Intracellular and extracellular moesins differentially regulate Src activity and ß-catenin translocation to the nucleus in breast cancer cells.

It is increasingly recognized that a single protein can have multiple, sometimes paradoxical, roles in cell functions as well as pathological conditions depending on its cellular locations. Here we report that moesins (MSNs) in the intracellular and extracellular domains present opposing roles in pro-tumorigenic signaling in breast cancer cells. Using live cell imaging with fluorescence resonance energy transfer (FRET)- and green fluorescent protein (GFP)-based biosensors, we investigated the molecular mechanism underlying the cellular location-dependent effect of MSN on Src and ß-catenin signaling in MDA-MB-231 breast cancer cells. Inhibition of intracellular MSN decreased the activities of Src and FAK, whereas overexpression of intracellular MSN increased them. By contrast, extracellular MSN decreased the activities of Src, FAK, and RhoA, as well as ß-catenin translocation to the nucleus. Consistently, Western blotting and MTT-based analysis showed that overexpression of intracellular MSN elevated the expression of oncogenic genes, such as p-Src, ß-catenin, Lrp5, MMP9, Runx2, and Snail, as well as cell viability, whereas extracellular MSN suppressed them. Conditioned medium derived from MSN-overexpressing mesenchymal stem cells or osteocytes showed the anti-tumor effects by inhibiting the Src activity and ß-catenin translocation to the nucleus as well as the activities of FAK and RhoA and MTT-based cell viability. Conditioned medium derived from MSN-inhibited cells increased the Src activity, but it did not affect the activities of FAK and RhoA. Silencing CD44 and/or FN1 in MDA-MB-231 cells blocked the suppression of Src activity and ß-catenin accumulation in the nucleus by extracellular MSN. Collectively, the results suggest that cellular location-specific MSN is a strong regulator of Src and ß-catenin signaling in breast cancer cells, and that extracellular MSN exerts tumor-suppressive effects via its interaction with CD44 and FN1.

Enhancement of electrode performance through surface modification using carbon nanotubes and porous gold nanostructures.

Recently, the demand for the sensitive detection of nanomaterials and biomolecules has been increasing for evaluating the toxicity of nanomaterials and early diagnosis of diseases. Although many studies have developed new detection assays, these are heavily influenced by the capabilities of the detection equipment. Therefore, the aim of the present study was to improve electrode performance by modifying the surface of the detection electrode using a simple method. Electrode surface modification was performed using carbon nanotubes (CNT) and porous gold nanostructures (NS) with excellent electrical and chemical properties. Through the simple physical deposition of CNT and electrochemical reduction of NS, the increasement of the electrode surface area was achieved. Because of the CNTs attached to the electrodes at the first step, the metal ions constituting the NS can adhere well to the electrodes. Nanoparticles with a porous structure can be generated through electrochemical reduction (cyclic voltammetry) of metal ions attached to electrodes. Consequently, the surface area of the electrode increased and electrochemical performance was improved (confirmed by atomic force microscopy, Nyquist plot and Bode plot). To quantitatively confirm the improvement of electrode performance according to the surface change through the proposed treatment technique, DNA was detected. Unlike previous surface modification studies, the developed surface treatment technique can be applied to a variety of detection equipment. To confirm this, the detection was performed using two detection devices with different operating principles. DNA detection using the two types of equipment confirmed that the detection limit was increased by approximately 1000-fold through applying a simple surface treatment. In addition, this method is applicable to detect various sizes of nanomaterials. The method proposed in this study is simple and has the advantage that it can be applied to various devices and various materials.

Fluid flow-induced activation of subcellular AMPK and its interaction with FAK and Src.

