Ultrasensitive Quantification of microRNA Copy Number in Individual Extracellular Vesicles Using DNA Tetrahedron-Based Single-Molecule Imaging.

The ultrasensitive detection of microRNAs (miRNAs) in extracellular vesicles (EVs) can accurately reflect the progress and metastasis of miRNA-mediated intercellular communication, providing an unprecedented opportunity for liquid biopsy. However, due to the low abundance and high heterogeneity of miRNAs in EVs, the ultrasensitive quantification and establishment of a distribution model for miRNA within native EVs remain challenging. Here, we have developed a DNA tetrahedron-based single-molecule fluorescence imaging strategy to overcome this challenge.

Demethylzeylasteral inhibits oxidative phosphorylation complex biogenesis by targeting LRPPRC in lung cancer.

Targeted inhibition of mitochondrial oxidative phosphorylation (OXPHOS) complex generation is an emerging and promising cancer treatment strategy, but limited targets and specific inhibitors have been reported. Leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) is an atypical RNA-binding protein that regulates the stability of all 13 mitochondrial DNA-encoded mRNA (mt-mRNA) and thus participates in the synthesis of the OXPHOS complex. LRPPRC is also a prospective therapeutic target for lung adenocarcinoma, serving as a promising target for OXPHOS inhibition.

Deep learning in single-molecule imaging and analysis: recent advances and prospects.

Single-molecule microscopy is advantageous in characterizing heterogeneous dynamics at the molecular level. However, there are several challenges that currently hinder the wide application of single molecule imaging in bio-chemical studies, including how to perform single-molecule measurements efficiently with minimal run-to-run variations, how to analyze weak single-molecule signals efficiently and accurately without the influence of human bias, and how to extract complete information about dynamics of interest from single-molecule data. As a new class of computer algorithms that simulate the human brain to extract data features, deep learning networks excel in task parallelism and model generalization, and are well-suited for handling nonlinear functions and extracting weak features, which provide a promising approach for single-molecule experiment automation and data processing.

Effect of water-soluble fullerenes on macrophage surface ultrastructure revealed by scanning ion conductance microscopy.

C-fullerenes have unique potential in antiviral, drug delivery, photodynamic therapy and other biomedical applications. However, little is known about their effects on macrophage surface morphology and ultrastructure. Here by using contact-free scanning ion conductance microscopy (SICM), we investigated the effects of two water-soluble fullerenes on the surface ultrastructure and function of macrophages.

Coaxial illumination module of the stimulated-emission-depletion nanoscope.

Stimulated-emission-depletion (STED) nanoscope achieves super-resolution imaging by using a donut-shaped depletion beam to darken the fluorophores around the excitation spot. As an important factor determining the resolution of imaging, the coaxiality between the excitation and the depletion beam is required to be maintained at the nanoscale, which is often degraded by various interference such as ambient vibration and temperatures etc. Here, we propose a specially designed STED illumination module to guarantee the coaxiality between the two beams while modulating the phase of the depletion beam.

Surface-Engineered Gold Nanoclusters for Stimulated Emission Depletion and Correlated Light and Electron Microscopy Imaging.

Stimulated emission depletion (STED) nanoscopy is an emerging super-resolution imaging platform for the study of the cellular structure. Developing suitable fluorescent probes of small size, good photostability, and easy functionalization is still in demand. Herein, we introduce a new type of surface-engineered gold nanoclusters (Au NCs) that are ultrasmall (1.

Fluorescence live cell imaging revealed wogonin targets mitochondria.

Scutellaria baicalensis is one of the widely used Chinese traditional medicines, and wogonin is one of major active components in it. However, the mechanism of action of wogonin has largely remained unclear. In this work, we designed a fluorescent probe, namely ATTO565-WGN, by conjugating wogonin with the fluorophore ATTO565 based on Mannich reaction via a flexible chain linker.

Exploiting the robust network structure of zein/low-acyl gellan gum nanocomplexes to create Pickering emulsion gels with favorable properties.

Zein/low-acyl gellan gum (GG) composite particles (ZGPs) were fabricated to stabilize Pickering emulsions (termed "ZGPEs"). The wettability of ZGPs was manipulated simply by adjusting the concentration of GG. The effects of GG concentration, oil fraction and pH on ZGPEs were systematically evaluated by confocal laser scanning microscopy (CLSM), cryo-scanning electron microscopy (cryo-SEM), dynamic light scattering technique, stimulated emission depletion (STED) nanoscopy and rheology.

