Nano-fishnet formation of silk controlled by Arginine density.

Silk fiber is renowned for its superb mechanical properties, such as over 7 times the toughness of Kevlar 49 Fibre. As the spider silk is tougher than any man-made fiber, there is a lot to be learned from spider silk. Recently, it has been reported that a large portion of the properties of silk is from naturally formed nano-fishnet structures of silk, but neither its formation mechanism nor its formation condition has been explained. Here, we show how the formation and disappearance of nano-fishnet of silk is determined by humidity, and how the humidity-dependency of nano-fishnet formation can be overcome by changing density of Arginine through sequence mutation. We demonstrate that the nano-fishnet-structured silk exhibits higher strength and toughness than its counterparts. This information on controllable nano-fishnet formation of silk is expected to pave the way for development of protein and synthetic fiber design. STATEMENT OF SIGNIFICANCE: Silk fibers are a very interesting material in that it exhibits superb mechanical properties such as 7 times the toughness of Kevlar 49 Fibre, despite being only composed of proteins. Therefore, it is important that we understand the principle of its high mechanical properties so that it may be applied in designing synthetic fibers. Recently, it has been reported that a large portion of its mechanical property comes from its nano-fishnet structures, but no detailed explanation on the condition or mechanism of formation. Through molecular dynamic simulations, we simulated the nano-fishnet formation of silk and analyzed the condition and mechanism behind it, and showed how the formation of nano-fishnet structures could be controlled by changing the density of Arginine residues. Our study provides information on fiber enhancement mechanism that could be applied to synthetic and protein fiber design.

Conformation Control of Amyloid Filaments by Repeated Thermal Perturbation.

We report that repeated thermal perturbation by thermal cycling (TC) accelerates the formation rate of amyloid filaments at microliter volumes (10-200 µL) and produces a new conformation of zigzag-shaped filaments. The amyloid filaments have been synthesized under different TC conditions, such as temperature variations (ΔT = 0-86 °C) and the number of cycles (C# = 30-90). In particular, the filament formation was promoted by TC with ΔT ≥ 30 °C. This indicates that the change in binding energy of ß-sheets and the breakage of disulfide bonds induced by TC with large ΔT contributed to the increased filament growth. This molecular interaction was investigated by molecular dynamics simulation. We also found that TC leads to the formation of amyloid filaments with peculiar conformation (zigzag-shaped filaments). Moreover, key structural parameters (tortuosity, segment length, and joint angle) of the amyloid filaments could be fine-tuned by selecting certain ΔT conditions. Taken together, we confirmed that the TC not only promotes the formation of amyloid filaments but also affects the conformational changes of the filaments.

Predicting the self-assembly film structure of class II hydrophobin NC2 and estimating its structural characteristics.

Hydrophobins are fungal proteins that can mediate water surface tension by forming amphiphilic self-assembly structures in hydrophobic-hydrophilic interfaces. Hydrophobins are known to self-assemble into two forms depending on their class: class I hydrophobins aggregate into a functional amyloid rodlet, while class II hydrophobins aggregate into a regularly patterned monolayer. Owing to its unique properties, hydrophobin has been considered as a biocompatible nanomaterial for various applications and there have been several attempts to engineer hydrophobins to enhance their function. Recently, a chimeric hydrophobin named NChi2 was found to be able to self-assemble into both rodlet and monolayer forms depending on the incubating environment. Although this remarkable feature suggests that NChi2 can function as a versatile bionanomaterial for various applications, only little information about the protein, such as its assembly structure or its characteristics, is provided. To investigate the extraordinary behavior of NChi2, it seems to be a prerequisite to first understand the characteristics of its parent hydrophobins, namely class I EAS and class II NC2. Here, we conducted a preliminary study on predicting the self-assembly structure of class II hydrophobin NC2 and estimating its structural characteristics by employing several computational methods. From the results, we found that NC2 shows stronger surface activity than HFBII, while its assembly structure is weaker than that of HFBII. We hope that this research serves as a foundation to further investigate the structural characteristics of a unique hydrophobin NChi2 in future studies.

Bioinspired Micro Glue Threads Fabricated by Liquid Bridge-to-Solidification as an Effective Sensing Platform.