AMP-activated protein kinase (AMPK) is a metabolic energy sensor that plays a critical role in cancer cell survival and growth. While the physical microenvironment is believed to influence tumor growth and progression, its role in AMPK regulation remains largely unknown. In the present study, we evaluated AMPK response to mechanical forces and its interaction with other mechano-responsive signaling proteins, FAK and Src. Using genetically encoded biosensors that can detect AMPK activities at different subcellular locations (cytosol, plasma membrane, nucleus, mitochondria, and Golgi apparatus), we observed that AMPK responds to shear stress in a subcellular location-dependent manner in breast cancer cells (MDA-MB-231). While normal epithelial cells (MCF-10A) also similarly responded to shear stress, they are less sensitive to shear stress compared to MDA-MB-231 cells. Inhibition of FAK and Src significantly decreased the basal activity level of AMPK at all five subcellular locations in MDA-MB-231 cells and selectively blocked shear stress-induced AMPK activation. Moreover, testing with cytoskeletal drugs revealed that myosin II might be the critical mediator of shear stress-induced AMPK activation in MDA-MB-231 cells. These findings suggest that breast cancer cells and normal epithelial cells may have different mechanosensitivity in AMPK signaling and that FAK and Src as well as the myosin II-dependent signaling pathway are involved in subcellular AMPK mechanotransduction in breast cancer cells.

Mechanotransduction of mitochondrial AMPK and its distinct role in flow-induced breast cancer cell migration.

The biophysical microenvironment of the tumor site has significant impact on breast cancer progression and metastasis. The importance of altered mechanotransduction in cancerous tissue has been documented, yet its role in the regulation of cellular metabolism and the potential link between cellular energy and cell migration remain poorly understood. In this study, we investigated the role of mechanotransduction in AMP-activated protein kinase (AMPK) activation in breast cancer cells in response to interstitial fluid flow (IFF). Additionally, we explored the involvement of AMPK in breast cancer cell migration. IFF was applied to the 3D cell-matrix construct. The subcellular signaling activity of Src, FAK, and AMPK was visualized in real-time using fluorescent resonance energy transfer (FRET). We observed that breast cancer cells (MDA-MB-231) are more sensitive to IFF than normal epithelial cells (MCF-10A). AMPK was activated at the mitochondria of MDA-MB-231 cells by IFF, but not in other subcellular compartments (i.e., cytosol, plasma membrane, and nucleus). The inhibition of FAK or Src abolished flow-induced AMPK activation in the mitochondria of MDA-MB-231 cells. We also observed that global AMPK activation reduced MDA-MB-231 cell migration. Interestingly, specific AMPK inhibition in the mitochondria reduced cell migration and blocked flow-induced cell migration. Our results suggest the linkage of FAK/Src and mitochondria-specific AMPK in mechanotransduction and the differential role of AMPK in breast cancer cell migration depending on its subcellular compartment-specific activation.

Length-Dependent Manifestation of Vibration Modes Regulates a Specific Intermediate Morphology of Aß17-42 in Different Environments.

Various cytotoxic mechanisms for neurodegenerative disease are induced by specific conformations of Aß intermediates. The efforts to understand the diverse intermediate forms of amyloid oligomers have been focused on understanding the aggregation mechanism of specific morphologies for Aß intermediates. However, these are still not easy tasks to be accomplished because the diverse conformations of Aß intermediates can be altered during the aggregation process, even though the same Aß monomers are present. Thus, efforts to reveal the conformational change mechanism could be a fundamental process to understand the formation of diverse Aß intermediate conformations. Here, we evaluate the conformational characteristics of Aß17-42 fibrillar oligomers in different environments according to the length. We observed that Aß fibrillar oligomers optimize their inherent hydrogen bonds and configurational entropy to stabilize their structure according to the simulation time and their length increase. In addition, we revealed the role of the expressed vibration mode shape in the fibrillar oligomers' elongation and deformation processes. Our results suggest that limitations in amyloid oligomer growth and transformations of their morphologies can be regulated and controlled by modifying the vibration features.

Metal ions affect the formation and stability of amyloid ß aggregates at multiple length scales.

