Structure determination using high-order spatial correlations in single-particle X-ray scattering.

Single-particle imaging using X-ray free-electron lasers (XFELs) is a promising technique for observing nanoscale biological samples under near-physiological conditions. However, as the sample's orientation in each diffraction pattern is unknown, advanced algorithms are required to reconstruct the 3D diffraction intensity volume and subsequently the sample's density model. While most approaches perform 3D reconstruction via determining the orientation of each diffraction pattern, a correlation-based approach utilizes the averaged spatial correlations of diffraction intensities over all patterns, making it well suited for processing experimental data with a poor signal-to-noise ratio of individual patterns. Here, a method is proposed to determine the 3D structure of a sample by analyzing the double, triple and quadruple spatial correlations in diffraction patterns. This ab initio method can reconstruct the basic shape of an irregular unsymmetric 3D sample without requiring any prior knowledge of the sample. The impact of background and noise on correlations is investigated and corrected to ensure the success of reconstruction under simulated experimental conditions. Additionally, the feasibility of using the correlation-based approach to process incomplete partial diffraction patterns is demonstrated. The proposed method is a variable addition to existing algorithms for 3D reconstruction and will further promote the development and adoption of XFEL single-particle imaging techniques.

Molecular size dependence on achievable resolution from XFEL single-particle 3D reconstruction.

Single-particle analysis using x-ray free-electron lasers (XFELs) is a novel method for obtaining structural information of samples in a state close to nature. In particular, it is suitable for observing the inner structure of large biomolecules by taking advantage of the high transmittance of x-rays. However, systematic studies on the resolution achievable for large molecules are lacking. In this study, the molecular size dependence of the resolution of a three-dimensional (3D) structure resulting from XFEL single-particle reconstruction is evaluated using synthetic data. Evidently, 3D structures of larger molecules can be restored with higher detail (defined relative to the molecular sizes) than smaller ones; however, reconstruction with high absolute resolution (defined in nm-1) is challenging. Our results provide useful information for the experimental design of 3D structure reconstruction using coherent x-ray diffraction patterns of single-particles.

Successful treatment of relapsing polychondritis with circumferential bronchial wall thickening including the tracheomembranous area with tumor necrosis factor-α inhibitor.

This is a case of a 55-year-old man who presented with cough and anterior chest pain. Tracheal biopsy confirmed the diagnosis of relapsing polychondritis (RP). Although the patient had circumferential bronchial wall thickening extending to the tracheomembranous area and was positive foRPR3-ANCA, he did not meet the diagnostic criteria for granulomatosis with polyangiitis. The patient was refractory to prednisolone + methotrexate + azathioprine and responded to adalimumab, a biologic tumor necrosis factor-α inhibitor effective in RP refractory cases. Herein, we report a rare case of RP with circumferential bronchial wall thickening extending to the tracheomembranous area.

Light-Driven Liquid Conveyors: Manipulating Liquid Mobility and Transporting Solids on Demand.

The intelligent transport of materials at interfaces is essential for a wide range of processes, including chemical microreactions, bioanalysis, and microfabrication. Both passive and active methods have been used to transport droplets, among which light-based techniques have attracted much attention because they are noncontact, safe, reversible, and controllable. However, conventional light-driven systems also involve challenges related to low transport ability and instability. Because of these shortcomings, technologies that can transport and manipulate droplets and microsolids on the same surface have yet to be realized. The present work demonstrates a light-driven system referred to as a liquid conveyor that enables the transport of both water droplets and microsolids. After the incorporation of an azobenzene-based molecular motor capable of undergoing photoisomerization into the surface liquid layer of this system, an isomerization gradient was induced by exposure to ultraviolet light emitting diodes that induced flow in this layer. Various parameters were optimized, including the concentration of the molecular motor compound, the light intensity, the viscosity of the liquid layer, and the droplet volume. This process eventually achieved the horizontal transport of droplets in any direction at varied rates. As a consequence of the limited heat buildup, the lack of droplet deformation, and extremely small contact angle hysteresis in this system, microsolids on droplets were also transported. This liquid conveyor is a promising platform for high-throughput omni-liquid/solid manipulation in the fields of biotechnology, chemistry, and mechanical engineering.

