Probing interactions between human lung adenocarcinoma A549 cell and its aptamers at single‐molecule resolution

Because cell‐specific aptamers have high potential for biomedical applications, investigation of the interaction between cell and its aptamers may be of key importance for an improved understanding of biochemical processes. Herein, the interaction between human lung adenocarcinoma A549 cell and its four aptamers was explored using single‐molecule force spectroscopy (SMFS). The values of the unbinding force varied from 117.1 to 171.0 pN at the loading rate of 1.8 × 10⁵ pN/s. Based on the dependence of singe molecule force on the atomic force microscopy loading rate, the corresponding kinetic parameters were obtained. The results revealed two activation barriers and two transient states in the unbinding process of aptamer/cell interaction. More importantly, the binding sites on A549 cells with its four aptamers were defined to be different using SMFS and flow cytometry. This work demonstrated that SMFS can be used as a powerful tool for exploring the aptamer/cell binding behavior at the single‐molecule level, and may provide valuable information for the design and application of aptamer probes. Copyright © 2014 John Wiley & Sons, Ltd.     

miR‐129‐2‐3p directly targets SYK gene and associates with the risk of ischaemic stroke in a Chinese population

Spleen tyrosine kinase (SYK) gene has been identified as novel susceptibility locus for ischaemic stroke (IS) previously. However, regulation of SYK gene remains unknown in IS. In this study, we aimed to identify miRNAs that might be involved in the development of IS by targeting SYK gene. miRNAs were firstly screened by bioinformatics predicting tool. The expression levels of SYK gene were detected by qRT‐PCR and western blotting, respectively, after miRNA transfection. Luciferase reporter assay was applied to investigate the direct binding between miRNAs and target gene. miRNA levels were detected by miRNA TaqMan assays in the blood cells of 270 IS patients and 270 control volunteers. Results suggest that SYK gene might be a direct target of miR‐129‐2‐3p. The blood level of miR‐129‐2‐3p was significantly lower in IS patients (P < 0.05), and negatively associated with the risk of IS (adjusted OR: 0.88; 95% CI: 0.80‐0.98; P = 0.021) by multivariable logistic regression analysis. The blood levels of SYK gene were significantly higher in IS patients, and miR‐129‐2‐3p expression was negatively correlated with mean platelet volume. In summary, our study suggests that miR‐129‐2‐3p might be involved in the pathogenesis of IS through interrupting SYK expression and the platelet function, and further investigation is needed to explore the underlying mechanism.     

The microvesicle/CD36 complex triggers a prothrombotic phenotype in patients with non‐valvular atrial fibrillation

The mechanisms responsible for platelet activation, the prothrombotic state, in non‐valvular atrial fibrillation (NVAF) are still obscure. Microvesicles (MVs) can transfer various messages to target cells and may be helpful for exploring the detailed mechanisms. We aimed to investigate the possible mechanisms by which proatherogenic factors of NVAF contribute to platelet activation. Two hundred and ten patients with NVAF were stratified as being at ‘low to moderate risk’ or ‘high risk’ for stroke according to the CHADS2 score. Levels of platelet‐derived MVs (PMVs) and platelet activation were examined. CD36‐positive or CD36‐deficient human platelets were stimulated by MVs isolated from NVAF patients with or without various inhibitors in vitro. Levels of PMVs and platelet activation markers enhanced significantly in high‐risk patients. The MVs isolated from plasma of NVAF patients bound to platelet CD36 and activated platelets by phosphorylating the mitogen‐activated protein kinase 4/Jun N‐terminal kinase 2 (MKK4/JNK2) pathways. However, CD36 deficiency protected against MV‐induced activation of platelets. We reveal a possible mechanism of platelet activation in NVAF and suggest that the platelet CD36 might be an effective target in preventing the prothrombotic state in NVAF.     

