Development of ethynyl-2′-deoxyuridine chemical probes for cell proliferation

A common method of evaluating cellular proliferation is to label DNA with chemical probes. 5-Ethynyl-2′-deoxyuridine (EdU) is a widely utilized chemical probe for labeling DNA, and upon incorporation, EdU treatment of cells is followed by a reaction with a small molecule fluorescent azide to allow detection. The limitations when using EdU include cytotoxicity and a reliance on nucleoside active transport mechanisms for entry into cells. Here we have developed six novel EdU pro-labels that consist of EdU modified with variable lipophilic acyl ester moieties. This pro-label:chemical probe relationship parallels the prodrug:drug relationship that is employed widely in medicinal chemistry. EdU and EdU pro-labels were evaluated for their labeling efficacy and cytotoxicity. Several EdU pro-label analogues incorporate into DNA at a similar level to EdU, suggesting that nucleoside transporters can be bypassed by the pro-labels. These EdU pro-labels also had reduced toxicity compared to EdU.     

尾巨桉液流特征分析

目前尾巨桉( Eucalyptus urophylla × E. grandis)在南部大面积种植,尤其是在广西,其水分利用效率对森林可持续发展和水资源管理的影响越来越受到关注,因此了解其水分利用特征具有一定的意义。该文通过Granier热扩散探针法(TDP)对广西黄冕国有林场4~5年生尾巨桉人工林液流密度(SFD)的年变化规律、不同个体变化及其与环境因子的关系进行了研究。结果表明：尾巨桉年平均日液流密度为830.1 L.m-2.d-1;从尾巨桉日液流密度的年变化来看,最大值不超过2000 L.m-2.d-1,与相似研究比较,该研究得到的结果偏低。不同直径尾巨桉SFD具有相似的变化趋势,胸径相近其液流密度也大致相同,但胸径相差很大时,其液流密度相差也大,相差最大可达1300 L.m-2.d-1,这主要与不同生长状况的植物根系从土壤吸收水分能力不同有关。相关研究表明光合有效辐射和水汽压亏缺是树木冠层蒸腾的主要动力,该研究也发现树干液流密度与水汽压亏缺( VPD)、光合有效辐射( PAR)在年变化上有很好的同步性,主要表现出夏秋季节较高、春冬季节较低的现象。 SFD与PAR的关系比较显著,与VPD、空气温度( AT)、土壤温度( ST)有一定的关系,但与空气相对湿度( RH)和土壤湿度( SM)没有呈现规律。环境因子和植物生物学特征是树干液流密度主要的影响因素,进一步探讨尾巨桉如何响应这些因子的变化显得尤为重要。     

Design and synthesis of a new fluorescent probe for cascade detection of Zn2+ and H2PO4− in water and targeted imaging of living cells

Design and synthesis of new fluorescence probes with good water‐solubility is of great importance to better understanding the significant role of ions which are related to biology and the environment. As important ions, zinc ion (Zn²⁺) and dihydrogen phosphate ion (H₂PO₄^?) display essential roles in living systems, and quantitative detection of these ions in water is still a challenge. In order to consider the significant role of the galactose moiety in the design of a water‐soluble fluorescence sensor, herein, we have developed a novel probe, Gal‐AQTF, for the cascade detection of Zn²⁺ and H₂PO₄^? with excellent selectivity in water. Through the introduction of the galactose moiety onto the sensor AQTF, which has been reported earlier by us, the water‐solubility, cell compatibility and targeting ability were enhanced. Gal‐AQTF has been successfully applied in the imaging of the living cells of HepG2 and A549, and illustrated good selectivity for the HepG2 cells which overly express the asialoglycoprotein (ASGP) receptor.     

Development of a highly sensitive fluorescence probe for peptidyl arginine deiminase (PAD) activity

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of arginine residues to citrulline residues. Aberrant levels of PAD activity are associated with various diseases, such as rheumatoid arthritis, Alzheimer’s disease, and multiple sclerosis, so there is a need for simple and convenient high-throughput screening systems to discover PAD inhibitors as candidate therapeutic agents. Here, we report a highly sensitive off/on-type fluorescence probe for PAD activity based on the donor-excited photoinduced electron transfer (d-PeT) mechanism, utilizing the specific cycloaddition reaction between the benzil group of the probe and the ureido group of the PAD product, citrulline, under acidic conditions. We synthesized and functionally evaluated a series of probes bearing substituents on the benzil phenyl group, and found that 4MEBz-FluME could successfully detect citrulline with higher sensitivity and broader dynamic range than our previously reported fluorescence probe, FGME. Moreover, we succeeded in establishing multiple assay systems for PAD subtypes activities, including PAD2 and PAD4, with 4MeBz-FluME thanks to its high sensitivity. We expect that our fluorescence probes will become a powerful tool for discovering PAD inhibitors of several subtypes. Thus, it should be suitable for high-throughput screening of chemical libraries for inhibitors of PADs.     

