Simple D‑A‑D Structural Bisbithiophenyl Diketopyrrolopyrrole as Efficient Bioimaging and Photothermal Agents

Design and synthesis of biocompatible and multifunctional photothermal agents is crucial for effective cancer phototherapy. In order to achieve this ambition, simple D-A-D structural bisbithiophenyl diketopyrrolopyrrole (TDPP) was fabricated. In this molecule, the donor, 2-thiophenylboric acid, was conjugated via Suzuki coupling reaction, which could expand the emission wavelength to the red region of the spectrum. TDPP could self-assemble into stable and uniform nanoparticles (TDPP NPs) in assistant of amphiphilic Pluronic F-127 polymer. Exposing TDPP NPs (100 μg/mL) aqueous dispersion to 638 nm (0.61 W/cm²) laser irradiation resulted in a temperature elevation of approximately 30 °C within 5 min, which is high enough for inducing the cytotoxicity and tumor inhibition. Because of the bathochromic shift absorption of TDPP NPs in water, TDPP NPs could also act as a contrast agent for near-infrared fluorescence imaging (NIRF) to visualize the drug distribution in vivo. Coupled with the infrared thermal imaging properties of the photothermal agent, TDPP NPs were proven to be a multifunctional theranostic agent for dual-modal imaging-guided phototherapy

Comprehensive Identification of Long Non-coding RNAs in Purified Cell Types from the Brain Reveals Functional LncRNA in OPC Fate Determination

<div><p>Long non-coding RNAs (lncRNAs) (> 200 bp) play crucial roles in transcriptional regulation during numerous biological processes. However, it is challenging to comprehensively identify lncRNAs, because they are often expressed at low levels and with more cell-type specificity than are protein-coding genes. In the present study, we performed <i>ab initio</i> transcriptome reconstruction using eight purified cell populations from mouse cortex and detected more than 5000 lncRNAs. Predicting the functions of lncRNAs using cell-type specific data revealed their potential functional roles in Central Nervous System (CNS) development. We performed motif searches in ENCODE DNase I digital footprint data and Mouse ENCODE promoters to infer transcription factor (TF) occupancy. By integrating TF binding and cell-type specific transcriptomic data, we constructed a novel framework that is useful for systematically identifying lncRNAs that are potentially essential for brain cell fate determination. Based on this integrative analysis, we identified lncRNAs that are regulated during Oligodendrocyte Precursor Cell (OPC) differentiation from Neural Stem Cells (NSCs) and that are likely to be involved in oligodendrogenesis. The top candidate, <i>lnc-OPC</i>, shows highly specific expression in OPCs and remarkable sequence conservation among placental mammals. Interestingly, <i>lnc-OPC</i> is significantly up-regulated in glial progenitors from experimental autoimmune encephalomyelitis (EAE) mouse models compared to wild-type mice. OLIG2-binding sites in the upstream regulatory region of <i>lnc-OPC</i> were identified by ChIP (chromatin immunoprecipitation)-Sequencing and validated by luciferase assays. Loss-of-function experiments confirmed that <i>lnc-OPC</i> plays a functional role in OPC genesis. Overall, our results substantiated the role of lncRNA in OPC fate determination and provided an unprecedented data source for future functional investigations in CNS cell types. We present our datasets and analysis results <i>via</i> the interactive genome browser at our laboratory website that is freely accessible to the research community. This is the first lncRNA expression database of collective populations of glia, vascular cells, and neurons. We anticipate that these studies will advance the knowledge of this major class of non-coding genes and their potential roles in neurological development and diseases.</p></div

Overview of study diagram and evaluation of lncRNA expression.

<p>(A) Schematic of the data integration and experiment validation. (B) Box plots illustrate expression level distributions of lncRNAs detected in Astrocytes, Neurons, and OPC cells (Red: lncRNAs that are also detected in cortex tissue samples; Blue: lncRNAs that are not detected in cortex tissue samples. Any lncRNAs with expression level of FPKM > 5 were excluded to allow the plot to be presented at a suitable scale. (C) Venn diagram shows lncRNAs detected in purified cell samples (red) or tissue samples (blue). (D) Cell-type specificity of the expression patterns of lncRNAs. Shown are the distributions (represented as a density curve) of specificity scores calculated for each gene across cell types, for coding genes (blue) and lncRNAs (red). The specificity score was calculated with a previously proposed index, which varies from 0 for housekeeping genes to 1 for cell-type specific genes.</p

Loss-of-function experimental validation of <i>lnc-OPC</i> function in OPC formation.