Biophysical insight into the heparin‐peptide interaction and its modulation by a small molecule

The heparin‐protein interaction plays a vital role in numerous physiological and pathological processes. Not only is the binding mechanism of these interactions poorly understood, studies concerning their therapeutic targeting are also limited. Here, we have studied the interaction of the heparin interacting peptide (HIP) from Tat (which plays important role in HIV infections) with heparin. Isothermal titration calorimetry binding exhibits distinct biphasic isotherm with two different affinities in the HIP‐heparin complex formation. Overall, the binding was mainly driven by the nonionic interactions with a small contribution from ionic interactions. The stoichiometric analysis suggested that the minimal site for a single HIP molecule is a chain of 4 to 5 saccharide molecules, also supported by docking studies. The investigation was also focused on exploiting the possibility of using a small molecule as an inhibitor of the HIP‐heparin complex. Quinacrine, because of its ability to mimic the HIP interactions with heparin, was shown to successfully modulate the HIP‐heparin interactions. This result demonstrates the feasibility of inhibiting the disease relevant heparin‐protein interactions by a small molecule, which could be an effective strategy for the development of future therapeutic agents.     

Characterizing the S‐layer structure and anti‐S‐layer antibody recognition on intact Tannerella forsythia cells by scanning probe microscopy and small angle X‐ray scattering

Tannerella forsythia is among the most potent triggers of periodontal diseases, and approaches to understand underlying mechanisms are currently intensively pursued. A ~22‐nm‐thick, 2D crystalline surface (S‐) layer that completely covers Tannerella forsythia cells is crucially involved in the bacterium–host cross‐talk. The S‐layer is composed of two intercalating glycoproteins (TfsA‐GP, TfsB‐GP) that are aligned into a periodic lattice. To characterize this unique S‐layer structure at the nanometer scale directly on intact T. forsythia cells, three complementary methods, i.e., small‐angle X‐ray scattering (SAXS), atomic force microscopy (AFM), and single‐molecular force spectroscopy (SMFS), were applied. SAXS served as a difference method using signals from wild‐type and S‐layer‐deficient cells for data evaluation, revealing two possible models for the assembly of the glycoproteins. Direct high‐resolution imaging of the outer surface of T. forsythia wild‐type cells by AFM revealed a p4 structure with a lattice constant of ~9.0 nm. In contrast, on mutant cells, no periodic lattice could be visualized. Additionally, SMFS was used to probe specific interaction forces between an anti‐TfsA antibody coupled to the AFM tip and the S‐layer as present on T. forsythia wild‐type and mutant cells, displaying TfsA‐GP alone. Unbinding forces between the antibody and wild‐type cells were greater than with mutant cells. This indicated that the TfsA‐GP is not so strongly attached to the mutant cell surface when the co‐assembling TfsB‐GP is missing. Altogether, the data gained from SAXS, AFM, and SMFS confirm the current model of the S‐layer architecture with two intercalating S‐layer glycoproteins and TfsA‐GP being mainly outwardly oriented. Copyright © 2013 John Wiley & Sons, Ltd.     

Depletion interactions and modulation of DNA‐intercalators binding: Opposite behavior of the “neutral” polymer poly(ethylene‐glycol)

In this work we have investigated the role of high molecular weight poly(ethylene‐glycol) 8000 (PEG 8000) in modulating the interactions of the DNA molecule with two hydrophobic compounds: Ethidium Bromide (EtBr) and GelRed (GR). Both compounds are DNA intercalators and are used here to mimic the behavior of more complex DNA ligands such as chemotherapeutic drugs and proteins whose domains intercalate DNA. By means of single‐molecule stretching experiments, we have been able to show that PEG 8000 strongly shifts the binding equilibrium between the intercalators and the DNA even at very low concentrations (1% in mass). Additionally, microcalorimetry experiments were performed to estimate the strength of the interaction between PEG and the DNA ligands. Our results suggest that PEG, depending on the system under study, may act as an “inert polymer” with no enthalpic contribution in some processes but, on the other hand, it may as well be an active (non‐neutral) osmolyte in the context of modulating the activity of the reactants and products involved in DNA‐ligand interactions. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 227–233, 2016.     

