Covalent Protein Labeling by SpyTag–SpyCatcher in Fixed Cells for Super‐Resolution Microscopy

Labeling proteins with high specificity and efficiency is a fundamental prerequisite for microscopic visualization of subcellular protein structures and interactions. Although the comparatively small size of epitope tags makes them less perturbative to fusion proteins, they require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag–SpyCatcher system as an epitope‐like tag for fluorescent labeling of intracellular proteins in fixed cells for both conventional and super‐resolution microscopy. We also applied this method to endogenous proteins by gene editing, demonstrating its high labeling efficiency and capability for isoform‐specific labeling.     

Super‐Resolution Imaging and Quantitative Analysis of Membrane Protein/Lipid Raft Clustering Mediated by Cell‐Surface Self‐Assembly of Hybrid Nanoconjugates

Super‐resolution imaging was used to quantify organizational changes in the plasma membrane after treatment with hybrid nanoconjugates. The nanoconjugates crosslinked CD20 on the surface of malignant B cells, thereby inducing apoptosis. Super‐resolution images were analyzed by using pair‐correlation analysis to determine cluster size and to count the average number of molecules in the clusters. The role of lipid rafts was investigated by pre‐treating cells with a cholesterol chelator and actin destabilizer to prevent lipid raft formation. Lipid raft cluster size correlated with apoptosis induction after treatment with the nanoconjugates. Lipid raft clusters had radii of ～200 nm in cells treated with the hybrid nanoconjugates. Super‐resolution images provided precise molecule location coordinates that could be used to determine density of bound conjugates, cluster size, and number of molecules per cluster.     

Super‐resolution Imaging of Amyloid Structures over Extended Times by Using Transient Binding of Single Thioflavin T Molecules

Oligomeric amyloid structures are crucial therapeutic targets in Alzheimer's and other amyloid diseases. However, these oligomers are too small to be resolved by standard light microscopy. We have developed a simple and versatile tool to image amyloid structures by using thioflavin T without the need for covalent labeling or immunostaining. The dynamic binding of single dye molecules generates photon bursts that are used for fluorophore localization on a nanometer scale. Thus, photobleaching cannot degrade image quality, allowing for extended observation times. Super‐resolution transient amyloid binding microscopy promises to directly image native amyloid by using standard probes and record amyloid dynamics over minutes to days. We imaged amyloid fibrils from multiple polypeptides, oligomeric, and fibrillar structures formed during different stages of amyloid‐β aggregation, as well as the structural remodeling of amyloid‐β fibrils by the compound epi‐gallocatechin gallate.     

Microdomain Formation Controls Spatiotemporal Dynamics of Cell‐Surface Glycoproteins

The effect of galectin‐mediated microdomain formation on the spatiotemporal dynamics of glycosylated membrane proteins in human microvascular endothelial cells (HMEC‐1) was studied qualitatively and quantitatively by high‐resolution fluorescence microscopy and artificially mimicked by metabolic glycoprotein engineering. Two types of membrane proteins, sialic acid‐bearing proteins (SABPs) and mucin‐type proteins (MTPs), were investigated. For visualization they were metabolically labeled with azido sugars and then coupled to a cyclooctyne‐conjugated fluorescent dye by click chemistry. Both spatial (diffusion) and temporal (residence time) dynamics of SABPs and MTPs on the membrane were investigated after treatment with exogenous galectin‐1 or ‐3. Strong effects of galectin‐mediated lattice formation were observed for MTPs (decreased spatial mobility), but not for SABPs. Lattice formation also strongly decreased the turnover of MTPs (increased residence time on the cell membrane). The effects of galectin‐mediated crosslinking was accurately mimicked by streptavidin‐mediated crosslinking of biotin‐tagged glycoproteins and verified by single‐molecule tracking. This technique allows the induction of crosslinking of membrane proteins under precisely controlled conditions, thereby influencing membrane residence time and the spatial dynamics of glycans on the cell membrane in a controlled way.     