Green Superlubricity Enabled by Only One Water Droplet on Plant Oil-Infused Surfaces.

The increase in energy loss due to friction and the use of large amounts of lubricants to improve it are major challenges we face from a global environmental perspective. Pitcher-plant-inspired liquid-infused surfaces (LISs) are emerging super-repellent surfaces against liquids. However, their coefficient of friction (CoF) against solids is higher than that of conventional lubricant surfaces. Herein, we demonstrate superlubricity with a single water droplet placed on a LIS holding oleic acid, a component of plant oil. When a water droplet is placed on the fluid layer, the CoF under reciprocating and rotating friction is 0.012 and 0.0098, respectively. A force in the direction opposite to the loading due to the Laplace pressure on the droplet and an autonomous positional movement of the water accompanied by the optimization of surface energy maintain the fluid-lubrication state and prevent direct contact between the surface and the friction material, resulting in a decrease of the dependence of the CoF on the friction velocity. The key technology here will not only present a novel strategy for preparing LISs against solids but also serve as a step toward a sustainable green strategy for friction reduction and lubrication, which would greatly reduce energy loss and environmental degradation.

Photo-Induced Crawling Motion of Azobenzene Crystals on Modified Gold Surfaces.

Photo-induced crawling motion of a crystal of 3,3'-dimethylazobenzene (DMAB) on gold surfaces having different surface properties and various patterns was studied. DMAB crystals crawl continuously when exposed to UV and visible lights simultaneously from different directions. On a gold surface functionalized by a thiol having a hydroxyl group at the terminal (16-hydroxy-1-hexadecanethiol (HOC16SH)), the crystals crawled with a relatively high velocity (ca. 4 µm min-1), and they changed the crystal shape while keeping a distinct crystal face. On a gold surface functionalized by a thiol having an alkyl chain terminal (1-hexadecanethiol (C16SH)), the crawling was observed with a slower velocity (ca. 1.5 µm min-1). However, the shape of the crystals became a droplet-like shape soon after the irradiation started, and the shape persisted during the motion. Light intensity dependence of the crawling velocity of the droplet-like crystal on this surface showed that UV light has stronger dependence for the motion than the visible light. On a substrate with a stripe pattern of alternating C16SH-modified gold and hexadecyltrimethylsilane (HDTMS)-modified glass, crystals crawled only on the surface of the C16SH-modified gold, which may be due to the wettability hysteresis at the surface. On a substrate with a stripe pattern of HOC16SH-modified gold and HDTMS-modified glass, crystals were attracted to the gold side. On a gold substrate with a periodic pattern of different height (ca. 50 nm) but having a uniform treatment with C16SH, crystals crawled up and down the steps without significant disturbance at the boundary of the step. Therefore, wettability of the surface has a greater impact on controlling the motion of the crystal than the surface structure. The present results not only unveil the crawling behavior on various surfaces but also offer a guide to controlling the motion toward applications for novel carriage vehicles to transport molecules/objects on a surface.

HLA-B52 allele in giant cell arteritis may indicate diffuse large-vessel vasculitis formation: a retrospective study.