Quantification of heparin's antimetastatic effect by single‐cell force spectroscopy

In circulation, cancer cells induce platelet activation, leading to the formation of a cancer cell‐encircling platelet cloak which facilitates each step of the metastatic cascade. Since cancer patients treated with the anticoagulant heparin showed reduced metastasis rates and improved survival, it is supposed that heparin suppresses the cloak's formation by inhibiting the interaction between platelet's adhesion molecule P‐selectin with its ligands on cancer cells. To quantify this heparin effect, we developed a single‐cell force spectroscopy approach and quantified the adhesion (maximum adhesion force [F_A] and detachment work [W_D]) between platelets and human non‐small cell lung cancer cells (A549). A configuration was used in which A549 cells were glued to tipless cantilevers and force‐distance (F‐D) curves were recorded on a layer of activated platelets. The concentration‐response relationship was determined for heparin at concentrations between 1 and 100 U/mL. Sigmoid dose‐response fit revealed half‐maximal inhibitory concentration (IC₅₀) values of 8.01 U/mL (F_A) and 6.46 U/mL (W_D) and a maximum decrease of the adhesion by 37.5% (F_A) and 38.42% (W_D). The effect of heparin on P‐selectin was tested using anti‐P‐selectin antibodies alone and in combination with heparin. Adding heparin after antibody treatment resulted in an additional reduction of 9.52% (F_A) and 7.12% (W_D). Together, we quantified heparin's antimetastatic effect and proved that it predominantly is related to the blockage of P‐selectin. Our approach represents a valuable method to investigate the adhesion of platelets to cancer cells and the efficiency of substances to block this interaction.     

Analysis of marker compounds with anti‐platelet aggregation effects in mailuoning injection using platelet binding assay combined with HPLC‐DAD‐ESI‐MS and solid‐phase extraction technique

Introduction – Mailuoning is prepared from a traditional formula of Chinese medicines and widely used as an antithrombotic agent. In this study, the platelet binding assay was used as a novel biospecific separation and analysis method to explore its active constituents, which could be considered as marker compounds for quality control. Objective – To establish a rapid and simple method to predict marker compounds in herbal medicine injection and evaluate the effects of those compounds. Material and methods – Platelets were used to bind and separate constituents. Binding constituents were analysed and taken as potential active compounds for further evaluation. Solid‐phase‐extraction was adopted to improve sensitivity. HPLC‐DAD and ESI‐MS were used to determine the binding constituents. Results – Five compounds were extracted through the platelet binding process and identified as neochlorogenic acid, caffeic acid, isochlorogenic acid and their isomers. Caffeic acid was selected for the flow cytometric assay to test its effect on platelets activation, which was determined by CD62P (P‐selectin) expression. The results indicated that caffeic acid could significantly inhibit platelet activation while chlorogenic acid did not. Conclusion – Caffeic acid could be considered as a marker compound of Mailuoning injection due to its anti‐platelet effect. The study also suggested that platelet binding assay combined with some preconcentration technique could be efficiently used to predict anti‐platelet compounds in complicated herbal medicines. Copyright © 2010 John Wiley & Sons, Ltd.     

