FGFR2-BRD4 Axis Regulates Transcriptional Networks of Histone 3 Modification and Synergy Between Its Inhibitors and PD-1/PD-L1 in a TNBC Mouse Model.

Epigenetic reprogramming is an independent mode of gene expression that often involves changes in the transcription and chromatin structure due to tumor initiation and development. In this study, we developed a specifically modified peptide array and searched for a recognized epigenetic reader. Our results demonstrated that BRD4 is not only an acetylation reader but of propionylation as well. We also studied the quantitative binding affinities between modified peptides and epigenetic regulators by isothermal titration calorimetry (ITC). Furthermore, we introduced the Fgfr2-S252W transgenic mouse model to confirm that this acetylation is associated with the activation of c-Myc and drives tumor formation. Targeted disruption of BRD4 in Fgfr2-S252W mouse tumor cells also confirmed that BRD4 is a key regulator of histone 3 acetylation. Finally, we developed a tumor slice culture system and demonstrated the synergy between immune checkpoint blockade and targeted therapy in triple-negative breast cancer (TNBC). These data extend our understanding of epigenetic reprogramming and epigenetics-based therapies.

Desuccinylation-Triggered Peptide Self-Assembly: Live Cell Imaging of SIRT5 Activity and Mitochondrial Activity Modulation.

Mimicking nature's ability to orchestrate molecular self-assembly in living cells is important yet challenging. Molecular self-assembly has found wide applications in cellular activity control, drug delivery, biomarker imaging, etc. Nonetheless, examples of suborganelle-confined supramolecular self-assembly are quite rare and research in this area remains challenging. Herein, we have presented a new strategy to program supramolecular self-assembly specifically in mitochondria by leveraging on a unique enzyme SIRT5. SIRT5 is a mitochondria-localized enzyme belonging to a family of NAD+-dependent histone deacetylases. Accumulating studies suggest that SIRT5 is involved in regulating diverse biological processes, such as reactive oxygen defense, fatty acid metabolism, and apoptosis. In this study, we designed a novel class of succinylated peptide precursors that can be transformed into self-assembling building blocks through SIRT5 catalysis, leading to the formation of supramolecular nanofibers in vitro and in living cells. The increased hydrophobicity arising from self-assembly remarkably enhanced the fluorescence of nitrobenzoxadiazole (NBD) in the nanofibers. With this approach, we have enabled activity-based imaging of SIRT5 in living cells for the first time. Moreover, SIRT5-mediated peptide self-assembly was found to depolarize mitochondria membrane potential and promote ROS formation. Coincubation of the peptide with three different chemotherapeutic agents significantly boosted the anticancer activities of these drugs. Our work has thus illustrated a new way of mitochondria-confined peptide self-assembly for SIRT5 imaging and potential anticancer treatment.

Reaction-Based Off-On Near-infrared Fluorescent Probe for Imaging Alkaline Phosphatase Activity in Living Cells and Mice.

Alkaline phosphatases are a group of enzymes that play important roles in regulating diverse cellular functions and disease pathogenesis. Hence, developing fluorescent probes for in vivo detection of alkaline phosphatase activity is highly desirable for studying the dynamic phosphorylation in living organisms. Here, we developed the very first reaction-based near-infrared (NIR) probe (DHXP) for sensitive detection of alkaline phosphatase activity both in vitro and in vivo. Our studies demonstrated that the probe displayed an up to 66-fold fluorescence increment upon incubation with alkaline phosphatases, and the detection limit of our probe was determined to be 0.07 U/L, which is lower than that of most of alkaline phosphatase probes reported in literature. Furthermore, we demonstrated that the probe can be applied to detecting alkaline phosphatase activity in cells and mice. In addition, our probe possesses excellent biocompatibility and rapid cell-internalization ability. In light of these prominent properties, we envision that DHXP will add useful tools for investigating alkaline phosphatase activity in biomedical research.

Investigation of the Subcellular Neurotoxicity of Amyloid-ß Using a Device Integrating Microfluidic Perfusion and Chemotactic Guidance.

Alzheimer's disease (AD) is a neurodegenerative disorder with the histopathological hallmark of extracellular accumulation of amyloid-ß (Aß) peptide in brain senile plaques. Though many studies have shown the neural toxicity from various forms of Aß peptides, the subcellular mechanisms of Aß peptide are still not well understood, partially due to the technical challenges of isolating axons or dendrites from the cell body for localized investigation. In this study, the subcellular toxicity and localization of Aß peptides are investigated by utilizing a microfluidic compartmentalized device, which combines physical restriction and chemotactic guidance to enable the isolation of axons and dendrites for localized pharmacological studies. It is found that Aß peptides induced neuronal death is mostly resulted from Aß treatment at cell body or axonal processes, but not at dendritic neurites. Simply applying Aß to axons alone induces significant hyperactive spiking activity. Dynamic transport of Aß aggregates is only observed between axon terminal and cell body. In addition to differential cellular uptake, more Aß-peptide secretion is detected significantly from axons than from dendritic side. These results clearly demonstrate the existence of a localized mechanism in Aß-induced neurotoxicity, and can potentially benefit the development of new therapeutic strategies for AD.

Fluorescent Probes for Single-Step Detection and Proteomic Profiling of Histone Deacetylases.