Natural‐Product‐Like Spiroketals and Fused Bicyclic Acetals as Potential Therapeutic Agents for B‐Cell Chronic Lymphocytic Leukaemia

B‐cell chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia in the Western world for which no curative treatments are currently available. Purine nucleotide analogues and alkylating agents feature frequently in combination regimens to treat the malignant state, but their use has not led to any significant improvement in patient survival. Consequently, there still remains a need for alternative small‐molecule chemotherapeutics. Natural products are an unparalleled source of drug leads, and an unending inspiration for the design of small‐molecule libraries for drug discovery. The screening of focused libraries of natural‐product‐like spiroketal and fused bicyclic acetal small molecules against primary CLL cells has led to the identification of a small series of novel and potent cytotoxic agents towards primary CLL cells. The validation of the activity of these molecules is delineated through a series of synthesis and screening iterations, whereas preliminary mode of action studies positively indicate their ability to induce cell death via an apoptotic pathway with no evidence of necrosis to further support their potential as novel chemotherapeutic agents.     

Fabrication of nanodiamond‐based composite monolithic column and its application in separation of small molecules

A nanodiamond‐based composite monolithic column was fabricated by redox initiation for high‐performance liquid chromatography. In the fabrication process, functionalized nanodiamond was used as the functional monomer, dipentaerythritol hexaacrylate and 1,10‐decanediol diacrylate as cross‐linking agents, polyethylene glycol 400 and 1‐propanol as coporogens, and dibenzoyl peroxide and N,N‐dimethyl aniline as initiators. Compared to polymer monolithic columns without nanodiamond, a nanodiamond‐based composite monolithic column prepared under the same conditions exhibited relatively high resolution and efficiency. Characterizations of the resulting nanocomposite were carried out, including scanning electron microscopy, mercury intrusion porosimetry, nitrogen adsorption–desorption isotherm measurement, and thermogravimetric analysis. The ND‐based composite monolith exhibited a uniform and reticular skeleton microstructure, thermal stability, and mechanical stability. In addition, the nanodiamond‐based composite monolithic column was used to separate a series of small molecules with good resolution and reproducibility in high‐performance liquid chromatography. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43776.     

Advancing Small‐Molecule‐Based Chemical Biology with Next‐Generation Sequencing Technologies

Next‐generation‐sequencing (NGS) technologies enable us to obtain extensive information by deciphering millions of individual DNA sequencing reactions simultaneously. The new DNA‐sequencing strategies exceed their precursors in output by many orders of magnitude, resulting in a quantitative increase in valuable sequence information that could be harnessed for qualitative analysis. Sequencing on this scale has facilitated significant advances in diverse disciplines, ranging from the discovery, design, and evaluation of many small molecules and relevant biological mechanisms to maturation of personalized therapies. NGS technologies that have recently become affordable allow us to gain in‐depth insight into small‐molecule‐triggered biological phenomena and empower researchers to develop advanced versions of small molecules. In this review we focus on the overlooked implications of NGS technologies in chemical biology, with a special emphasis on small‐molecule development and screening.     

Super‐Tough and Highly‐Ductile Poly(l‐lactic acid)/Thermoplastic Polyurethane/Epoxide‐Containing Ethylene Copolymer Blends Prepared by Reactive Blending

Ethylene‐methyl acrylate‐glycidyl methacrylate copolymer (E‐MA‐GMA) is employed to improve the impact toughness of poly(l ‐lactic acid) (PLLA)/thermoplastic polyurethane (TPU) blends by reactive melt‐blending. The reaction and miscibility between the components are confirmed by Fourier transform infrared spectroscopy, dynamic mechanical analysis, and differential scanning calorimetry. A super‐tough PLLA/TPU/E‐MA‐GMA multiphase blend (75/10/15) exhibits a significantly improved impact strength of 77.77 kJ m^?2, which is more than 17 times higher than that of PLLA/TPU (90/10) blend. A co‐continuous‐like TPU phase structure involving E‐MA‐GMA phase at the etched cryo‐fractured surface and the high‐orientated matrix deformation at the impact‐fractured surface are observed by scanning electron microscopy. The high‐orientated matrix deformation induced by the co‐continuous TPU phase structure is responsible for the super toughness of PLLA/TPU/E‐MA‐GMA blends.     

Ultrastructural Imaging of Salmonella–Host Interactions Using Super‐resolution Correlative Light‐Electron Microscopy of Bioorthogonal Pathogens

The imaging of intracellular pathogens inside host cells is complicated by the low resolution and sensitivity of fluorescence microscopy and by the lack of ultrastructural information to visualize the pathogens. Herein, we present a new method to visualize these pathogens during infection that circumvents these problems: by using a metabolic hijacking approach to bioorthogonally label the intracellular pathogen Salmonella Typhimurium and by using these bioorthogonal groups to introduce fluorophores compatible with stochastic optical reconstruction microscopy (STORM) and placing this in a correlative light electron microscopy (CLEM) workflow, the pathogen can be imaged within its host cell context Typhimurium with a resolution of 20 nm. This STORM‐CLEM approach thus presents a new approach to understand these pathogens during infection.     

