石蜡包埋组织中组蛋白的提取分离和翻译后修饰的定量分析

组蛋白翻译后修饰(HPTMs)参与基因转录调控,其异常与肿瘤等重大疾病的发生发展密切相关。石蜡包埋组织是当前疾病研究的重要样本资源,对肿瘤机制和标志物研究具有重要意义。目前基于质谱的蛋白质组学技术已成为HPTMs分析的有力工具,而针对福尔马林固定石蜡包埋(FFPE)组织样品的HPTMs分析还十分有限。该研究发展了一种基于高效液相色谱-串联质谱的FFPE组织样本HPTMs分离分析新方法。通过研究并优化组蛋白的提取策略,建立了FFPE组织样本脱蜡水化处理、蛋白质提取与聚丙烯酰胺凝胶电泳分离相结合的组蛋白提取和分离方法。通过研究FFPE切片数量、组蛋白化学衍生化方法等对组蛋白鉴定的影响,确定了组蛋白处理的具体步骤。通过HPLC分离结合非依赖性采集模式的质谱分析,鉴定了组蛋白修饰的类型、位点和丰度。最后,将优化的实验方法应用于FFPE临床样本的HPTMs分析,鉴定了2例人乳腺浸润性癌和癌旁正常组织的HPTMs图谱,均获得了100种以上的不同组蛋白修饰形式的多肽。定量分析了他们的差异性水平,通过主成分降维分析,发现浸润性癌和癌旁正常组织之间组蛋白修饰丰度存在明显的差异,且差异性具有一定的规律,特别是涉及转录调控的组蛋白修饰与乳腺癌的预后和治疗靶点具有相关性,进而探讨了乳腺癌中异常HPTMs的生物学意义。该研究对临床石蜡样本中组蛋白修饰的分离分析以及肿瘤表观遗传标志物的检测进行了有益的探索。     

Simultaneous Dual Encoding of Three‐Dimensional Structures by Light‐Induced Modular Ligation

A highly efficient strategy for the simultaneous dual surface encoding of 2D and 3D microscaffolds is reported. The combination of an oligo(ethylene glycol)‐based network with two novel and readily synthesized monomers with photoreactive side chains yields two new photoresists, which can be used for the fabrication of microstructures (by two‐photon polymerization) that exhibit a dual‐photoreactive surface. By combining both functional photoresists into one scaffold, a dual functionalization pattern can be obtained by a single irradiation step in the presence of adequate reaction partners based on a self‐sorting mechanism. The versatility of the approach is shown by the dual patterning of halogenated and fluorescent markers as well as proteins. Furthermore, we introduce a new ToF–SIMS mode (“delayed extraction”) for the characterization of the obtained microstructures that combines high mass resolution with improved lateral resolution.     

Site‐Specific Dual Labeling of Proteins on Cysteine Residues with Chlorotetrazines

Dual‐labeled biomolecules constitute a new generation of bioconjugates with promising applications in therapy and diagnosis. Unfortunately, the development of these new families of biologics is hampered by the technical difficulties associated with their construction. In particular, the site specificity of the conjugation is critical as the number and position of payloads can have a dramatic impact on the pharmacokinetics of the bioconjugate. Herein, we introduce dichlorotetrazine as a trivalent platform for the selective double modification of proteins on cysteine residues. This strategy is applied to the dual labeling of albumin with a macrocyclic chelator for nuclear imaging and a fluorescent probe for fluorescence imaging.     

Red‐ and Green‐Emitting Iridium(III) Complexes for a Dual Barometric and Temperature‐Sensitive Paint