Histone deacetylases (HDACs) play important roles in regulating various physiological and pathological processes. Developing fluorescent probes capable of detecting HDAC activity can help further elucidate the roles of HDACs in biology. In this study, we first developed a set of activity-based fluorescent probes by incorporating the Kac residue and the O-NBD group. Upon enzymatic removal of the acetyl group in the Kac residue, the released free amine reacted intramolecularly with the O-NBD moiety, resulting in turn-on fluorescence. These designed probes are capable of detecting HDAC activity in a continuous fashion, thereby eliminating the extra step of fluorescence development. Remarkably, the amount of turn-on fluorescence can be as high as 50-fold, which is superior to the existing one-step HDAC fluorescent probes. Inhibition experiments further proved that the probes can serve as useful tools for screening HDAC inhibitors. Building on these results, we moved on and designed a dual-purpose fluorescent probe by introducing a diazirine photo-cross-linker into the probe. The resulting probe was not only capable of reporting enzymatic activity but also able to directly identify and capture the protein targets from the complex cellular environment. By combining a fluorometric method and in-gel fluorescence scanning technique, we found that epigenetic readers and erasers can be readily identified and differentiated using a single probe. This is not achievable with traditional photoaffinity probes. In light of the prominent properties and the diverse functions of this newly developed probe, we envision that it can provide a robust tool for functional analysis of HDACs and facilitate future drug discovery in epigenetics.

Peptide-Decorated Gold Nanoparticles as Functional Nano-Capping Agent of Mesoporous Silica Container for Targeting Drug Delivery.

A stimuli-responsive drug delivery system (DDS) with bioactive surface is constructed by end-capping mesoporous silica nanoparticles (MSNs) with functional peptide-coated gold nanoparticles (GNPs). MSNs are first functionalized with acid-labile α-amide-ß-carboxyl groups to carry negative charges, and then capped with positively charged GNPs that are decorated with oligo-lysine-containing peptide. The resulting hybrid delivery system exhibits endo/lysosomal pH triggered drug release, and the incorporation of RGD peptide facilitates targeting delivery to αvß3 integrin overexpressing cancer cells. The system can serve as a platform for preparing diversified multifunctional nanocomposites using various functional inorganic nanoparticles and bioactive peptides.

The rational design of a peptide-based hydrogel responsive to H2S.

The development of hydrogels that are responsive to external stimuli in a well-controlled manner is important for numerous biomedical applications. Herein we reported the first example of a hydrogel responsive to hydrogen sulphide (H2S). H2S is an important gasotransmitter whose deregulation has been associated with a number of pathological conditions. Our hydrogel design is based on the functionalization of an ultrashort hydrogelating peptide sequence with an azidobenzyl moiety, which was reported to react with H2S selectively under physiological conditions. The resulting peptide was able to produce hydrogels at a concentration as low as 0.1 wt%. It could then be fully degraded in the presence of excess H2S. We envision that the novel hydrogel developed in this study may provide useful tools for biomedical research.

An iminocoumarin benzothiazole-based fluorescent probe for imaging hydrogen sulfide in living cells.

Hydrogen sulfide (H2S) has recently been identified as the third gaseous signaling molecule that is involved in regulating many important cellular processes. We report herein a novel fluorescent probe for detecting H2S based on iminocoumarin benzothiazole scaffold. The probe displayed high sensitivity and around 80-fold increment in fluorescence signal after reacting with H2S under physiological condition. The fluorescent intensity of the probe was linearly related to H2S concentration in the range of 0-100 µM with a detection limit of 0.15 µM (3σ/slope). The probe also showed excellent selectivity towards H2S over other biologically relevant species, including ROS, RSS and RNS. Its selectivity for H2S is 32 folds higher than other reactive sulfur species. Furthermore, the probe has been applied for imaging H2S in living cells. Cell imaging experiments demonstrated that the probe is cell-permeable and can be used to monitor the alteration of H2S concentrations in living cells. We envisage that this probe can provide useful tools to further elucidate the biological roles of H2S.

Site-specific immobilization of biomolecules by a biocompatible reaction between terminal cysteine and 2-cyanobenzothiazole.

We report herein a new site-specific microarray immobilization method based on a biocompatible reaction between terminal cysteine and 2-cyanobenzothiazole (CBT). This immobilization strategy has been successfully applied to anchor small molecules, peptides and proteins onto microarrays.

Microwave-controlled facile synthesis of well-defined PbS hexapods.

Controlled synthesis of well-defined PbS nanostructures in terms of size and shape has been strongly motivated by their potential applications ranging from solar photovoltaics to near-infrared optics. Hereby, we report a facile microwave-assistant method for ultrafast fabrication of PbS nanostructures, by which uniform PbS hexapods with six arms stretching along six (100) directions of the crystal seeds have been easily synthesized within minutes. Various morphologies including rectangle plates, uniform cubes as well as nanoparticles were obtained by tuning the parameters for the formation of PbS nanocrystals. The results reveal that both concentration and feed ratio of precursors determine the growth of PbS nanocrystals significantly. And higher initial precursor concentration favors the formation of the hexapod structures. The process of crystal growth is monitored through scanning electron microscopy of PbS from different durations of the reaction. This controlled ultrafast synthesis of PbS structures at nanometer and micrometer scale with various morphologies may be promising in large scale fabrication of nanostructures. Based on the systematically study of the growth process, a possible mechanism for the formation of the hexapod-like structure is discussed.

One-step template-free synthesis of monoporous polymer microspheres with uniform sizes via microwave-mediated dispersion polymerization.

One-step facile synthesis of monoporous polymer microspheres via microwave-controlled dispersion polymerization is introduced. This template-free method employing the dispersion polymerization of styrene under microwave irradiation induces directly the formation of uniform monoporous polymer microspheres, with controllable morphologies and sizes, which can be tuned by simply adjusting parameters for the synthesis. A comparison to conventional heating indicates that microwave irradiation plays a vital role in the formation of this novel morphology.

Microwave synthesis of zinc sulfite and porous zinc oxide microrods.

ZnSO(3) microrods with uniform size and shape can be prepared under microwave irradiation at much higher speed than a conventional heating bath, usually in minutes. The annealing of ZnSO(3) microrods produced porous ZnO microrods with pore size between 50-200 nm.