Helicobacter pylori urease activates blood platelets through a lipoxygenase‐mediated pathway

The bacterium Helicobacter pylori causes peptic ulcers and gastric cancer in human beings by mechanisms yet not fully understood. H. pylori produces urease which neutralizes the acidic medium permitting its survival in the stomach. We have previously shown that ureases from jackbean, soybean or Bacillus pasteurii induce blood platelet aggregation independently of their enzyme activity by a pathway requiring platelet secretion, activation of calcium channels and lipoxygenase‐derived eicosanoids. We investigated whether H. pylori urease displays platelet‐activating properties and defined biochemical pathways involved in this phenomenon. For that the effects of purified recombinant H. pylori urease (HPU) added to rabbit platelets were assessed turbidimetrically. ATP secretion and production of lipoxygenase metabolites by activated platelets were measured. Fluorescein‐labelled HPU bound to platelets but not to erythrocytes. HPU induced aggregation of rabbit platelets (ED₅₀ 0.28 μM) accompanied by ATP secretion. No correlation was found between platelet activation and ureolytic activity of HPU. Platelet aggregation was blocked by esculetin (12‐lipoxygenase inhibitor) and enhanced ～3‐fold by indomethacin (cyclooxygenase inhibitor). A metabolite of 12‐lipoxygenase was produced by platelets exposed to HPU. Platelet responses to HPU did not involve platelet‐activating factor, but required activation of verapamil‐inhibitable calcium channels. Our data show that purified H. pylori urease activates blood platelets at submicromolar concentrations. This property seems to be common to ureases regardless of their source (plant or bacteria) or quaternary structure (single, di‐ or tri‐chain proteins). These properties of HPU could play an important role in pathogenesis of gastrointestinal and associated cardiovascular diseases caused by H. pylori.     

Molecular basis of the head‐to‐tail assembly of giant muscle proteins obscurin‐like 1 and titin

Large filament proteins in muscle sarcomeres comprise many immunoglobulin‐like domains that provide a molecular platform for self‐assembly and interactions with heterologous protein partners. We have unravelled the molecular basis for the head‐to‐tail interaction of the carboxyl terminus of titin and the amino‐terminus of obscurin‐like‐1 by X‐ray crystallography. The binary complex is formed by a parallel intermolecular β‐sheet that presents a novel immunoglobulin‐like domain‐mediated assembly mechanism in muscle filament proteins. Complementary binding data show that the assembly is entropy‐driven rather than dominated data by specific polar interactions. The assembly observed leads to a V‐shaped zipper‐like arrangement of the two filament proteins.     

Newly Identified HNP‐F from Human Neutrophil Peptide‐1 Promotes Hemostasis

Active hemostatic agents can play a crucial role in saving patients’ lives during surgery. Active hemostats have several advantages including utilization of natural blood coagulation and biocompatibility. Among them, although human neutrophil peptide‐1 (HNP‐1) has been previously reported with the hemostatic mechanism, which part of HNP‐1 facilitates the hemostatic activity is not known. Here, a partial peptide (HNP‐F) promoting hemostasis, originating from HNP‐1, has been newly identified by the blood coagulation ability test. HNP‐F shows the best hemostatic effect between the anterior half and posterior half of peptides. Moreover, microscopic images show platelet aggregation and an increase in the concentration of platelet factor 4, and the scanning electron microscope image of platelets support platelet activation by HNP‐F. Thromboelastography indicates decreased clotting time and increased physical properties of blood clotting. Mouse liver experiments demonstrate improved hemostatic effect by treatment of peptide solution. Cell viability and hemolysis assays confirm the HNP‐F's biosafety. It is hypothesized that the surface charge and structure of HNP‐F could be favorable to interact with fibrinogen or thrombospondin‐1. Collectively, because HNP‐F as an active peptide hemostat has many advantages, it could be expected to become a potent hemostatic biomaterial, additive or pharmaceutical candidate for various hemostatic applications.     