A new dual luminescent sensitive paint for barometric pressure and temperature (T) is presented. The green‐emitting iridium(III) complex [Ir(ppy)₂(carbac)] (ppy=2‐phenylpyridine; carbac=1‐(9H‐carbazol‐9‐yl)‐5,5‐dimethylhexane‐2,4‐dione) was applied as a novel probe for T along with the red‐emitting complex [Ir(btpy)₃], (btpy=2‐(benzo[b]thiophene‐2‐yl)pyridine) which functions as a barometric (in fact oxygen‐sensitive) probe. Both iridium complexes were dissolved in different polymer materials to achieve optimal responses. The probe [Ir(ppy)₂(carbac)] was dispersed in gas‐blocking poly(acrylonitrile) microparticles in order to suppress any quenching of its luminescence by oxygen. The barometric probe [Ir(btpy)₃], in turn, was incorporated in a cellulose acetate butyrate film which exhibits good permeability for oxygen. The effects of temperature on the response of the oxygen probe can be corrected by simultaneous optical determination of T, as the poly(acrylonitrile) microparticles containing the temperature indicator are incorporated into the film. The phosphorescent signals of the probes for T and barometric pressure, respectively, can be separated by optical filters due to the ≈75 nm difference in their emission maxima. The dual sensor is applicable to luminescence lifetime imaging of T and barometric pressure. It is the first luminescent dual sensor material for barometric pressure/T based exclusively on the use of Ir^III complexes in combination with luminescence lifetime imaging.     

A Reversible Dual Mode Chemodosimeter for the Detection of Cyanide Ions in Natural Sources

Herein, we report the synthesis of two indolium probes 1 and 2 based on anthracene and pyrene derivatives and their interactions with various anions. Of these probes, the pyrene conjugate 2 acts as a dual colorimetric and fluorescent chemodosimeter for the selective and sensitive detection of cyanide ions. The detection limit of probe 2 for CN^? ions was found to be 10 ppb (30 nM ). The nature of interaction has been thoroughly studied through various techniques such as ¹H NMR and IR spectroscopy, HRMS, and isothermal calorimetric (ITC) studies. These studies confirm that probe 2 forms a 1,2‐adduct in the presence of CN^? ions. Kinetic studies using probe 2 showed the completion of the reaction within 15 s with a rate constant of k′=0.522±0.063 s^?1_. This probe can be coated on a solid surface (dipstick) and a polymer matrix for the on‐site analysis and quantification of endogenous cyanide ions in natural sources such as Indian almonds.     

Lanthanide‐Coordinated Semiconducting Polymer Dots Used for Flow Cytometry and Mass Cytometry

Simultaneous monitoring of biomarkers as well as single‐cell analyses based on flow cytometry and mass cytometry are important for investigations of disease mechanisms, drug discovery, and signaling‐network studies. Flow cytometry and mass cytometry are complementary to each other; however, probes that can satisfy all the requirements for these two advanced technologies are limited. In this study, we report a probe of lanthanide‐coordinated semiconducting polymer dots (Pdots), which possess fluorescence and mass signals. We demonstrated the usage of this dual‐functionality probe for both flow cytometry and mass cytometry in a mimetic cell mixture and human peripheral blood mononuclear cells as model systems. The probes not only offer high fluorescence signal for use in flow cytometry, but also show better performance in mass cytometry than the commercially available counterparts.     

Atomic Force Microscopy for Molecular Structure Elucidation

Using scanning probe microscopy techniques, at low temperatures and in ultrahigh vacuum, individual molecules adsorbed on surfaces can be probed with ultrahigh resolution to determine their structure and details of their conformation, configuration, charge states, aromaticity, and the contributions of resonance structures. Functionalizing the tip of an atomic force microscope with a CO molecule enabled atomic‐resolution imaging of single molecules, and measurement of their adsorption geometry and bond‐order relations. In addition, by using scanning tunneling microscopy and Kelvin probe force microscopy, the density of the molecular frontier orbitals and the electric charge distribution within molecules can be mapped. Combining these techniques yields a high‐resolution tool for the identification and characterization of individual molecules. The single‐molecule sensitivity and the possibility of atom manipulation to induce chemical reactions with the tip of the microscope open up unique applications in chemistry, and differentiate scanning probe microscopy from conventional methods for molecular structure elucidation. Besides being an aid for challenging cases in natural product identification, atomic force microscopy has been shown to be a powerful tool for the investigation of on‐surface reactions and the characterization of radicals and molecular mixtures. Herein we review the progress that high‐resolution scanning probe microscopy with functionalized tips has made for molecular structure identification and characterization, and discuss the challenges it will face in the years to come.     