Amyloid ß (Aß) aggregates, which are a hallmark for neurodegenerative disease, are formed through a self-assembly process such as aggregation of Aß peptide chains. This aggregation process depends on the solvent conditions under which the proteins are aggregated. Nevertheless, the underlying mechanism of the ionic effect on the formation and stability of amyloid aggregates has not been fully understood. Here, we report how metal ions play a role in the formation and stability of Aß aggregates at different length scales, i.e. oligomers and fibrils. It is shown that the metal (i.e. zinc or copper) ion increases the stability of Aß oligomers, whereas the metal ion reduces the stability of Aß fibrils. In addition, we found that zinc ions are able to more effectively destabilize fibril structures than copper ions. Metal ion-mediated (de)stabilization of Aß oligomers (or fibrils) is attributed to the critical effect of the metal ion on the ß-sheet rich crystalline structure of the amyloid aggregate and the status of hydrogen bonds within the aggregate. Our study sheds light on the role of the metal ion in stabilizing the amyloid oligomers known as a toxic agent (to functional cells), which is consistent with clinical observation that high concentrations of metal ions are found in patients suffering from neurodegenerative diseases.

Double amplified colorimetric detection of DNA using gold nanoparticles, enzymes and a catalytic hairpin assembly.

The authors describe an isothermal and ultrasensitive colorimetric DNA assay that consists of two amplification stages using enzymes and a catalytic hairpin assembly (CHA). The first step consists in the selective amplification of DNA using Klenow fragment and nicking enzyme. The second step consists in the amplification of the optical signal by using a catalytic hairpin assembly. After two amplification steps, the DNA reaction induces the aggregation of the red gold nanoparticles to give a blue color shift. The degree of aggregation can be quantified by measurement of the ratio of the UV-vis absorbances of the solutions at 620 and 524 nm which are the wavelengths of the aggregated gold nanoparticles and bare gold nanoparticles. The detection limit is as low as 3.1 fM. Due to the use of a specific enzyme, only the desired DNAs will be detected. The method can be applied to the determination of DNA of various lengths. Despite the presence of large amounts of wildtype DNA, it can readily detect a target DNA. Conceivably, the technique has a large potential because of its high sensitivity and selectivity. Graphical abstract Schematic presentation of DNA detection using gold nanoparticles (AuNP), enzymes and catalytic hairpin assembly (CHA). Effective DNA detection is achieved through the aggregation of AuNPs which is caused by DNA amplification using enzymes and signal amplification using CHA.

Structural analysis of oligomeric and protofibrillar Aß amyloid pair structures considering F20L mutation effects using molecular dynamics simulations.

Aß amyloid proteins are involved in neuro-degenerative diseases such as Alzheimer's, Parkinson's, and so forth. Because of its structurally stable feature under physiological conditions, Aß amyloid protein disrupts the normal cell function. Because of these concerns, understanding the structural feature of Aß amyloid protein in detail is crucial. There have been some efforts on lowering the structural stabilities of Aß amyloid fibrils by decreasing the aromatic residues characteristic and hydrophobic effect. Yet, there is a lack of understanding of Aß amyloid pair structures considering those effects. In this study, we provide the structural characteristics of wildtype (WT) and phenylalanine residue mutation to leucine (F20L) Aß amyloid pair structures using molecular dynamics simulation in detail. We also considered the polymorphic feature of F20L and WT Aß pair amyloids based on the facing ß-strand directions between the amyloid pairs. As a result, we were able to observe the varying effects of mutation, polymorphism, and protofibril lengths on the structural stability of pair amyloids. Furthermore, we have also found that opposite structural stability exists on a certain polymorphic Aß pair amyloids depending on its oligomeric or protofibrillar state, which can be helpful for understanding the amyloid growth mechanism via repetitive fragmentation and elongation mechanism. Proteins 2017; 85:580-592. © 2016 Wiley Periodicals, Inc.

Characterizing Structural Stability of Amyloid Motif Fibrils Mediated by Water Molecules.

In biological systems, structural confinements of amyloid fibrils can be mediated by the role of water molecules. However, the underlying effect of the dynamic behavior of water molecules on structural stabilities of amyloid fibrils is still unclear. By performing molecular dynamics simulations, we investigate the dynamic features and the effect of interior water molecules on conformations and mechanical characteristics of various amyloid fibrils. We find that a specific mechanism induced by the dynamic properties of interior water molecules can affect diffusion of water molecules inside amyloid fibrils, inducing their different structural stabilities. The conformation of amyloid fibrils induced by interior water molecules show the fibrils' different mechanical features. We elucidate the role of confined and movable interior water molecules in structural stabilities of various amyloid fibrils. Our results offer insights not only in further understanding of mechanical features of amyloids as mediated by water molecules, but also in the fine-tuning of the functional abilities of amyloid fibrils for applications.

