Chromophore-Assisted Proximity Labeling of DNA Reveals Chromosomal Organization in Living Cells

The spatial arrangement of chromosome within the nucleus is linked to genome function and gene expression regulation. Existing genome-wide mapping methods often rely on chemically crosslinking DNA with protein baits, which raises concerns of artifacts being introduced during cell fixation. By genetically targeting a photosensitizer protein to specific subnuclear locations, we achieved blue-light-activated labeling of local DNA with a bioorthogonal functional handle for affinity purification and sequence identification through next-generation sequencing. When applied to the nuclear lamina in human embryonic kidney 293T cells, it revealed lamina-associated domains (LADs) that cover 37.6 % of the genome. These LADs overlap with heterochromatin hallmarks and are depleted with CpG islands. This simple labeling method avoids the harsh treatment of chemical crosslinking and is generally applicable to the genome-wide high-resolution mapping of the spatial chromosome organization in living cells.     

3D-ASL灌注成像与静息态功能MRI在癫痫诊断及鉴别中的价值

选取123例癫痫患者作为研究组,同期69例健康体检者作为对照组,均行三维动脉自旋标记技术(3D-ASL)灌注成像与静息态功能MRI(rs-FMRI)检查,研究二者在癫痫病诊断及鉴别中的价值.结果发现,研究组全面发作脑血流量(CBF)、低频振幅(ALFF)＜部分发作和对照组,全面发作血清脑源性神经细胞营养因子(BDNF)...     

蛋白质荧光标记中协同效应的高效液相色谱

以牛血清白蛋白和胰岛素为研究对象, 经1,5-I-AEDANS衍生后, 采用高效液相色谱(HPLC)进行分离分析, 发现两者的衍生存在明显的协同作用.     

两位置交换问题魔角旋转核磁共振时域信号的准确解

已知两位置交换分子运动速率对于静态¹³CNMR谱的影响是由一简单代数关系决定,它已成为分析固体中分子运动的常规方法.慢速的魔角旋转可以保留边带,所产生的边带型原则上也可用来分析分子运动,只是缺乏象静态谱那样的简单的关系,以至于在实际应用上是不可行的.本文给出作者所发现的存在分子运动时魔角旋转核磁共振自由诱导衰减信号的解析表达式的理论推导,提供分析固体分子运动的理论谱计算公式.我们选择二甲矾(dimethylsulfone)作为模型化合物,记录其不同温度下¹³CNMR谱,又计算了两个甲基做两位置交换运动的理论谱.理论谱与实验谱定性一致.定量上,边带强度存在一些差别,但边带宽度符合很好.通过理论谱与实验谱的比较,得到交换速率与温度的对应关系.虽然本文限于化学位移各向异性相互作用两位置交换问题,但容易推广到偶极作用多位置交换问题.     

Investigation in thorium phosphate by NMR II-phosphorus dipolar networks

With through space and through bond experiments in two-dimensional NMR we analyze the transformation from the thorium phosphate-hydrogen phosphate hydrate (TPHPH) to the β form of the thorium phosphate diphosphate (β-TPD) in relation with the phosphorus networks. These techniques are complementary: the through space coupling gives an insight on the dipolar phosphorus networks while the through bond coupling is particularly efficient in the detection of the P₂O₇ groups. With these experiments we show that in a first step, by heating the precursor TPHPH above 250 °C, it transforms into an form of TPD. This transformation is due to the complete condensation of hydrogen phosphate groups HPO₄ into P₂O₇ entities. By heating -TPD above 950 °C it transforms into its well-known β form. The form is characterized by a hygroscopic behavior: some water molecules are present near the P₂O₇ groups that makes non-equivalent their phosphorus nuclei. PO₄ dipolar networks are always present in the form. The main effect of these PO₄ and P₂O₇ units is to give the system a channel structure and the water enters in them.     