A Dual‐Response DNA Probe for Simultaneously Monitoring Enzymatic Activity and Environmental pH Using a Nanopore

Both protease overexpression and local pH changes are key signatures of cancer. However, the sensitive detection of protease activities and the accurate measurement of pH in a tumor environment remain challenging. Here, we develop a dual‐response DNA probe that can simultaneously monitor protease activities and measure the local pH by translocation through α‐hemolysin (αHL). The DNA probe bears a short peptide containing phenylalanine at a pre‐designed position. Enzymatic cleavage of the peptide either exposes or removes the N‐terminal phenylalanine that can form a complex with cucurbit[7]uril. Translocation of the DNA hybrid through αHL generates current signatures that can be used to quantify protease activities. Furthermore, the current signatures possess a pH‐dependent pattern that reflects the local pH. Our results demonstrate that the versatile DNA probe may be further explored for simultaneously measuring multiple parameters of a complex system such as single cells in the future.     

Super‐quenched Molecular Probe Based on Aggregation‐Induced Emission and Photoinduced Electron Transfer Mechanisms for Formaldehyde Detection in Human Serum

Energy transfer between fluorescent dyes and quenchers is widely used in the design of light‐up probes. Although dual quenchers are more effective in offering lower background signals and higher turn‐on ratios than one quencher, such probes are less explored in practice as they require both quenchers to be within the proximity of the fluorescent core. In this contribution, we utilized intramolecular motion and photoinduced electron transfer (PET) as quenching mechanisms to build super‐quenched light‐up probes based on fluorogens with aggregation‐induced emission. The optimized light‐up probe possesses negligible background and is able to detect not only free formaldehyde (FA) but also polymeric FA, with an unprecedented turn‐on ratio of >4900. We envision that this novel dual quenching strategy will help to develop various light‐up probes for analyte sensing.     

Identification of Tumor Initiating Cells with a Small‐Molecule Fluorescent Probe by Using Vimentin as a Biomarker

Tumor initiating cells (TICs) have been implicated in clinical relapse and metastasis of a variety of epithelial cancers, including lung cancer. While efforts toward the development of specific probes for TIC detection and targeting are ongoing, a universal TIC probe has yet to be developed. We report the first TIC‐specific fluorescent chemical probe, TiY, with identification of the molecular target as vimentin, a marker for epithelial‐to‐mesenchymal transition (EMT). TiY selectively stains TICs over differentiated tumor cells or normal cells, and facilitates the visualization and enrichment of functionally active TICs from patient tumors. At high concentration, TiY also shows anti‐TIC activity with low toxicity to non‐TICs. With the unexplored target vimentin, TiY shows potential as a first universal probe for TIC detection in different cancers.     

Rational Design of an “OFF–ON” Phosphorescent Chemodosimeter Based on an Iridium(III) Complex and Its Application for Time‐Resolved Luminescent Detection and Bioimaging of Cysteine and Homocysteine

Biothiols, such as cysteine (Cys) and homocysteine (Hcy), play very crucial roles in biological systems. Abnormal levels of these biothiols are often associated with many types of diseases. Therefore, the detection of Cys (or Hcy) is of great importance. In this work, we have synthesized an excellent “OFF‐ON” phosphorescent chemodosimeter 1 for sensing Cys and Hcy with high selectivity and naked‐eye detection based on an Ir^III complex containing a 2,4‐dinitrobenzenesulfonyl (DNBS) group within its ligand. The “OFF‐ON” phosphorescent response can be assigned to the electron‐transfer process from Ir^III center and C^N ligands to the DNBS group as the strong electron‐acceptor, which can quench the phosphorescence of probe 1 completely. The DNBS group can be cleaved by thiols of Cys or Hcy, and both the ³M LCT and ³LC states are responsible for the excited‐state properties of the reaction product of probe 1 and Cys (or Hcy). Thus, the phosphorescence is switched on. Based on these results, a general principle for designing “OFF‐ON” phosphorescent chemodosimeters based on heavy‐metal complexes has been provided. Importantly, utilizing the long emission‐lifetime of phosphorescence signal, the time‐resolved luminescent assay of 1 in sensing Cys was realized successfully, which can eliminate the interference from the short‐lived background fluorescence and improve the signal‐to‐noise ratio. As far as we know, this is the first report about the time‐resolved luminescent detection of biothiols. Finally, probe 1 has been used successfully for bioimaging the changes of Cys/Hcy concentration in living cells.     