Spiders synthesize their web using a liquid bridge-to-solidification mechanism at the end of their glands. Inspired by this process, in this work, we fabricated micro-glue threads (µGTs, polymer microwires) by a simple "pinch and spread" process using just two fingertips. The µGTs exhibited excellent tensile strength (∼50 GPa), comparable to those of spider silk and biological fibers. The chemical, physical, and mechanical properties of the µGTs were investigated, and it was confirmed that the thickness of the µGTs could be controlled by ethanol treatment in varying concentrations. Moreover, electrically conductive µGTs were easily fabricated by simply mixing them with various nanomaterials such as gold nanoparticles, zinc oxide nanowires, and reduced graphene oxide (rGO). Interestingly, the conductive µGTs, fabricated using rGO, exhibited remarkable electrical conductivity (0.45 µS) compared to those fabricated using other materials. The conductive µGTs are applicable not only to NO2 gas sensing but also as electrical fuselike materials that melt when the humidity increases. Collectively, the results present µGTs as cost-effective, simple, and versatile materials, which enables their application in a variety of sensors.

Spider silk with weaker bonding resulting in higher strength and toughness through progressive unfolding and load transfer.

The superior mechanical properties of silk is known to come partly from its hydrogen bonds, which is determined by its amino acid sequences. Hydrogen bonds are one of the main sources of strength of silk fiber, yet the toughest silk fibers have amino acids sequences that results in lesser number of hydrogen bonds than other silk fibers. In this work, we show how such silk fiber with lower number of hydrogen bonds may result in fiber with higher toughness by investigating the process of how hydrogen bond characteristics of silk are translated into its mechanical properties. From the tensile pulling tests via molecular dynamics simulations on silk fiber with varying number of hydrogen bonds, the mechanism of how weaker bonded silk results in higher strength and toughness by synergic effect with the characteristic progressive unfolding and load transfer of silk fiber is explained. The results provide new perspectives on how silk and other fibers should be designed to achieve higher toughness.

Effects of the Hydrophobicity of Key Residues on the Characteristics and Stability of Glucose Oxidase on a Graphene Surface.

Glucose oxidase (GOx) is one of the most widely investigated enzymes in the field of bioelectrochemistry. It is mainly used for the detection of glucose in solutions and enzyme-based biofuel cells. On the basis of the combination of GOx with graphene, novel nanodevices exceeding conventional limits can be developed. To develop a hybrid enzyme-graphene nanodevice with a good performance, it is important that GOx is deposited well on the graphene surface while maintaining its structure and not impeding the oxidation activity of the GOx. In this study, we propose a method to improve the stability of GOx and secure its immobility on the graphene sheet and its glucose-binding affinity by single-point mutation of GOx using molecular dynamics simulations. We confirm that the structural stability, immobility, and substrate binding affinity of GOx can be modified by changing the hydrophobicity of a key residue. We demonstrate that biosensors or biofuel cells can be redesigned and their properties can be improved by using molecular dynamics simulation.

The Formation Mechanism of Segmented Ring-Shaped Aß Oligomers and Protofibrils.

A clear understanding of amyloid formation with diverse morphologies is critical to overcoming the fatal disease amyloidosis. Studies have revealed that monomer concentration is a crucial factor for determining amyloid morphologies, such as protofibrils, annular, or spherical oligomers. However, gaining a complete understanding of the mechanism of formation of the various amyloid morphologies has been limited by the lack of experimental devices and insufficient knowledge. In this study, we demonstrate that the monomer concentration is an essential factor in determining the morphology of beta-amyloid (Aß) oligomers or protofibrils. By computational and experimental approaches, we investigated the strategies for structural stabilization of amyloid protein, the morphological changes, and amyloid aggregation. In particular, we found unprecedented conformations, e.g., single bent oligomers and segmented ring-shaped protofibrils, the formation of which was explained by the computational analysis. Our findings provide insight into the structural features of amyloid molecules formed at low concentrations of monomer, which will help determine the clinical targets (in therapy) to effectively inhibit amyloid formation in the early stages of the amyloid growth phase.

Twenty-Eight-Day Repeated Inhalation Toxicity Study of Aluminum Oxide Nanoparticles in Male Sprague-Dawley Rats.