A microcantilever-based silver ion sensor using DNA-functionalized gold nanoparticles as a mass amplifier.

Silver ions have been used to sterilize many products, however, it has recently been demonstrated that silver ions can be toxic. This toxicity has been studied over many years with the lethal concentration at 10 µM. Silver ions can accumulate through the food chain, causing serious health problems in many species. Hence, there is a need for a commercially available silver ion sensor, with high detection sensitivity. In this work, we develop an ultra-sensitive silver ion sensor platform, using cytosine based DNA and gold nanoparticles as the mass amplifier. We achieve a lower detection limit for silver ions of 10 pM; this detection limit is one million times lower than the toxic concentration. Using our sensor platform we examine highly selective characteristics of other typical ions in water from natural sources. Furthermore, our sensor platform is able to detect silver ions in a real practical sample of commercially available drinking water. Our sensor platform, which we have termed a 'MAIS' (mass amplifier ion sensor), with a simple detection procedure, high sensitivity, selectivity and real practical applicability has shown potential as an early toxicity assessment of silver ions in the environment.

Mechanical and vibrational characterization of amyloid-like HET-s nanosheets based on the skewed plate theory.

Pathological amyloidogenic prion proteins have a toxic effect on functional cells in the human cerebrum because of poor degradability and the tendency to accumulate in an uncontrolled manner under physiological conditions. HET-s, a fungal prion protein, is known to undergo conformational variations from fibrillar to nanosheet structures during a change from low to high pH conditions. It has been said that this conformational change can lead to self-propagation by nucleating on the lateral surface of singlet fibrils. Efforts have been made toward the mechanical characterization of fibrillar amyloids, but a global understanding of amyloid-like HET-s nanosheet structures is lacking. In this study, we analyzed the mechanical and vibrational characteristics of the skewed HET-s nanosheet structures that developed under neutral pH conditions by performing various molecular dynamics simulations. By applying the skewed plate theory to HET-s nanosheets for various length scales with numerous pores inside the structures, we found that the skewed HET-s nanosheet structure has mechanical properties comparable to those of previously reported biological film materials and nanomaterials. Considering the inherent characteristics of structural stability, our observation provides valuable and detailed structural information on skewed amyloid-like HET-s nanosheets.

Detection of Silver Ions Using Dielectrophoretic Tweezers-Based Force Spectroscopy.

Understanding of the interactions of silver ions (Ag+) with polynucleotides is important not only to detect Ag+ over a wide range of concentrations in a simple, robust, and high-throughput manner but also to investigate the intermolecular interactions of hydrogen and coordinate interactions that are generated due to the interplay of Ag+, hydrogen ions (H+), and polynucleotides since it is critical to prevent adverse environmental effects that may cause DNA damage and develop strategies to treat this damage. Here, we demonstrate a novel approach to simultaneously detect Ag+ satisfying the above requirements and examine the combined intermolecular interactions of Ag+-polycytosine and H+-polycytosine DNA complexes using dielectrophoretic tweezers-based force spectroscopy. For this investigation, we detected Ag+ over a range of concentrations (1 nM to 100 µM) by quantifying the rupture force of the combined interactions and examined the interplay between the three factors (Ag+, H+, and polycytosine) using the same assay for the detection of Ag+. Our study provides a new avenue not only for the detection of heavy metal ions but also for the investigation of heavy metal ions-polynucleotide DNA complexes using the same assay.

Sodium chloride's effect on self-assembly of diphenylalanine bilayer.

Understanding self-assembling peptides becomes essential in nanotechnology, thereby providing a bottom-up method for fabrication of nanostructures. Diphenylalanine constitutes an outstanding building block that can be assembled into various nanostructures, including two-dimensional bilayers or nanotubes, exhibiting superb mechanical properties. It is known that the effect of the ions is critical in conformational and chemical interactions of bilayers or membranes. In this study, we analyzed the effect of sodium chloride on diphenylalanine bilayer using coarse-grained molecular dynamics simulations, and calculated the bending Young's modulus and the torsional modulus by applying normal modal analysis using an elastic network model. The results showed that sodium chloride dramatically increases the assembling efficiency and stability, thereby promising to allow the precise design and control of the fabrication process and properties of bio-inspired materials. © 2016 Wiley Periodicals, Inc.