Immobilization of active α‐chymotrypsin on RF‐plasma‐functionalized polymer surfaces

Various polymeric surfaces (polyester, polyethylene, polystyrene) were functionalized under oxygen and dichlorosilane‐RF‐cold‐plasma environments and were employed as substrates for further in situ derivatization reactions and immobilization of α‐Chymotrypsin. The nature and morphology of the derivatized substrates and the substrates with immobilized enzymes were analyzed using survey and high‐resolution X‐ray photoelectron spectroscopy, attenuated total reflectance‐fourier transform infrared (ATR‐FTIR), laser desorption fourier transform ion cyclotron resonance mass spectrometry, chemical derivatization, and atomic force microscopy (AFM) techniques. It was demonstrated that the tacticity of the polystyrene substrate did not notably influence the activity of the immobilized enzyme, however, spacer molecules intercalated between the polymeric substrates (e.g., polyethylene) and the enzyme significantly increased the enzyme activity (comparable with that of the free enzyme). Computer‐aided conformational modeling of the substrate‐spacer systems indicated that the longer the spacer chain, the greater the mobility of the enzyme. It is suggested that the greater mobility of the enzyme molecules is responsible for the enhanced activity. It has also been shown that the stability of the immobilized enzyme systems was good; they retained their activity during several washing/assay cycles. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 1783–1796, 2000     

Progress and Prospects of Pyrrole‐Imidazole Polyamide–Fluorophore Conjugates as Sequence‐Selective DNA Probes

Recently, the versatility of N‐methylpyrrole (Py)‐N‐methylimidazole (Im) polyamide conjugates, which have been developed from the DNA‐binding antibiotics distamycin A and netropsin, has been shown. These synthetic small molecules can permeate cells to bind with duplex DNA in a sequence‐specific manner, and hence can influence gene expression in vivo. Accordingly, several reports demonstrating the sequence specificity and biological activity of Py‐Im polyamides have accumulated. However, the benefits of Py‐Im polyamides, in particular those conjugated with fluorophores, has been overlooked. Moreover, clear directions for the employment of these attractive artificial small molecules have not yet been shown. Here, we present a detailed overview of the current and prospective applications of Py‐Im polyamide–fluorophore conjugates, including sequence‐specific recognition with fluorescence emission properties, and their potential roles in biological imaging.     

Genome‐Wide Assessment of the Binding Effects of Artificial Transcriptional Activators by High‐Throughput Sequencing

One of the major goals in DNA‐based personalized medicine is the development of sequence‐specific small molecules to target the genome. SAHA‐PIPs belong to such class of small molecule. In the context of the complex eukaryotic genome, the differential biological effects of SAHA‐PIPs are unclear. This question can be addressed by identifying the binding regions across the genome; however, it is a challenge to enrich small‐molecule‐bound DNA without chemical crosslinking. Here, we developed a method that employs high‐throughput sequencing to map the binding area of small molecules throughout the chromatinized human genome. Analysis of the sequenced data confirmed the presence of specific binding sites for SAHA‐PIPs from the enriched sequence reads. Mapping the binding sites and enriched regions on the human genome clarifies the reason for the distinct biological effects of SAHA‐PIP. This approach will be useful for identifying the function of other small molecules on a large scale.     

Au‐nanoparticle‐decorated SnO2 nanorod sensor with enhanced xylene‐sensing performance

In this work, pure and Au‐nanoparticle‐decorated SnO₂ nanorods were prepared via a one‐step hydrothermal method combined with a facile deposition process, and then characterized by X‐ray diffraction, scanning electron microscopy, transmission electron microscopy, high‐resolution transmission electron microscopy, and X‐ray photoelectron spectroscopy. The xylene‐sensing performance of the nanorods was also investigated. The results show that Au nanoparticles with an average diameter of 4‐6 nm were immobilized on the surface of rutile SnO₂ nanorods. As the amount of Au increased, the surface‐adsorbed oxygen species gradually increased. The 12 mol% Au‐SnO₂ nanorods exhibited the highest response to 50 ppm xylene, a lower operating temperature (279 to 255°C), favorable selectivity, and fast response‐recovery speed (3 and 4 seconds, respectively). These properties made the fabricated nanorods good candidates for xylene detection. The possible mechanism for the enhanced xylene‐sensing performance is discussed.     