ADLOC: An Aptamer‐Displacement Assay Based on Luminescent Oxygen Channeling

Functional nucleic acids, such as aptamers and allosteric ribozymes, can sense their ligands specifically, thereby undergoing structural alterations that can be converted into a detectable signal. The direct coupling of molecular recognition to signal generation enables the production of versatile reporters that can be applied as molecular probes for various purposes, including high‐throughput screening. Here we describe an unprecedented type of a nucleic acid‐based sensor system and show that it is amenable to high‐throughput screening (HTS) applications. The approach detects the displacement of an aptamer from its bound protein partner by means of luminescent oxygen channeling. In a proof‐of‐principle study we demonstrate that the format is feasible for efficient identification of small drug‐like molecules that bind to a protein target, in this case to the Sec7 domain of cytohesin. We extended the approach to a new cytohesin‐specific single chain DNA aptamer, C10.41, which exhibits a similar binding behavior to cytohesins but has the advantage of being more stable and easier to synthesize and to modify than the RNA‐aptamer M69. The results obtained with both aptamers indicate the general suitability of the aptamer‐displacement assay based on luminescent oxygen channelling (ADLOC) for HTS. We also analyzed the potential for false positive hits and identified from a library of 18 000 drug‐like small molecules two compounds as strong singlet‐oxygen quenchers. With full automation and the use of commercially available plate readers, we estimate that the ADLOC‐based assay described here could be used to screen at least 100 000 compounds per day.     

Dual Fluorescent‐ and Isotopic‐Labelled Self‐Assembling Vancomycin for in vivo Imaging of Bacterial Infections

The increase of bacterial resistance demands rapid and accurate diagnosis of bacterial infections. Biosurface‐induced supramolecular assembly for diagnosis and therapy has received little attention in detecting bacterial infections. Herein we present a dual fluorescent‐nuclear probe based on self‐assembly of vancomycin (Van) on Gram‐positive bacteria for imaging bacterial infection. A Van‐ and rhodamine‐modified peptide derivative (Rho‐FF‐Van), as the imaging agent, binds to the terminal peptide of the methicillin‐resistant staphylococcus aureus (MRSA) and self‐assembles to form nanoaggregates on the surface of MRSA . In an in vivo myositis model, Rho‐FF‐Van results in a significant increased fluorescence signal at the MRSA infected site. Radiolabeled with iodine‐125, Rho‐FF‐Van shows strong radioactive signal in the MRSA ‐infected lungs in a murine model. This novel dual fluorescent and nuclear probe promises a new way for in vivo imaging of bacterial infections.     

BODIPY: A Highly Versatile Platform for the Design of Bimodal Imaging Probes

In molecular imaging, multimodal imaging agents can provide complementary information, for improving the accuracy of disease diagnosis or enhancing patient management. In particular, optical/nuclear imaging may find important preclinical and clinical applications. To simplify the preparation of dual‐labeled imaging agents, we prepared versatile monomolecular multimodal imaging probe (MOMIP) platforms containing both a fluorescent dye (BODIPY) and a metal chelator (polyazamacrocycle). One of the MOMIP was conjugated to a cyclopeptide (i.e., octreotide) and radiolabeled with ¹¹¹In. In vitro and in vivo studies of the resulting bioconjugate were conducted, highlighting the potential of these BODIPY‐based bimodal probes. This work also confirmed that the biovector and/or the bimodal probes must be chosen carefully, due to the impact of the MOMIP on the overall properties of the resulting imaging agent.     

Substrate Profiling of Glutathione S‐transferase with Engineered Enzymes and Matched Glutathione Analogues

The identification of specific substrates of glutathione S‐transferases (GSTs) is important for understanding drug metabolism. A method termed bioorthogonal identification of GST substrates (BIGS) was developed, in which a reduced glutathione (GSH) analogue was developed for recognition by a rationally engineered GST to label the substrates of the corresponding native GST. A K44G‐W40A‐R41A mutant (GST‐KWR) of the mu‐class glutathione S‐transferases GSTM1 was shown to be active with a clickable GSH analogue (GSH‐R1) as the cosubstrate. The GSH‐R1 conjugation products can react with an azido‐based biotin probe for ready enrichment and MS identification. Proof‐of‐principle studies were carried to detect the products of GSH‐R1 conjugation to 1‐chloro‐2,4‐dinitrobenzene (CDNB) and dopamine quinone. The BIGS technology was then used to identify GSTM1 substrates in the Chinese herbal medicine Ganmaocongji.     