Highly Efficient Photochromic Tungsten Oxide@PNIPAM Composite Spheres with a Fast Response.

Coloration efficiency and a fast response are important in developing materials for optical switching. A novel, highly efficient photochromic tungsten oxide@poly(-isopropylacrylamide) (PNIPAM) hybrid sphere is reported, whose colors can be rapidly converted between yellow and blue under different lights. The color change can be seen clearly even if the tungsten oxide content in the hybrid sphere is very low, exhibiting outstanding coloration efficiency of tungsten oxide.

Analyzing protein dynamics from fluorescence intensity traces using unsupervised deep learning network.

We propose an unsupervised deep learning network to analyze the dynamics of membrane proteins from the fluorescence intensity traces. This system was trained in an unsupervised manner with the raw experimental time traces and synthesized ones, so neither predefined state number nor pre-labelling were required. With the bidirectional Long Short-Term Memory (biLSTM) networks as the hidden layers, both the past and future context can be used fully to improve the prediction results and can even extract information from the noise distribution.

Fluorescent Polymer Dot-Based Multicolor Stimulated Emission Depletion Nanoscopy with a Single Laser Beam Pair for Cellular Tracking.

Stimulated emission depletion (STED) nanoscopy provides subdiffraction resolution while preserving the benefits of fluorescence confocal microscopy in live-cell imaging. However, there are several challenges for multicolor STED nanoscopy, including sophisticated microscopy architectures, fast photobleaching, and cross talk of fluorescent probes. Here, we introduce two types of nanoscale fluorescent semiconducting polymer dots (Pdots) with different emission wavelengths: CNPPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)]) Pdots and PDFDP (poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorene}---{2,5-bis (,'-diphenylamino)-1,4-phenylene}]) Pdots, for dual-color STED bioimaging and cellular tracking.

Analysis of the Diffusivity Change from Single-Molecule Trajectories on Living Cells.

With the wide application of live-cell single-molecule imaging and tracking of biomolecules at work, deriving diffusion state changes of individual molecules is of particular interest as these changes reflect molecular oligomerization or interaction with other cellular components and thus correlate with functional changes. We have developed a Rayleigh mixture distribution-based hidden Markov model method to analyze time-lapse diffusivity change of single molecules, especially membrane proteins, with unknown dynamic states in living cells. With this method, the diffusion parameters, including diffusion state number, state transition probability, diffusion coefficient, and state mixture ratio, can be extracted from the single-molecule diffusion trajectories accurately via easy computation.

Effects of pectin polydispersity on zein/pectin composite nanoparticles (ZAPs) as high internal-phase Pickering emulsion stabilizers.

In the present study, the properties of two apple sourced-pectin (AP-1 and AP-2) were comparative studied, and their influence on the formation of high internal-phase Pickering emulsions (HIPPEs) was investigated. Results showed that AP-2 has lower polydispersity index (PDI = 2.51) than AP-1.

Automated Stoichiometry Analysis of Single-Molecule Fluorescence Imaging Traces via Deep Learning.

The stoichiometry of protein complexes is precisely regulated in cells and is fundamental to protein function. Singe-molecule fluorescence imaging based photobleaching event counting is a new approach for protein stoichiometry determination under physiological conditions. Due to the interference of the high noise level and photoblinking events, accurately extracting real bleaching steps from single-molecule fluorescence traces is still a challenging task.

Quantitative Characterization of the Membrane Dynamics of Newly Delivered TGF-β Receptors by Single-Molecule Imaging.

The dynamics and stoichiometry of receptors newly delivered on the plasma membrane play a vital role in cell signal transduction, yet knowledge of this process is limited because of the lack of suitable analytical methods. Here we developed a new strategy that combines single-molecule imaging (SMI) and fluorescence recovery after photobleaching (FRAP), named FRAP-SMI, to monitor and quantify individual newly delivered and inserted transmembrane receptors on plasma membranes of living cells. Transforming-growth-factor-β type II receptor (TβRII), a typical serine/threoninekinase receptor, was studied with this method.

Nanoscale Distribution of Transforming Growth Factor Receptor on Post-Golgi Vesicle Revealed by Super-resolution Microscopy.