pH‐sensitive keratin‐based polymer hydrogel and its controllable drug‐release behavior

Using feather keratin as biocompatible and inexpensive natural biopolymer and methacrylic acid as a functional monomer, we prepared a pH‐sensitive feather‐keratin‐based polymer hydrogel (FKPGel) with grafted copolymerization. The obtained FKPGel was characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy. The swelling behavior and pH sensitivity of the FKPGel were investigated. When the small molecule (rhodamine B) and macromolecule (bovine serum albumin) were used as model drug molecules, the FKPGel exhibited controllable release behavior in vitro, and the hydrogels had pH sensitivity. For a small molecular drug, the cumulative release rate was 97% in 24 h at pH 8.4. For macromolecular drug, the cumulative release rate reached 89% at pH 7.4. Its release behavior could be controlled by the pH value. In summary, a simple method was found to reuse disused feathers. It is a kind of pH‐sensitive hydrogels to be applied in drug‐delivery systems. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41572.     

Three‐dimensional crystalline supramolecular nanostructures from self‐assembly of rod–coil molecules incorporating lateral carboxyl group in the middle of the rod segment

We report the synthesis and self‐assembling behaviour of coil–rod–coil molecules 1a–1c and 2a–2c , which incorporate lateral carboxyl or ester groups in the middle of the rod segment. The self‐assembling behaviour of these molecules was investigated in the bulk using differential scanning calorimetry, polarised optical microscopy and small‐angle X‐ray scattering. Our results reveal that hydrogen bonds strongly influence the self‐assembling behaviour of rod‐like building blocks. Molecules 1a–1c , which incorporate carboxyl groups in the middle of rod segments, self‐assemble into two‐dimensional (2‐D) columnar, three‐dimensional (3‐D) body‐centred tetragonal and 3‐D hexagonal close‐packed assemblies in the crystalline state. However, molecules 2a–2c , which contain ester groups in the centre of rod segments, self‐assemble into unexpected lamellar, hexagonal perforated lamellar and 2‐D columnar nanostructures in the bulk, indicating that hydrogen bonds impede intermolecular stacking in this rod–coil system. © 2015 Society of Chemical Industry     

Non‐Stick Droplets

Recently, many ways (sometimes inspired by nature) for achieving super‐hydrophobic surfaces have been proposed in the literature. On such surfaces, water makes a contact angle close to 180°, which produces spectacular properties: droplets do not stick and the surfaces repel water, which bounces when thrown on them. Here, we describe a way to reach the maximum possible contact angle, namely 180°, by texturing the liquid surface instead of the solid one, as it is done for super‐hydrophobic solids. It is shown that the contact between such a marble and the solid on which it is deposited is very small, which dramatically reduces the friction when these marbles move. High speeds are thus observed. Together with the fact that the marbles roll as they move, this produces spectacular changes in shape. But the marbles resist to these changes, which can be of interest for practical applications in microfluidics.     

A Combined High‐Resolution Mass Spectrometric and in silico Approach for the Characterisation of Small Ligands of β2‐Microglobulin

β₂‐Microglobulin (β₂‐m) is a protein responsible for a severe complication of long‐term hemodialysis, known as dialysis‐related amyloidosis, in which initial β₂‐m misfolding leads to amyloid fibril deposition, mainly in the skeletal tissue. Whereas much attention is paid to understanding the complex mechanism of amyloid formation, the evaluation of small molecules that may bind β₂‐m and possibly inhibit the aggregation process is still largely unexplored mainly because the protein lacks a specific active site. Based on our previous findings, we selected a pilot set of sulfonated molecules that are known to either bind or not to the protein, including binders that are anti‐amyloidogenic. We show how a complementary approach, using high‐resolution mass spectrometry and in silico studies, can offer rapid and precise information on affinity, as well as insight into the structural requisites that favour or disfavour the inhibitory activity. Overall, this approach can be used for predictive purposes and for a rapid screening of fibrillogenesis inhibitors.