Enantioselective Preparation of Arenes with β‐Stereogenic Centers: Confronting the 1,1‐Disubstituted Olefin Problem Using CuH/Pd Cooperative Catalysis

Arenes with β‐stereogenic centers are important substructures in pharmaceuticals and natural products. We have developed an asymmetric anti‐Markovnikov hydroarylation of 1,1‐disubstituted olefins by dual palladium and copper hydride catalysis as a convenient and general approach to access these substructures. This efficient one‐step process addresses several limitations of the traditional stepwise approaches. The use of cesium benzoate as a base and a common phosphine ligand for both the Cu‐ and Pd‐catalyzed processes were important discoveries that allow these challenging olefin substrates to be efficiently transformed. A variety of aryl bromide coupling partners, including numerous heterocycles, were coupled with 1,1‐disubstituted alkenes to generate arenes with β‐stereogenic centers.     

Development of a Sensitive Bioluminogenic Probe for Imaging Highly Reactive Oxygen Species in Living Rats

A sensitive bioluminogenic probe for highly reactive oxygen species (hROS), SO₃H‐APL, was developed based on the concept of dual control of bioluminescence emission by means of bioluminescent enzyme‐induced electron transfer (BioLeT) and modulation of cell‐membrane permeability. This probe enables non‐invasive visualization of physiologically relevant amounts of hROS generated deep inside the body of living rats for the first time. It is expected to serve as a practical analytical tool for investigating a wide range of biological functions of hROS in vivo. The design concept should be applicable to other in vivo bioluminogenic probes.     

An Activatable Lanthanide Luminescent Probe for Time‐Gated Detection of Nitroreductase in Live Bacteria

Herein we report the development of a turn‐on lanthanide luminescent probe for time‐gated detection of nitroreductases (NTRs) in live bacteria. The probe is activated through NTR‐induced formation of the sensitizing carbostyril antenna and resulting energy transfer to the lanthanide center. This novel NTR‐responsive trigger is virtually non‐fluorescent in its inactivated form and features a large signal increase upon activation. We show that the probe is capable of selectively sensing NTR in lysates as well as in live bacteria of the ESKAPE family which are clinically highly relevant multiresistant pathogens responsible for the majority of hospital infections. The results suggest that our probe could be used to develop diagnostic tools for bacterial infections.     

Light and pH dual‐sensitive biodegradable polymeric nanoparticles for controlled release of cargos

In this article, a light and pH dual‐sensitive block copolymer PEG‐b‐poly(MPC‐Azo/DEA) was facilely prepared for the first time by azide‐alkyne click chemistry between amphiphilic block copolymer bearing pendant alkynyl group poly(ethylene glycol)‐poly(5‐methyl‐5‐propargylxycarbonyl‐1,3‐dioxane‐2‐one) (PEG‐b‐poly(MPC)) and two azide‐containing compounds azobenzene derivative (Azo‐N₃) and 2‐azido‐1‐ethyl‐diethylamine (DEA‐N₃). Light response of the polymeric nanoparticles benefits from the azobenzene segments and pH responsiveness is attributed to DEA moieties. The prepared copolymer could self‐assemble into spherical micelle particles. The morphological changes of these particles in response to dual stimuli were investigated by UV/vis spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Nile Red (NR) was utilized as probe, and fluorescence spectroscopy was served as an evidence for the enhanced release of cargos from polymeric nanoparticles under combined stimulation. Anticancer drug, DOX was loaded into the nanoparticles and the loaded‐DOX could be released from these nanoparticles under dual stimuli. MTT assays further demonstrated that PEG‐b‐poly(MPC) and PEG‐b‐poly(MPC‐Azo/DEA) were of biocompatibility and low toxicity against HepG2 cells as well as SMCC‐7721 cells. More importantly, the prepared DOX‐loaded nanoparticles exhibited good anticancer ability for the two cells. The synthesized light and pH dual‐sensitive biodegradable polymeric nanoparticles were expected to be platforms for precisely controlled release of encapsulated molecules. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 1773–1783