A generalized knowledge‐based discriminatory function for biomolecular interactions

Several novel and established knowledge‐based discriminatory function formulations and reference state derivations have been evaluated to identify parameter sets capable of distinguishing native and near‐native biomolecular interactions from incorrect ones. We developed the r·m·r function, a novel atomic level radial distribution function with mean reference state that averages over all pairwise atom types from a reduced atom type composition, using experimentally determined intermolecular complexes in the Cambridge Structural Database (CSD) and the Protein Data Bank (PDB) as the information sources. We demonstrate that r·m·r had the best discriminatory accuracy and power for protein‐small molecule and protein‐DNA interactions, regardless of whether the native complex was included or excluded, from the test set. The superior performance of the r·m·r discriminatory function compared with seventeen alternative functions evaluated on publicly available test sets for protein‐small molecule and protein‐DNA interactions indicated that the function was not over optimized through back testing on a single class of biomolecular interactions. The initial success of the reduced composition and superior performance with the CSD as the distribution set over the PDB implies that further improvements and generality of the function are possible by deriving probabilities from subsets of the CSD, using structures that consist of only the atom types to be considered for given biomolecular interactions. The method is available as a web server module at http://protinfo.compbio.washington.edu . Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Differential effects of 24‐hydroxycholesterol and 27‐hydroxycholesterol on tyrosine hydroxylase and α‐synuclein in human neuroblastoma SH‐SY5Y cells

Evidence suggests that environmental and dietary factors may contribute to the pathogenesis of Parkinson’s disease (PD). High dietary intake of cholesterol is such a factor that has been shown to increase or decrease the risk of PD. However, because circulating cholesterol does not cross the blood–brain barrier, the mechanisms linking dietary cholesterol to the pathogenesis of PD remain to be understood. In contrast to cholesterol, the oxidized cholesterol metabolites (oxysterols), 24S‐hydroxycholesterol (24‐OHC) and 27‐hydroxycholesterol (27‐OHC), can cross the blood–brain barrier and may place the brain at risk of degeneration. In this study, we incubated the human neuroblastoma SH‐SY5Y cells for 24 h with 24‐OHC, 27‐OHC, or a mixture of 24‐OHC plus 27‐OHC, and have determined effects on tyrosine hydroxylase (the rate‐limiting enzyme in dopamine synthesis) levels, α‐synuclein levels, and apoptosis. We demonstrate that while 24‐OHC increases the levels of tyrosine hydroxylase, 27‐OHC increases levels of α‐synuclein, and induces apoptosis. Our findings show for the first time that oxysterols trigger changes in levels of proteins that are associated with the pathogenesis of PD. As steady state levels of 24‐OHC and 27‐OHC are tightly regulated in the brain, disturbances in these levels may contribute to the pathogenesis of PD.     

A fluorescent reporter for mapping cellular protein‐protein interactions in time and space

We introduce a fluorescent reporter for monitoring protein–protein interactions in living cells. The method is based on the Split‐Ubiquitin method and uses the ratio of two auto‐fluorescent reporter proteins as signal for interaction (SPLIFF). The mating of two haploid yeast cells initiates the analysis and the interactions are followed online by two‐channel time‐lapse microscopy of the diploid cells during their first cell cycle. Using this approach we could with high spatio‐temporal resolution visualize the differences between the interactions of the microtubule binding protein Stu2p with two of its binding partners, monitor the transient association of a Ran‐GTPase with its receptors at the nuclear pore, and distinguish between protein interactions at the polar cortical domain at different phases of polar growth. These examples further demonstrate that protein–protein interactions identified from large‐scale screens can be effectively followed up by high‐resolution single‐cell analysis.     

Relation between TLR4/NF‐κB signaling pathway activation by 27‐hydroxycholesterol and 4‐hydroxynonenal,and atherosclerotic plaque instability