Ratiometric assay of CARM1 activity using a FRET-based fluorescent probe

One of the regulatory mechanisms of epigenetic gene expression is the post-translational methylation of arginine residues, which is catalyzed by protein arginine methyltransferases (PRMTs). Abnormal expression of PRMT4/CARM1, one of the PRMTs, is associated with various diseases, including cancers. Here, we designed and synthesized a Förster resonance energy transfer (FRET)-based probe, FRC, which contains coumarin and fluorescein fluorophores at the N-terminus and C-terminus of a peptide containing an arginine residue within an appropriate amino acid sequence to serve as a substrate of CARM1; the two fluorophores act as a FRET donor and a FRET acceptor, respectively. Since trypsin specifically hydrolyzes the arginine residue, but not a monomethylarginine or dimethylarginine residue, CARM1 activity can be evaluated from the change of the coumarin/fluorescein fluorescence ratio of FRC in the presence of trypsin.     

Raman ChemLighter: Fiber optic Raman probe imaging in combination with augmented chemical reality

Raman spectroscopy using fiber optic probe combines non‐contacted and label‐free molecular fingerprinting with high mechanical flexibility for biomedical, clinical and industrial applications. Inherently, fiber optic Raman probes provide information from a single point only, and the acquisition of images is not straightforward. For many applications, it is highly crucial to determine the molecular distribution and provide imaging information of the sample. Here, we propose an approach for Raman imaging using a handheld fiber optic probe, which is built around computer vision–based assessment of positional information and simultaneous acquisition of spectroscopic information. By combining this implementation with real‐time data processing and analysis, it is possible to create not only fiber‐based Raman imaging but also an augmented chemical reality image of the molecular distribution of the sample surface in real‐time. We experimentally demonstrated that using our approach, it is possible to determine and to distinguish borders of different bimolecular compounds in a short time. Because the method can be transferred to other optical probes and other spectroscopic techniques, it is expected that the implementation will have a large impact for clinical, biomedical and industrial applications.      

一种基于配体肽的CRP荧光检测探针的研制

C反应蛋白（C-reaction protein,CRP）是反映机体炎症的有效标志物,早期检测是判断炎症相关疾病的关键,因此,研制CRP新型检测制剂具有重要意义。利用噬菌体表面展示技术对CRP特异性亲和配体进行了筛选,采用固相肽合成技术对目标配体进行了合成,并经生物标记、高效液相色谱法（high performance liquid chromatography,HPLC）分析及质谱（mass spectrometry,MS）鉴定,成功制得检测CRP的荧光探针。经3轮筛选、ELISA检测、重组噬菌体测序及序列比对后得到1个目标配体肽：S-P-H-N-R-S-N-L-V-Q-E-L;经肽合成及生物标记获得1种CRP荧光探针：FITC-（Acp）-S-P-H-N-R-S-N-L-V-Q-E-L。研究结果为CRP的有效检测提供了一种新型制剂。     

Dipolar relaxation in a lipid bilayer detected by a fluorescent probe, 4'-dimethylaminochalcone

The dynamic behavior of polar molecules in egg phosphatidylcholine (PC) bilayers has been studied using a membrane fluorescent probe, 4'-dimethylaminochalcone (DMAC). Time and spectrally resolved fluorescence spectroscopy of DMAC incorporated in PC liposomes, as compared to studies of the probe in organic solvents, shows the existence of two independent populations, associated with different extent and speed of dipolar solvent relaxation. The first DMAC population represents approximately 69% of the fluorescence-emitting molecules, has a short fluorescence decay time (0.32 ns) and undergoes Stokes shift of 80 nm. The remaining 31% fraction of DMAC molecules has a decay time of 0.74 ns and undergoes a high (106 nm) Stokes shift. A fraction of the shift, ca. 24 nm for the first and 46 nm for the second population, is attributed to the fast (<0.1 ns) rotational relaxation of nearby dipolar molecules, which might be water. This two-state model accounts well for the detailed fluorescence properties of DMAC in egg PC, i.e. its broadened steady-state spectrum, its average fluorescence quantum yield and its complex wavelength-dependent fluorescence decays.