p

Verification of new transcripts by northern blot.

<p>Balb/c MEF cells were infected with BAC derived Smith virus and harvested at indicated times post infection. Total RNA was separated by denaturing gel electrophoresis, transferred to nylon membrane and incubated with probes specific for S and AS transcripts. RNA integrity and loading was evaluated by inspecting 28S (not shown) and 18S rRNA bands under UV light after transfer to membrane. Transcripts in the <i>m15–16</i> (A), <i>m19-m20</i> (B), <i>M116</i> (C) and <i>M71-m74</i> (D) gene regions were analyzed (Due to smiling effects during gel electrophoresis for the image shown in 4A and C, the ladder was not accurate for inner lanes of the gel and the position of the ribosomal bands was therefore used to estimate the band sizes). Predicted genes (Rawlinson's annotation) are depicted as empty arrows, while thin black arrows show longest transcripts cloned in our cDNA library as well as clones used to generate probes (marked with *). 3′ ends of transcripts are marked with arrowheads. The nucleotide coordinates relative to Smith sequence (NC_004065.1) of isolated transcripts are given below thin arrows, while the names of the clones are written above. Thin gray lines show isolated transcripts that cannot be detected with the probe. Gray histograms showRNA-Seqreads aligned to MCMV genome. Maximal possible exposure times were used to ensure even low abundance transcripts are detected and are noted on the blots.</p

Top 20 host genes¹ upregulated in infection.

1<p>p<0.05 identified using SAMMate with EdgeR.</p><p>Genes associated with genetic networks identified by IPA are shown in bold.</p>2<p>Overlaps CXCL10 and CXCL11 so its upregulation may be due to this overlap.</p

Comparison of cDNA cloning and RNA-Seq data in relation to current genome annotation.

<p>Comparison of poly(A) cDNA library (green arrows) and RNA-Seq analysis of murine cytomegalovirus (gray histograms). The longest clone from each group of clones in the cDNA library is shown. ELAND alignments of RNA-Seq reads were loaded in Integrative Genomics Viewer and compared to NC_004065.1, (red arrows) and GU305914.1 (blue arrows). The data range for RNA-Seq data was set to 20–5000. Data is shown in 30 kb ranges with 1 kb overlap. Data is shown for genomic region spanning 119–230 kB of the MCMV genome.</p