Hydrophilic Labeling Reagents of Dipyrrylmethene-BF2 Dyes for Two-Photon Excited Fluorometry: Syntheses and Photophysical Characterization

Recently introduced bioaffinity assay technology, ArcDia TPX, is based on two-photon excited fluorescence (TPE) and it enables separation-free ultra-sensitive immunoassays from microvolumes. Here we present syntheses of novel two-photon excitable fluorescent labeling reagents which have been specially designed to be used as label molecules in the ArcDia TPX assay technique. The labeling reagents are based on dipyrrylmetheneboron difluoride (dipyrrylmethene-BF2) chromophore, which have been substituted with aryl, heteroaryl or arylalkenyl chemical groups to extend the pi-electron conjugation. These substitutions results in a series of dipyrrylmethene-BF2 fluorophores with different photophysical properties. Dipyrrylmethene-BF2 fluorophores have been further substituted with a dipeptide linker unit and finally activated as succinimidyl esters to enable specific coupling with primary amino groups. The dipeptide linker serves as a spacer arm between the label and a target, and enhances the solubility of the label in aqueous solutions. Study of the chemical and photophysical performance of the new labeling reagents is described. The new labeling reagents exhibit high fluorescence quantum yields, and molar absorption coefficients. The results show that the new labels with the hydrophilic dipeptide linker unit provide large two-photon excitation cross-sections, high fluorescence quantum efficiency and good solubility in aqueous solutions. The results suggest that the novel dipyrrylmethene-BF2 labels are highly applicable to bioaffinity assays based on two-photon excitation of fluorescence.     

Modifying LnHPDO3A Chelates for Improved T1 and CEST MRI Applications

The new ligand HPDO3MA [(R,R,R,R)-10-(2-hydroxypropyl)-α,α′,α′′-trimethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid] was designed to combine and optimize the chemical properties of the macrocyclic ligands HPDO3A and DOTMA. The presence of the methyl groups on the acetic pendant arms of HPDO3A is expected to rigidify the structure of the ligand and favor an increase of the kinetic inertness of the Ln complexes. ¹H NMR spectra of Eu(HPDO3MA) displayed the presence of two pairs of diastereoisomers: SAP (square antiprismatic) and TSAP (twisted square antiprismatic) isomers (56 and 44 %, respectively). In addition, ¹H and ¹⁷O relaxometric NMR studies of Gd(HPDO3MA) showed approximately a 10 % increase in relaxivity and a faster water exchange rate with respect to Gd(HPDO3A). Moreover, a detailed chemical exchange saturation transfer (CEST) characterization of Yb(HPDO3MA) displayed a sensitivity about two times larger than that of Yb(HPDO3A) both in phantom and in cell labeling experiments. Finally, the kinetic inertness of Yb(HPDO3MA) was measured to be twice as high as that of Yb(HPDO3A), with a dissociation half-life at physiological pH of about 2500 years.     

Enhanced Sensitivity to Local Dynamics in Peptides by Use of Temperature-Jump IR Spectroscopy and Isotope Labeling

Site-specific isotopic labeling of molecules is a widely used approach in IR spectroscopy to resolve local contributions to vibrational modes. The induced frequency shift of the corresponding IR band depends on the substituted masses, as well as on hydrogen bonding and vibrational coupling. The impact of these different factors was analyzed with a designed three-stranded β-sheet peptide and by use of selected ¹³C isotope substitutions at multiple positions in the peptide backbone. Single-strand labels give rise to isotopically shifted bands at different frequencies, depending on the specific sites; this demonstrates sensitivity to the local environment. Cross-strand double- and triple-labeled peptides exhibited two resolved bands that could be uniquely assigned to specific residues, the equilibrium IR spectra of which indicated only weak local-mode coupling. Temperature-jump IR laser spectroscopy was applied to monitor structural dynamics and revealed an impressive enhancement of the isotope sensitivity to both local positions and coupling between them, relative to that of equilibrium FTIR spectroscopy. Site-specific relaxation rates were altered upon the introduction of additional cross-strand isotopes. Likewise, the rates for the global β-sheet dynamics were affected in a manner dependent on the distinct relaxation behavior of the labeled oscillator. This study reveals that isotope labels provide not only local structural probes, but rather sense the dynamic complexity of the molecular environment.