It is now thought that atherosclerosis, although due to increased plasma lipids, is mainly the consequence of a complicated inflammatory process, with immune responses at the different stages of plaque development. Increasing evidence points to a significant role of Toll‐like receptor 4 (TLR4), a key player in innate immunity, in the pathogenesis of atherosclerosis. This study aimed to determine the effects on TLR4 activation of two reactive oxidized lipids carried by oxidized low‐density lipoproteins, the oxysterol 27‐hydroxycholesterol (27‐OH) and the aldehyde 4‐hydroxynonenal (HNE), both of which accumulate in atherosclerotic plaques and play a key role in the pathogenesis of atherosclerosis. Secondarily, it examined their potential involvement in mediating inflammation and extracellular matrix degradation, the hallmarks of high‐risk atherosclerotic unstable plaques. In human promonocytic U937 cells, both 27‐OH and HNE were found to enhance cell release of IL‐8, IL‐1β, and TNF‐α and to upregulate matrix metalloproteinase‐9 (MMP‐9) via TLR4/NF‐κB‐dependent pathway; these actions may sustain the inflammatory response and matrix degradation that lead to atherosclerotic plaque instability and to their rupture. Using specific antibodies, it was also demonstrated that these inflammatory cytokines increase MMP‐9 upregulation, thus enhancing the release of this matrix‐degrading enzyme by macrophage cells and contributing to plaque instability. These innovative results suggest that, by accumulating in atherosclerotic plaques, the two oxidized lipids may contribute to plaque instability and rupture. They appear to do so by sustaining the release of inflammatory molecules and MMP‐9 by inflammatory and immune cells, for example, macrophages, through activation of TLR4 and its NF‐κB downstream signaling.     

Molecular Targets for the Testing of COVID‐19

The pandemic outbreaks of coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), spread all over the world in a short period of time. Efficient identification of the infection by SARS‐CoV‐2 has been one of the most important tasks to facilitate all the following counter measurements in dealing with the infectious disease. In Taiwan, a COVID‐19 Open Science Platform adheres to the spirit of open science: sharing sources, data, and methods to promote progress in academic research while corroborating findings from various disciplines has established in mid‐February 2020, for collaborative research in support of the development of detection methods, therapeutics, and a vaccine for COVID‐19. Research priorities include infection control, epidemiology, clinical characterization and management, detection methods (including viral RNA detection, viral antigen detection, and serum antibody detection), therapeutics (neutralizing antibody and small molecule drugs), vaccines, and SARS‐CoV‐2 pathogenesis. In addition, research on social ethics and the law are included to take full account of the impact of the COVID‐19 virus.     

Nanomechanical interactions of phenylalanine-glycine nucleoporins studied by single molecule force-volume spectroscopy

Phenylalanine-glycine (FG)-repeat nucleoporins (Nups) form the major components of the selective gating mechanism in the nuclear pore complex (NPC). Hence, a primary requirement is to understand how they vacillate between preventing the access of passively diffusing molecules and promoting the translocation of receptor-bound cargo into the NPC. To shed light on such behavior, we have studied the nanomechanical properties of a cysteine-modified FG-rich C-terminal domain of hNup153 (i.e., cNup153) and its interactions with importin-beta. This is carried out using single molecule force spectroscopy (SMFS) with the atomic force microscope (AFM). In the absence of importin-beta, cNup153 is highly flexible and can be reversibly stretched and relaxed without any change to its intrinsic entropic elasticity, indicating a lack of intra-FG interactions, i.e., natively unfolded. Importin-beta-modified AFM tips reveal complex binding topologies with cNup153, and provide evidence for binding promiscuity in FG-receptor interactions. These differences suggest that cooperativity between FG-domains arises from FG-receptor interactions instead of FG-FG interactions. On a technical note, this work highlights an improved SMFS technique which involves pre-passivating the underlying substrate surface with polyethylene glycol to reduce undesirable AFM tip-surface effects. A high yield of acceptable data is subsequently obtained from the low surface coverage of target molecules by implementing SMFS measurements in force-volume (FV) mode.     