Aluminum oxide nanoparticles (Al2O3 NPs) are among the most widely used nanomaterials; however, relatively little information about their risk identification and assessment is available. In the present study, we aimed to investigate the potential toxicity of Al2O3 NPs following repeated inhalation exposure in male Sprague-Dawley rats. Rats were exposed to Al2O3 NPs for 28 days (5 days/week) at doses of 0, 0.2, 1, and 5 mg/m3 using a nose-only inhalation system. During the experimental period, we evaluated the clinical signs, body weight change, hematological and serum biochemical parameters, necropsy findings, organ weight, and histopathology findings. Additionally, we analyzed the bronchoalveolar lavage fluid (BALF), including differential leukocyte counts, and aluminum contents in the major organs and blood. Aluminum contents were the highest in lung tissues and showed a dose-dependent relationship in the exposure group. Histopathology showed alveolar macrophage accumulation in the lungs of rats in the 5 mg/m3 group during exposure and recovery. These changes tended to increase at the end of the recovery period. In the BALF analysis, total cell and neutrophil counts and lactate dehydrogenase, tumor necrosis factor-α, and interleukin-6 levels significantly increased in the 1 and 5 mg/m3 groups during exposure. Under the present experimental conditions, we suggested that the no-observed-adverse-effect level of Al2O3 NPs in male rats was 1 mg/m3, and the target organ was the lung.

Loading-device effects on the protein-unfolding mechanisms using molecular-dynamic simulations.

Experimental force spectroscopy has been effectively utilized for measuring structural characterization of biomolecules and mechanical properties of biomaterials. Specifically, atomic force microscopy (AFM) has been widely used to portray biomolecular characterization in single-molecule experiment by observing the unfolding behavior of the proteins. Not only the experimental techniques enable us to characterize globular protein, but computational methods like molecular dynamics (MD) also gives insight into understanding biomolecular structures. To better comprehend the behavior of biomolecules, conditions such as pulling velocities and loading rates are put to the test, yet there are still limitations in understanding the unfolding behavior of biomolecules with the effect of different loading devices. In this study, we performed an all-atom MD and steered molecular dynamics (SMD) simulations considering different loading device effects such as "soft" and "stiff" to characterize the anisotropic unfolding behavior of ubiquitin protein. We found out the anisotropic unfolding pathways of the protein through the broken number of hydrogen bonds and geometric secondary structures of the biomolecule. Our study provides the importance for usage of various loading-devices on biomolecules when analyzing the structural compositions and the characteristics of globular biomolecules.

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.

Mechanical features of various silkworm crystalline considering hydration effect via molecular dynamics simulations.

Silk materials are receiving significant attention as base materials for various functional nanomaterials and nanodevices, due to its exceptionally high mechanical properties, biocompatibility, and degradable characteristics. Although crystalline silk regions are composed of various repetitive motifs with differing amino acid sequences, how the effect of humidity works differently on each of the motifs and their structural characteristics remains unclear. We report molecular dynamics (MD) simulations on various silkworm fibroins composed of major motifs (i.e. (GAGAGS)n, (GAGAGA)n, and (GAGAGY)n) at varying degrees of hydration, and reveal how each major motifs of silk fibroins change at each degrees of hydration using MD simulations and their structural properties in mechanical perspective via steered molecular dynamics simulations. Our results explain what effects humidity can have on nanoscale materials and devices consisting of crystalline silk materials.

Targeting Hypoxia-Inducible Factor-1α/Pyruvate Dehydrogenase Kinase 1 Axis by Dichloroacetate Suppresses Bleomycin-induced Pulmonary Fibrosis.

Hypoxia has long been implicated in the pathogenesis of fibrotic diseases. Aberrantly activated myofibroblasts are the primary pathological driver of fibrotic progression, yet how various microenvironmental influences, such as hypoxia, contribute to their sustained activation and differentiation is poorly understood. As a defining feature of hypoxia is its impact on cellular metabolism, we sought to investigate how hypoxia-induced metabolic reprogramming affects myofibroblast differentiation and fibrotic progression, and to test the preclinical efficacy of targeting glycolytic metabolism for the treatment of pulmonary fibrosis. Bleomycin-induced pulmonary fibrotic progression was evaluated in two independent, fibroblast-specific, promoter-driven, hypoxia-inducible factor (Hif) 1A knockout mouse models and in glycolytic inhibitor, dichloroacetate-treated mice. Genetic and pharmacological approaches were used to explicate the role of metabolic reprogramming in myofibroblast differentiation. Hypoxia significantly enhanced transforming growth factor-ß-induced myofibroblast differentiation through HIF-1α, whereas overexpression of the critical HIF-1α-mediated glycolytic switch, pyruvate dehydrogenase kinase 1 (PDK1) was sufficient to activate glycolysis and potentiate myofibroblast differentiation, even in the absence of HIF-1α. Inhibition of the HIF-1α/PDK1 axis by genomic deletion of Hif1A or pharmacological inhibition of PDK1 significantly attenuated bleomycin-induced pulmonary fibrosis. Our findings suggest that HIF-1α/PDK1-mediated glycolytic reprogramming is a critical metabolic alteration that acts to promote myofibroblast differentiation and fibrotic progression, and demonstrate that targeting glycolytic metabolism may prove to be a potential therapeutic strategy for the treatment of pulmonary fibrosis.