End Capping Alters the Structural Characteristics and Mechanical Properties of Transthyretin (105-115) Amyloid Protofibrils.

Pathological amyloid proteins are associated with degenerative and neurodegenerative diseases. These amyloid proteins develop as oligomer, fibrillar, and plaque forms, due to the denatured and unstable status of the amyloid monomers. Specifically, the development of fibrillar amyloid proteins has been investigated through several experimental studies. To understand the generation of amyloid fibrils, environmental factors such as point mutations, pH, and polymorphic characteristics have been considered. Recently, amyloid fibril studies related to end-capping effects have been conducted to understand amyloid fibril development. However, atomic-level studies to determine the stability and mechanical properties of amyloid fibrils based on end capping have not been undertaken. In this study, we show that end capping alters the structural characteristics and conformations of transthyretin (TTR) amyloid fibrils by using molecular dynamics (MD) simulations. Variation in the structural conformations and characteristics of the TTR fibrils through end capping are observed, due to the resulting electrostatic energies and hydrophobicity characteristics. Moreover, the end capping changes the mechanical properties of TTR fibrils. Our results shed light on amyloid fibril formation under end-capping conditions.

Highly sensitive, direct and real-time detection of silver nanowires by using a quartz crystal microbalance.

For several decades, silver nanomaterials (AgNMs) have been used in various research areas and commercial products. Among the many AgNMs, silver nanowires (AgNWs) are one of the mostly widely used nanomaterials due to their high electrical and thermal conductivity. However, recent studies have investigated the toxicity of AgNWs. For this reason, it is necessary to develop a successful detection method of AgNWs for protecting human health. In this study, label-free, highly sensitive, direct, and real-time detection of AgNWs is performed for the first time. The detection mechanism is based on the resonance frequency shift upon the mass change from the hybridization between the probe DNA on the electrode and the linker DNA attached on AgNWs. The frequency shift is measured by using a quartz crystal microbalance. We are able to detect 1 ng ml-1 of AgNWs in deionized water in real-time. Moreover, our detection method can selectively detect AgNWs among other types of one-dimensional nanomaterials and can also be applied to detection in drinking water.

Ultra-sensitive detection of zinc oxide nanowires using a quartz crystal microbalance and phosphoric acid DNA.

Recent advancements of nanomaterials have inspired numerous scientific and industrial applications. Zinc oxide nanowires (ZnO NWs) is one of the most important nanomaterials due to their extraordinary properties. However, studies performed over the past decade have reported toxicity of ZnO NWs. Therefore, there has been increasing demand for effective detection of ZnO NWs. In this study, we propose a method for the detection of ZnO NW using a quartz crystal microbalance (QCM) and DNA probes. The detection method is based on the covalent interaction between ZnO NWs and the phosphoric acid group of single-stranded DNA (i.e., linker DNA), and DNA hybridization between the linker DNA and the probe DNA strand on the QCM electrode. Rapid, high sensitivity, in situ detection of ZnO NWs was demonstrated for the first time. The limit of detection was 10(-4) µg ml(-1) in deionized water, which represents a sensitivity that is 100000 times higher than the toxic ZnO NW concentration level. Moreover, the selectivity of the ZnO NW detection method was demonstrated by comparison with other types of nanowires and the method was able to detect ZnO NWs in tap water sensitively even after stored for 14 d in a refrigerator. The performance of our proposed method was sufficient to achieve detection of ZnO NW in the 'real-world' environment.

Mechanical behavior comparison of spider and silkworm silks using molecular dynamics at atomic scale.

