Dextran-Mimetic Quantum Dots for Multimodal Macrophage Imaging In Vivo, Ex Vivo, and In Situ.

Macrophages are white blood cells with diverse functions contributing to a healthy immune response as well as the pathogenesis of cancer, osteoarthritis, atherosclerosis, and obesity. Due to their pleiotropic and dynamic nature, tools for imaging and tracking these cells at scales spanning the whole body down to microns could help to understand their role in disease states. Here we report fluorescent and radioisotopic quantum dots (QDs) for multimodal imaging of macrophage cells in vivo, ex vivo, and in situ. Macrophage specificity is imparted by click-conjugation to dextran, a biocompatible polysaccharide that natively targets these cell types. The emission spectral band of the crystalline semiconductor core was tuned to the near-infrared for optical imaging deep in tissue, and probes were covalently conjugated to radioactive iodine for nuclear imaging. The performance of these probes was compared with all-organic dextran probe analogues in terms of their capacity to target macrophages in visceral adipose tissue using in vivo positron emission tomography/computed tomography (PET/CT) imaging, in vivo fluorescence imaging, ex vivo fluorescence, post-mortem isotopic analyses, and optical microscopy. All probe classes exhibited equivalent physicochemical characteristics in aqueous solution and similar in vivo targeting specificity. However, dextran-mimetic QDs provided enhanced signal-to-noise ratio for improved optical quantification, long-term photostability, and resistance to chemical fixation. In addition, the vascular circulation time for the QD-based probes was extended 9-fold compared with dextran, likely due to differences in conformational flexibility. The enhanced photophysical and photochemical properties of dextran-mimetic QDs may accelerate applications in macrophage targeting, tracking, and imaging across broad resolution scales, particularly advancing capabilities in single-cell and single-molecule imaging and quantification.

Functional analysis of low-grade glioma genetic variants predicts key target genes and transcription factors.

BACKGROUND: Large-scale genome-wide association studies (GWAS) have implicated thousands of germline genetic variants in modulating individuals' risk to various diseases, including cancer. At least 25 risk loci have been identified for low-grade gliomas (LGGs), but their molecular functions remain largely unknown. METHODS: We hypothesized that GWAS loci contain causal single nucleotide polymorphisms (SNPs) that reside in accessible open chromatin regions and modulate the expression of target genes by perturbing the binding affinity of transcription factors (TFs). We performed an integrative analysis of genomic and epigenomic data from The Cancer Genome Atlas and other public repositories to identify candidate causal SNPs within linkage disequilibrium blocks of LGG GWAS loci. We assessed their potential regulatory role via in silico TF binding sequence perturbations, convolutional neural network trained on TF binding data, and simulated annealing-based interpretation methods. RESULTS: We built an interactive website (http://education.knoweng.org/alg3/) summarizing the functional footprinting of 280 variants in 25 LGG GWAS regions, providing rich information for further computational and experimental scrutiny. We identified as case studies PHLDB1 and SLC25A26 as candidate target genes of rs12803321 and rs11706832, respectively, and predicted the GWAS variant rs648044 to be the causal SNP modulating ZBTB16, a known tumor suppressor in multiple cancers. We showed that rs648044 likely perturbed the binding affinity of the TF MAFF, as supported by RNA interference and in vitro MAFF binding experiments. CONCLUSIONS: The identified candidate (causal SNP, target gene, TF) triplets and the accompanying resource will help accelerate our understanding of the molecular mechanisms underlying genetic risk factors for gliomas.

Short-Wave Infrared Quantum Dots with Compact Sizes as Molecular Probes for Fluorescence Microscopy.

Materials with short-wave infrared (SWIR) emission are promising contrast agents for in vivo animal imaging, providing high-contrast and high-resolution images of blood vessels in deep tissues. However, SWIR emitters have not been developed as molecular labels for microscopy applications in the life sciences, which require optimized probes that are bright, stable, and small. Here, we design and synthesize semiconductor quantum dots (QDs) with SWIR emission based on HgxCd1-xSe alloy cores red shifted to the SWIR by epitaxial deposition of thin HgxCd1-xS shells with a small band gap. By tuning alloy composition alone, the emission can be shifted across the visible-to-SWIR (VIR) spectra while maintaining a small and equal size, allowing direct comparisons of molecular labeling performance across a broad range of wavelength. After coating with click-functional multidentate polymers, the VIR-QD spectral series has high quantum yield in the SWIR (14-33%), compact size (13 nm hydrodynamic diameter), and long-term stability in aqueous media during continuous excitation. We show that these properties enable diverse applications of SWIR molecular probes for fluorescence microscopy using conjugates of antibodies, growth factors, and nucleic acids. A broadly useful outcome is a 10-55-fold enhancement of the signal-to-background ratio at both the single-molecule level and the ensemble level in the SWIR relative to visible wavelengths, primarily due to drastically reduced autofluorescence. We anticipate that VIR-QDs with SWIR emission will enable ultrasensitive molecular imaging of low-copy number analytes in biospecimens with high autofluorescence.

Analysis of Tertiary Interactions between SART3 and U6 Small Nuclear RNA Using Modified Nanocapillaries.

We employed modified glass nanocapillaries to investigate interactions between the RNA-binding protein, known as cell carcinoma antigen recognized by T cells-3 (SART3), and the noncoding spliceosome component, U6 small nuclear RNA (snRNA), at the single-molecule level. We functionalized the nanocapillaries with U6 snRNA fragments, which were hybridized to DNA molecules and then covalently attached to the nanocapillary surface. When transported through the modified nanocapillaries, two different SART3-derived constructs, HAT-RRM1-RRM2 and RRM1-RRM2, exhibited resistive ionic current pulses with different dwell times, which represented their different binding affinities to tethered U6 snRNAs. The dissociation constants (KD), estimated from the bias voltage dependence of translocation events, were approximately 1.9 µM and 201 µM for HAT-RRM1-RRM2 and RRM1-RRM2, respectively. These values were comparable to corresponding values obtained with isothermal titration calorimetry, demonstrating that the modified glass nanocapillaries are applicable to analyses of protein-ligand interactions at the single-molecule level.

Labeling proteins inside living cells using external fluorophores for microscopy.

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.