Capping effects on polymorphic Aß16-21 amyloids depend on their size: A molecular dynamics simulation study.

Understanding Aß amyloid oligomers associated with neuro-degenerative diseases is needed due to their toxic characteristics and mediation of amyloid fibril growth. Depending on various physiological circumstances such as ionic strength, metal ion, and point-residue mutation, oligomeric amyloids exhibit polymorphic behavior and structural stabilities, i.e. showing different conformation and stabilities. Specifically, experimental and computational researchers have found that the capping modulates the physical and chemical properties of amyloids by preserving electrostatic energy interactions, which is one of the dominant factors for amyloid stability. Still, there is no detailed knowledge for the polymorphic amyloids with reflecting the terminal capping effects. In the present study, we investigated the role of terminal capping (i.e. N-terminal acetylation and C-terminal amidation) on polymorphic Aß16-21 amyloid oligomer and protofibrils via molecular dynamics (MD) simulations. We found that the capping effects have differently altered the conformation of polymorphic antiparallel-homo and -hetero Aß16-21 amyloid oligomer, but not Aß16-21 amyloid protofibrils. However, regardless of polymorphic composition of the amyloids, the capping induces the thermodynamic instabilities of Aß16-21 amyloid oligomers, but does not show any distinct affect on Aß16-21 amyloid protofibrils. Specifically, among the molecular mechanic factors, electrostatic energy dominantly contributes the thermodynamic stability of the Aß16-21 amyloids. We hope that our computation study about the role of the capping effects on the polymorphic amyloids will facilitate additional efforts to enhance degradation of amyloids and to design a selective drug in the future.

The distinct metabolic phenotype of lung squamous cell carcinoma defines selective vulnerability to glycolytic inhibition.

Adenocarcinoma (ADC) and squamous cell carcinoma (SqCC) are the two predominant subtypes of non-small cell lung cancer (NSCLC) and are distinct in their histological, molecular and clinical presentation. However, metabolic signatures specific to individual NSCLC subtypes remain unknown. Here, we perform an integrative analysis of human NSCLC tumour samples, patient-derived xenografts, murine model of NSCLC, NSCLC cell lines and The Cancer Genome Atlas (TCGA) and reveal a markedly elevated expression of the GLUT1 glucose transporter in lung SqCC, which augments glucose uptake and glycolytic flux. We show that a critical reliance on glycolysis renders lung SqCC vulnerable to glycolytic inhibition, while lung ADC exhibits significant glucose independence. Clinically, elevated GLUT1-mediated glycolysis in lung SqCC strongly correlates with high 18F-FDG uptake and poor prognosis. This previously undescribed metabolic heterogeneity of NSCLC subtypes implicates significant potential for the development of diagnostic, prognostic and targeted therapeutic strategies for lung SqCC, a cancer for which existing therapeutic options are clinically insufficient.

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.

HIF-1α-PDK1 axis-induced active glycolysis plays an essential role in macrophage migratory capacity.

In severely hypoxic condition, HIF-1α-mediated induction of Pdk1 was found to regulate glucose oxidation by preventing the entry of pyruvate into the tricarboxylic cycle. Monocyte-derived macrophages, however, encounter a gradual decrease in oxygen availability during its migration process in inflammatory areas. Here we show that HIF-1α-PDK1-mediated metabolic changes occur in mild hypoxia, where mitochondrial cytochrome c oxidase activity is unimpaired, suggesting a mode of glycolytic reprogramming. In primary macrophages, PKM2, a glycolytic enzyme responsible for glycolytic ATP synthesis localizes in filopodia and lammelipodia, where ATP is rapidly consumed during actin remodelling processes. Remarkably, inhibition of glycolytic reprogramming with dichloroacetate significantly impairs macrophage migration in vitro and in vivo. Furthermore, inhibition of the macrophage HIF-1α-PDK1 axis suppresses systemic inflammation, suggesting a potential therapeutic approach for regulating inflammatory processes. Our findings thus demonstrate that adaptive responses in glucose metabolism contribute to macrophage migratory activity.

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.

Intermittent induction of HIF-1α produces lasting effects on malignant progression independent of its continued expression.