Transforming growth factor-β (TGF-β) type II receptor (TβRII) plays a critical role in the initiation of TGF-β signaling pathway; therefore, the study of its synthesis and transport processes is of great important. In this work, we achieved super-resolution imaging of a new type of TβRII-containing post-Golgi vesicle by our home-built stimulated emission depletion (STED) microscope. We visualized the ring-shaped structure of these vesicles containing newly synthesized TβRII in the cytoplasm and characterized their size distribution from 300 to 1000 nm.

Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane.

Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane.

Single-molecule imaging reveals the stoichiometry change of β2-adrenergic receptors by a pharmacological biased ligand.

The stoichiometry of the β2-adrenergic receptor (β2AR) was determined using single-molecule fluorescence imaging in living cells. The results showed that β2AR mainly existed as monomers under physiological conditions and exhibited β-arrestin-dependent dimerization upon stimulation with the pharmacological biased ligand carvedilol. The association of β2AR dimerization with biased signalling is revealed.

Study of the interactions between endolysin and bacterial peptidoglycan on S. aureus by dynamic force spectroscopy.

The cell wall binding domain (CBD) of bacteriophage lysins can recognize target bacteria with extraordinary specificity through binding to bacterial peptidoglycan, thus it is a promising new probe to identify the corresponding bacterial pathogen. In this work, we used atomic force microscopy (AFM) based single-molecule force spectroscopy to investigate the interaction between the CBD of lysin PlyV12 (PlyV12C) and pathogenic bacterium Staphylococcus aureus (S. aureus).

Mammalian actin-binding protein 1/HIP-55 is essential for the scission of clathrin-coated pits by regulating dynamin-actin interaction.

Actin and dynamin work cooperatively to drive the invagination and scission of clathrin-coated pits (CCPs). However, little is known about the mechanism that orchestrates the spatiotemporal recruitment of dynamin and actin. Here, we have identified the mammalian actin-binding protein 1 (mAbp1; also called HIP-55 or SH3P7), which could bind to clathrin, actin, as well as dynamin, as an adaptor that links the dynamic recruitment of dynamin and actin for the scission of CCPs.

Analysis of the steps in single-molecule photobleaching traces by using the hidden markov model and maximum-likelihood clustering.

The step analysis of single-molecule photobleaching data offers a new approach for studying protein stoichiometry under physiological conditions. As such, it is important to develop suitable algorithms that can accurately extract the step events from the noisy single-molecule data. Herein, we report a HMM method that combines maximum-likelihood clustering for initializing the emission-probability distribution of the HMMs with an extended silhouette clustering criterion for estimating the state number of single molecules.

Electron transport characteristics of the dimeric 1,4-benzenedithiol junction.

Understanding the electron transport between single molecules connected through weak interaction is of great importance for molecular electronics. In this paper, we report measurements of the conductivity of the dimeric 1,4-benzenedithiol (BDT) junction using the scanning tunneling microscopy (STM)-based current-displacement I(s) method. The conductance was measured to be 6.

Single-molecule dynamics of site-specific labeled transforming growth factor type II receptors on living cells.

We achieved single-molecule imaging and tracking of the transforming growth factor type II receptor (TβRII) that was labeled by an organic dye via a genetically encoded unnatural amino acid (UAA) and the copper-free click chemistry. The stoichiometry, mobility and dimerization kinetics of individual TβRII molecules were determined.

Conformational dynamics of an ATP-binding DNA aptamer: a single-molecule study.

Nucleic acid aptamers are single-stranded RNA or DNA molecules that bind to their targets with high specificity and affinity. Although their biomedical applications have been booming, it is still debatable whether an aptamer recognizes its target through "induced fit" or "conformational selection", a central question in molecular recognition. To address this question, an ATP-binding DNA aptamer was selected as a model system and the conformational properties of this aptamer with and without the presence of ATP were investigated by single-pair Förster resonance energy transfer (spFRET) spectroscopy.

Atomic force microscopy study of the effect of HER 2 antibody on EGF mediated ErbB ligand-receptor interaction.

Unlabelled: HER2, a member of the epidermal growth factor receptor (ErbB) family, is over-expressed in many cancers. Trastuzumab and Pertuzumab are two monoclonal antibodies targeting different extracellular domains of HER2 for cancer therapy. As Pertuzumab binds to the dimerization arm of HER2, it can block HER2 heterodimerization and in turn ErbB signaling.