<p>(A) Changes in <i>lnc-OPC</i> expression upon differentiation of OPCs from NSCs. <i>lnc-OPC</i> expression in NSCs without differentiation was set to 1. The expression of <i>lnc-OPC</i> increased upon differentiation of OPCs from NSCs. (B) Efficiency of knockdown of <i>lnc-OPC</i> expression by shRNAs in puromycin-selected NSCs, as evaluated by qPCR. Two shRNAs, sh-lnc-OPC2 and sh-lnc-OPC3, succeeded in knocking down <i>lnc-OPC</i> expression when compared to control (sh-Luc). <i>lnc-OPC</i> expression in control was set to 100%. (C) Expression of OPC markers in control (sh-Luc) and <i>lnc-OPC</i>-depleted cultures (two constructs were used: sh-lnc-OPC2 and sh-lnc-OPC3) upon differentiation of OPCs from NSCs. The expression of OPC markers in controls were set to 1. Knockdown of <i>lnc-OPC</i> reduced the expression of PLP, CNP, and MBP. For A-C, experiments were performed in triplicate and error bars indicate Standard Error. <i>t</i>-test analysis ** <i>p</i>< 0.01. (D) Immunostaining analysis of OPC formation in control (sh-Luc) and <i>lnc-OPC</i>-depleted cultures (two constructs were used: sh-lnc-OPC2 and sh-lnc-OPC3). Images were taken after differentiation for 5 days. Blue: DAPI, Red: O4. Scale bar, 100 μm. (E) Quantification of OPC differentiation from puromycin-selected NSCs was measured as the percentage of O4+ cells. Results are from three independent experiments and 10 randomly selected microscopy fields were counted each time. <i>t</i>-test analysis * <i>p</i>< 0.05.</p

OLIG2 binds to upstream regulatory region of <i>lnc-OPC</i> and controls its expression.

<p>(A) Read mapping signal tracks of <i>lnc-OPC</i>. The upstream regulatory regions of <i>lnc-OPC</i> cloned into luciferase reporter constructs are indicated by grey bars. OLIG2 ChIP-qPCR targeted regions are indicated by red bars. OLIG2-binding sites revealed by OLIG2 ChIP-Seq are indicated by black bars. OLIG2-binding sites predicted by motif search are indicated by green bars. The ENCODE-annotated promoter region is indicated by a purple bar. (B, C) Detection of OLIG2-binding sites in the upstream regulatory region of <i>lnc-OPC</i> by OLIG2 ChIP-qPCR. Two pairs of primers were used. Enrichment over genomic input DNA and fold changes over IgG control were calculated. Experiments were performed in triplicate and error bars indicate Standard Error. <i>t</i>-test analysis ** <i>p</i>< 0.01. (D) The effect of OLIG2 on luciferase expression is represented as changes in relative luciferase activity. The 293FT cells were cotransfected with the luciferase reporter plasmids containing different lengths of the <i>lnc-OPC</i> regulatory region, along with an OLIG2- or GFP-expressing construct. Empty pGL4.11 vector was used as a control. The luciferase activity of cells cotransfected with GFP and luciferase reporter plasmids were used as controls and set to 1. Luciferase activity for each sample was normalized to GFP-transfected controls. Experiments were performed in triplicate and error bars indicate Standard Error. <i>t</i>-test analysis * <i>p</i>< 0.05.</p

Comparative genomics analysis of <i>lnc-OPC</i> revealed its evolutionary history.

<p>(A) Signal tracks of the expression of two TEs at the <i>lnc-OPC</i> locus in ESCs. RepeatMask annotation (bottom) shows two MERVL located in the last intron of <i>lnc-OPC</i>. The height of the bar above the repeat elements corresponds to (1 –%divergence). (B) The structure of <i>lnc-OPC</i> is shown. (C) Conservation displayed as Phylip score. (D) Pairwise alignments of mouse-rat and mouse-human <i>lnc</i>-OPC. (E) Multiple alignments of conservation of <i>lnc-OPC</i> in 60 vertebrates shown in the UCSC browser.</p

Predicting putative functions of lncRNAs.

<p>(A) Shown is a heatmap representing an association matrix of lncRNAs and functional terms. Columns represent lnc-OPC and lncRNAs that are known to be expressed in brain and have known functions in the literature. Rows represent selected gene ontology terms and MsigDB gene sets. Color depth represents NES (normalized enrichment score) calculated by GSEA, indicating the strength of association. (B) RNA-Seq signal tracks for lncRNA <i>Tunar</i> across cell types are shown. All tracks are set to the same scale for easy comparison of expression levels. (C) GO enrichment analysis of co-expression modules identified by WGCNA. Only the most significant GO Biological Process terms are displayed for modules with > 100 members.</p

Developmental regulation of lncRNA expression by TFs.

<p>(A) (Left) The distribution of <i>cis</i>-regulatory module size in promoter regions (2 kb upstream and 1 kb downstream of TSS) of TF genes, lncRNAs, coding non-TF genes, and random intergenic regions. (Right) The distribution is illustrated with box plots. (B) (Left) Representative examples of dynamic DHSs enriched in ESC and three other available ENCODE datasets related to CNS development. (Right) Sequence motifs associated with dynamic DHSs enriched in each dataset. Top enriched representative TF motifs from <i>de novo</i> motif analysis are shown. The proportion of <i>cis</i>-regulatory sequences within dynamic DHS regions containing at least one instance of each motif is shown to the right of the motif, which is significantly enriched compared to the expected random background frequency of the motif (shown in parentheses).</p

Global identification of lncRNAs regulated during oligodendrogenesis.

<p>(A) Heatmap depicting protein-coding genes and lncRNAs that are significantly differentially expressed (Fold change > 2 and FDR < 0.05) between NSCs and OPCs. Two biological replicates are represented in the plot. (B) Example RNA-Seq tracks for three lncRNAs that are enriched in the OPC lineage.</p