Dysregulation of hypoxia-inducible transcription factors HIF-1α and HIF-2α correlates with poor prognosis in human cancers; yet, divergent and sometimes opposing activities of these factors in cancer biology have been observed. Adding to this complexity is that HIF-1α apparently possesses tumor-suppressing activities, as indicated by the loss-of-function mutations or even homozygous deletion of HIF1A in certain human cancers. As a step towards understanding this complexity, we employed 8-week intermittent induction of a stable HIF-1α variant, HIF1α(PP), in various cancer cell lines and examined the effects on malignant progression in xenografts of immunocompromised mice in comparison to those of HIF2α(PP). Although 8-week treatment led to eventual loss of HIF1α(PP) expression, treated osteosarcoma U-2 OS cells acquired tumorigenicity in the subcutaneous tissue. Furthermore, the prior treatment resulted in widespread invasion of malignant glioma U-87 MG cells in the mouse brain and sustained growth of U-118 MG glioma cells. The lasting effects of HIF-1α on malignant progression are specific because neither HIF2α(PP) nor ß-galactosidase yielded similar effects. By contrast, transient expression of HIF1α(PP) in U-87 MG cells or constitutive expression of HIF1α(PP) but not HIF2α(PP) in a patient-derived glioma sphere culture inhibited tumor growth and spread. Our results indicate that intermittent induction of HIF-1α produces lasting effects on malignant progression even at its own expense.

Complex role of HIF in cancer: the known, the unknown, and the unexpected.

Tumor hypoxia has long been recognized as a driving force of malignant progression and therapeutic resistance. The discovery of hypoxia-inducible transcription factors (HIFs) has greatly advanced our understanding of how cancer cells cope with hypoxic stress by maintaining bioenergetics through the stimulation of glycolysis. Until recently, however, it remained perplexing why proliferative cancer cells opt for aerobic glycolysis, an energy-inefficient process of glucose metabolism. Furthermore, the role of HIF in cancer has also become complex. In this review, we highlight recent groundbreaking findings in cancer metabolism, put forward plausible explanations to the complex role of HIF, and underscore remaining issues in cancer biology.

ITF2 prevents activation of the ß-catenin-TCF4 complex in colon cancer cells and levels decrease with tumor progression.

BACKGROUND & AIMS: Immunoglobulin transcription factor 2 (ITF2) was believed to promote neoplastic transformation via activation of ß-catenin. However, ITF2 recently was reported to suppress colon carcinogenesis. We investigated the roles of ITF2 in colorectal cancer cell lines and tumor formation and growth in mice. METHODS: Levels of ITF2, ß-catenin, and c-Myc were measured in 12 human colorectal tumor samples and by immunohistochemistry. ITF2 regulation of ß-catenin and T-cell factor (TCF) were analyzed using luciferase reporter, reverse-transcription quantitative polymerase chain reaction, flow cytometry, and immunoblot analyses. Mice were given subcutaneous injections of human colorectal cancer cell lines that stably express ITF2, small hairpin RNAs to reduce levels of ITF2, or control plasmids; xenograft tumor growth was assessed. Human colorectal carcinoma tissue arrays were used to associate levels of ITF2 expression and clinical outcomes. RESULTS: Levels of ß-catenin, cMyc, and ITF2 were increased in areas of human colon adenomas and carcinomas, compared with nontumor areas of the same tissues. ITF2 levels were reduced and cMyc levels were increased in areas of carcinoma, compared with adenoma. In human colorectal cancer cell lines, activation of the ß-catenin-TCF4 complex and expression of its target genes were regulated negatively by ITF2. ITF2 inhibited formation of the ß-catenin-TCF4 complex by competing with TCF4 for ß-catenin binding. Stable transgenic expression of ITF2 in human colorectal cancer cell lines reduced their proliferation and tumorigenic potential in mice, whereas small hairpin RNA knockdown of ITF2 promoted growth of xenograft tumors in mice. In an analysis of colorectal tumor tissue arrays, loss of ITF2 from colorectal tumor tissues was associated with poor outcomes of patients. A gene set enrichment analysis supported the negative correlation between the level of ITF2 and activity of the ß-catenin-TCF4 complex. CONCLUSIONS: In human colorectal cancer cell lines and tissue samples, ITF2 appears to prevent activation of the ß-catenin-TCF4 complex and transcription of its gene targets. Loss of ITF2 promotes the ability of colorectal cancer cells to form xenograft tumors, and is associated with tumor progression and shorter survival times of patients.