Spider and silkworm silk proteins have received much attention owing to their inherent structural stability, biodegradability, and biocompatibility. These silk protein materials have various mechanical characteristics such as elastic modulus, ultimate strength and fracture toughness. While the considerable mechanical characteristics of the core crystalline regions of spider silk proteins at the atomistic scale have been investigated through several experimental techniques and computational studies, there is a lack of comparison between spider and silkworm fibroins in the atomistic scale. In this study, we investigated the differences between the mechanical characteristics of spider and silkworm fibroin structures by applying molecular dynamics and steered molecular dynamics. We found that serine amino acids in silkworm fibroins not only increased the number of hydrogen bonds, but also altered their structural characteristics and mechanical properties.

Role of miR-222-3p in c-Src-Mediated Regulation of Osteoclastogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs that play a mostly post-transcriptional regulatory role in gene expression. Using RAW264.7 pre-osteoclast cells and genome-wide expression analysis, we identified a set of miRNAs that are involved in osteoclastogenesis. Based on in silico analysis, we specifically focused on miR-222-3p and evaluated its role in osteoclastogenesis. The results show that the inhibitor of miR-222-3p upregulated the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and tartrate-resistant acid phosphatase (TRAP), while its mimicking agent downregulated their mRNA levels. Western blot analysis showed that its inhibitor increased the protein levels of TRAP and cathepsin K, while its mimicking agent decreased their levels. Genome-wide mRNA expression analysis in the presence and absence of receptor activator of nuclear factor κ-B ligand (RANKL) predicted c-Src as a potential regulatory target of miR-222-3p. Live cell imaging using a fluorescence resonance energy transfer (FRET) technique revealed that miR-222-3p acted as an inhibitor of c-Src activity, and a partial silencing of c-Src suppressed RANKL-induced expression of TRAP and cathepsin K, as well as the number of multi-nucleated osteoclasts and their pit formation. Collectively, the study herein demonstrates that miR-222-3p serves as an inhibitor of osteoclastogenesis and c-Src mediates its inhibition of cathepsin K and TRAP.

Effects of lysine residues on structural characteristics and stability of tau proteins.

Pathological amyloid proteins have been implicated in neuro-degenerative diseases, specifically Alzheimer's, Parkinson's, Lewy-body diseases and prion related diseases. In prion related diseases, functional tau proteins can be transformed into pathological agents by environmental factors, including oxidative stress, inflammation, Aß-mediated toxicity and covalent modification. These pathological agents are stable under physiological conditions and are not easily degraded. This un-degradable characteristic of tau proteins enables their utilization as functional materials to capturing the carbon dioxides. For the proper utilization of amyloid proteins as functional materials efficiently, a basic study regarding their structural characteristic is necessary. Here, we investigated the basic tau protein structure of wild-type (WT) and tau proteins with lysine residues mutation at glutamic residue (Q2K) on tau protein at atomistic scale. We also reported the size effect of both the WT and Q2K structures, which allowed us to identify the stability of those amyloid structures.

Morphology and mechanical properties of multi-stranded amyloid fibrils probed by atomistic and coarse-grained simulations.

Amyloid fibrils are responsible for pathogenesis of various diseases and exhibit the structural feature of an ordered, hierarchical structure such as multi-stranded helical structure. As the multi-strandedness of amyloid fibrils has recently been found to be highly correlated with their toxicity and infectivity, it is necessary to study how the hierarchical (i.e. multi-stranded) structure of amyloid fibril is formed. Moreover, although it has recently been reported that the nanomechanics of amyloid proteins plays a key role on the amyloid-induced pathogenesis, a critical role that the multi-stranded helical structure of the fibrils plays in their nanomechanical properties has not fully characterized. In this work, we characterize the morphology and mechanical properties of multi-stranded amyloid fibrils by using equilibrium molecular dynamics simulation and elastic network model. It is shown that the helical pitch of multi-stranded amyloid fibril is linearly proportional to the number of filaments comprising the amyloid fibril, and that multi-strandedness gives rise to improving the bending rigidity of the fibril. Moreover, we have also studied the morphology and mechanical properties of a single protofilament (filament) in order to understand the effect of cross-ß structure and mutation on the structures and mechanical properties of amyloid fibrils. Our study sheds light on the underlying design principles showing how the multi-stranded amyloid fibril is formed and how the structure of amyloid fibrils governs their nanomechanical properties.