BACKGROUND: This study aimed to identify new characteristics of elderly onset large-vessel vasculitis (EOLVV) by focusing on human leucocyte antigen (HLA) genotype, polymyalgia rheumatica (PMR), and affected vascular lesions observed on positron emission tomography/computed tomography (PET/CT) imaging. METHODS: We retrospectively studied 65 consecutive Japanese patients with large-vessel vasculitis (LVV) who had extracranial vasculitis lesions and underwent PET/CT imaging. PET/CT images were assessed using the semi-quantitative PET visual score of each affected vessel, and the PET vascular activity score (PETVAS) and number of affected vessels were calculated. Subjects were subsequently grouped based on age at onset, superficial temporal artery (STA) involvement, and presence of PMR and compared each group according to HLA genotype. Unsupervised hierarchical cluster analysis was used to identify the patients with similar characteristics in terms of affected vascular lesions detected through PET/CT imaging. The clinical characteristics and PET/CT findings of the population newly identified in this study were examined. RESULTS: Twenty-seven patients with EOLVV did not meet the American College of Rheumatology 1990 criteria for giant cell arteritis (GCA) and Takayasu arteritis and were considered as unclassified EOLVV (UEOLVV). The unsupervised hierarchical cluster analysis revealed that UEOLVV with PMR and large-vessel GCA (LV-GCA) formed a cluster of LVV with GCA features (i.e., PMR and/or STA involvement) when restricted to patients who were HLA-B52-positive. Patients who were HLA-B52-positive with LVV and GCA features had similar clinical characteristics and patterns of affected vessels and presented with diffuse LVV lesions. HLA-B52-positive patients who had LVV with GCA features also presented with higher PETVAS, more affected vessels, and lower rates of biologics usage and relapse compared to HLA-B52-positive patients with TAK. CONCLUSIONS: Patients who had UEOLVV with PMR had similar characteristics to patients with LV-GCA. Patients who were HLA-B52-positive and had LVV with GCA features presented with diffuse vascular lesions and may comprise a core population of Japanese patients with EOLVV. The findings of HLA-B52 positivity and diffusely affected vessels in patients with EOLVV can be considered as suspicious findings of LV-GCA.

Effect of Surface Properties on the Photo-Induced Crawling Motion of Azobenzene Crystals on Glass Surfaces.

Photo-induced crawling motion of a crystal of 3,3'-dimethylazobenzene (DMAB) on a glass substrate having different surface properties was studied. When exposed to UV and visible lights simultaneously from different directions, crystals crawl continuously on a glass surface. On a hydrophilic surface, the crystals crawled faster than those on other surfaces but crystals showed spreading while they moved. On hydrophobic surfaces, on the other hand, the crystals showed little shape change and slower crawling motion. The contact angles of the liquid phase of DMAB on surface-modified glass substrates showed positive correlation with the water contact angles. The interaction of melted azobenzene with glass surfaces plays an important role for the crawling motion. We proposed models to explain the asymmetric condition that leads to the directional motion. Specifically by considering the penetration length of UV and visible light sources, it was successfully shown that the depth of light penetration is different at the position of a crystal. This creates a nonequilibrium condition where melting and crystallization are predominant in the same crystal.

Association of elevated serum soluble CD226 levels with the disease activity and flares of systemic lupus erythematosus.

CD226 is an activating receptor expressed on the cell surface of natural killer cells and T cells. Although CD226 polymorphism is known to be involved in systemic lupus erythematosus (SLE), the involvement of soluble CD226 (sCD226) in SLE is still unknown. In the present study, we measured serum sCD226 levels using an enzyme-linked immunosorbent assay in 58 SLE patients and 33 healthy controls (HCs) and evaluated their associations with SLE Disease Activity Index 2000 (SLEDAI-2K), clinical manifestations, laboratory data, and the cumulative probability of flare. Serum sCD226 levels showed no significant differences between SLE patients and HCs. However, sCD226 levels were significantly elevated in active SLE patients with a SLEDAI-2K score of ≥ 20 compared with HCs. In SLE patients, sCD226 levels were significantly correlated with SLEDAI-2K scores and anti-dsDNA antibody titers. Moreover, the cumulative probability of flare was markedly higher in patients with high sCD226 than in those with low sCD226. In patients with neuropsychiatric involvement, sCD226 levels were elevated and reflected neuropsychiatric disease activity. These findings indicate that serum sCD226 levels are associated with disease activity and flares of SLE. Thus, it may be a useful biomarker for SLE, and its monitoring allows for more precise SLE management.