Human estrogen sulfotransferase and its related fluorescently labeled decapeptides specifically interact with oxidized low‐density lipoprotein

Estrogen sulfotransferase (SULT1E) mainly catalyzes the sulfation of estrogens, which are known to prevent the pathogenesis of atherosclerosis. Recently, we found that peptides with a YKDG sequence specifically bind to oxidized low‐density lipoprotein (Ox‐LDL), which plays a major role in the pathogenesis of atherosclerosis. Here, we investigated the interaction between human SULT1E1 (hSULT1E1), which has a YKEG sequence (residues 61–64) unlike other human SULTs, and Ox‐LDL. Results from polyacrylamide gel electrophoresis and western blotting demonstrated that hSULT1E1 specifically binds to Ox‐LDL and its major lipid component (lysophosphatidylcholine; LPC), and platelet‐activating factor (PAF), which bears a marked resemblance to LPC in terms of structure and activity. Moreover, an N‐terminally fluorescein isothiocyanate (FITC)‐labeled decapeptide (MIYKEGDVEK; FITC‐hSULT1E1‐P10) corresponding to residues 59–68 of hSULT1E1 specifically binds to Ox‐LDL, LPC, and PAF. Unveiling the specific interaction between hSULT1E1 and Ox‐LDL, LPC, and PAF provides important information regarding the mechanisms underlying various diseases caused by Ox‐LDL, LPC, and PAF, such as atherosclerosis. In addition, FITC‐hSULT1E1‐P10 could be used as an efficient fluorescent probe for the detection of Ox‐LDL, LPC, and PAF, which could facilitate the mechanistic study, identification, diagnosis, prevention, and treatment of atherosclerosis.     

Mass spectrometry‐based secretome analysis of non‐small cell lung cancer cell lines

Tyrosine kinase inhibitors, such as erlotinib, display reliable responses and survival benefits for the treatment of human non‐small cell lung cancer (NSCLC) patients. However, primary or acquired resistance limits their therapeutic success. In this study, we conducted in‐depth mass spectrometric analyses of NSCLC cell secretomes. To identify secreted proteins that are differentially regulated in erlotinib‐sensitive (PC‐9) and ‐resistant (PC‐9ER) NSCLC cell lines, SILAC experiments were performed. On average, 900 proteins were identified in each sample with low variations in the numbers of identified proteins. Fourteen proteins were found to be differently regulated among erlotinib‐sensitive and ‐resistant NSCLC cell lines, with five proteins (tissue‐type plasminogen activator, epidermal growth factor receptor, urokinase‐type plasminogen activator, platelet‐derived growth factor D, and myeloid‐derived growth factor) showing the most prominent regulation. Tissue‐type plasminogen activator (t‐PA) was up to 10‐times upregulated in erlotinib‐resistant NSCLC cells compared with erlotinib‐sensitive cells. T‐PA is an established tumor marker for various cancer types and seems to be a promising prognostic marker to differentiate erlotinib‐sensitive from erlotinib‐resistant NSCLC cells. To gain further insights into t‐PA‐regulated pathways, a t‐PA variant was expressed in E. coli cells and its interactions with proteins secreted from erlotinib‐sensitive and ‐resistant NCSLC cells were studied by a combined affinity enrichment chemical cross‐linking/mass spectrometry (MS) approach. Fourteen proteins were identified as potential t‐PA interaction partners, deserving a closer inspection to unravel the mechanisms underlying erlotinib resistance in NSCLC cells.     

Super‐beacons: Open‐source probes with spontaneous tuneable blinking compatible with live‐cell super‐resolution microscopy

Localization‐based super‐resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non‐fluorescent states. These transitions are commonly regulated by high‐intensity illumination, imposing constrains to imaging hardware and producing sample photodamage. Here, we propose single‐molecule self‐quenching as a mechanism to generate spontaneous photoswitching. To demonstrate this principle, we developed a new class of DNA‐based open‐source super‐resolution probes named super‐beacons, with photoswitching kinetics that can be tuned structurally, thermally and chemically. The potential of these probes for live‐cell compatible super‐resolution microscopy without high‐illumination or toxic imaging buffers is revealed by imaging interferon inducible transmembrane proteins (IFITMs) at sub‐100 nm resolutions.