Increased Proportion of CD226+ B Cells Is Associated With the Disease Activity and Prognosis of Systemic Lupus Erythematosus.

Background: CD226, an activating receptor expressed on the surface of natural killer (NK) cells and T cells, is also seen on B cells and CD226 polymorphism is associated with systemic lupus erythematosus (SLE). Because the specific roles of CD226+ B cells in SLE are still unknown, we investigated the association of CD226+ B cells with SLE. Methods: We measured CD226 expression on B cells and its subsets using flow cytometry in 48 SLE patients and 24 healthy controls (HCs). We assessed the relationships between CD226+ B cells and SLE Disease Activity Index 2000 (SLEDAI-2K), clinical manifestations, laboratory data, and prognosis after 12 months. Results: The proportions of CD226+ cells in whole B cells and all its subsets were significantly higher in SLE patients than HCs. In SLE patients, the proportions of CD226+ B cells and CD226+ switched-memory (SM) B cells were significantly correlated with SLEDAI-2K scores and anti-dsDNA antibody titers, and negatively correlated with serum complement levels. Moreover, basal percentages of CD226+ B cells and CD226+ SM B cells were low in patients who were in Lupus Low Disease Activity State after 12 months. In patients with renal involvement, the proportion of CD226+ B cells increased. Additionally, the proportion of CD226+ B cells was higher in patients who were not in complete renal remission after 12 months. Conclusions: Increased proportion of CD226+ B cells was associated with disease activity and prognosis of SLE. CD226+ B cells may be a useful biomarker for the management of SLE.

In Situ Observation of Desorption and Direct Electron Transfer Reaction of Cytochrome c on Bare ITO Electrode with Electrochemical Slab Optical Waveguide Spectroscopy.

Cyclic voltammograms (CVs) of cytochrome c (cytc) on the bare ITO were measured with every 10 times of the continuous the solution exchange (SE) processes at the same time of slab optical waveguide (SOWG) spectral observation, and it was proved that direct electron transfer (DET) reaction functionality of cytc adsorbed on the bare ITO electrode was kept after 100 times SE processes. The existence of three kinds of cytc molecules which were weakly adsorbed, strongly adsorbed and immobilized on the bare ITO electrode below a monolayer coverage was indicated from the change in the Soret band absorbance at 408 nm due to the desorption reaction of cytc with In Situ observation by electrochemically controlled SOWG spectroscopy. As the actual procedure, 100 times of the SE process in the SOWG cell by hands induced the gradual decrease of the absorbance due to cytc desorption, and with every 10 times of the SE process SOWG absorption spectra were obtained. The SOWG absorbance decay curve was well fitted with two components exponential equation depending on the SE process numbers showing that around 31.6% of the cytc molecules adsorbed on bare ITO electrode were finally immobilized.

Parameter optimization for 3D-reconstruction from XFEL diffraction patterns based on Fourier slice matching.

Single-particle analysis (SPA) by X-ray free electron laser (XFEL) is a novel method that can observe biomolecules and living tissue that are difficult to crystallize in a state close to nature. To reconstruct three-dimensional (3D) molecular structure from two-dimensional (2D) XFEL diffraction patterns, we have to estimate the incident beam angle to the molecule for each pattern to assemble the 3D-diffraction intensity distribution using interpolation, and retrieve the phase information. In this study, we investigated the optimal parameter sets to assemble the 3D-diffraction intensity distribution from simulated 2D-diffraction patterns of ribosome. In particular, we examined how the parameters need to be adjusted for diffraction patterns with different binning sizes and beam intensities to obtain the highest resolution of molecular structure phase retrieved from the 3D-diffraction intensity. We found that resolution of restored molecular structure is sensitive to the interpolation parameters. Using the optimal parameter set, a linear oversampling ratio of around four is found to be sufficient for correct angle estimation and phase retrieval from the diffraction patterns of SPA by XFEL.

Single-particle XFEL 3D reconstruction of ribosome-size particles based on Fourier slice matching: requirements to reach subnanometer resolution.

Three-dimensional (3D) structures of biomolecules provide insight into their functions. Using X-ray free-electron laser (XFEL) scattering experiments, it was possible to observe biomolecules that are difficult to crystallize, under conditions that are similar to their natural environment. However, resolving 3D structure from XFEL data is not without its challenges. For example, strong beam intensity is required to obtain sufficient diffraction signal and the beam incidence angles to the molecule need to be estimated for diffraction patterns with significant noise. Therefore, it is important to quantitatively assess how the experimental conditions such as the amount of data and their quality affect the expected resolution of the resulting 3D models. In this study, as an example, the restoration of 3D structure of ribosome from two-dimensional diffraction patterns created by simulation is shown. Tests are performed using the diffraction patterns simulated for different beam intensities and using different numbers of these patterns. Guidelines for selecting parameters for slice-matching 3D reconstruction procedures are established. Also, the minimum requirements for XFEL experimental conditions to obtain diffraction patterns for reconstructing molecular structures to a high-resolution of a few nanometers are discussed.

Three-dimensional reconstruction for coherent diffraction patterns obtained by XFEL.

The three-dimensional (3D) structural analysis of single particles using an X-ray free-electron laser (XFEL) is a new structural biology technique that enables observations of molecules that are difficult to crystallize, such as flexible biomolecular complexes and living tissue in the state close to physiological conditions. In order to restore the 3D structure from the diffraction patterns obtained by the XFEL, computational algorithms are necessary as the orientation of the incident beam with respect to the sample needs to be estimated. A program package for XFEL single-particle analysis based on the Xmipp software package, that is commonly used for image processing in 3D cryo-electron microscopy, has been developed. The reconstruction program has been tested using diffraction patterns of an aerosol nanoparticle obtained by tomographic coherent X-ray diffraction microscopy.

In situ Observation of Direct Electron Transfer Reaction of Cytochrome c Immobilized on ITO Electrode Modified with 11-{2-[2-(2-Methoxyethoxy)ethoxy]ethoxy}undecylphosphonic Acid Self-assembled Monolayer Film by Electrochemical Slab Optical Waveguide Spectroscopy.

To immobilize cytochrome c (cyt.c) on an ITO electrode while keeping its direct electron transfer (DET) functionality, the ITO electrode surface was modified with 11-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}undecylphosphonic acid (CH3O (CH2CH2O)3C11H22PO(OH)2, M-EG3-UPA) self-assembled monolayer (SAM) film. After a 100-times washing process to exchange a phosphate buffer saline solution surrounding cyt.c and ITO electrode to a fresh one, an in situ observation of visible absorption spectral change with slab optical waveguide (SOWG) spectroscopy showed that 87.7% of the cyt.c adsorbed on the M-EG3-UPA modified ITO electrode remained on the ITO electrode. The SOWG absorption spectra corresponding to oxidized and reduced cyt.c were observed with setting the ITO electrode potential at 0.3 and -0.3 V vs. Ag/AgCl, respectively, while probing the DET reaction between cyt.c and ITO electrode occurred. The amount of cyt.c was evaluated to be about 19.4% of a monolayer coverage based on the coulomb amount in oxidation and reduction peaks on cyclic voltammetry (CV) data. The CV peak current maintained to be 83.4% compared with the initial value for a M-EG3-UPA modified ITO electrode after 60 min continuous scan with 0.1 V/s between 0.3 and -0.3 V vs. Ag/AgCl.

Analysis of the Interfacial Molecular Behavior of a Lubrication Film of n-Dodecane Containing Stearic Acid under Lubricating Conditions by Sum Frequency Generation Spectroscopy.

The molecular behavior of n-dodecane with added stearic acid at a friction interface was studied using sum frequency generation (SFG) spectroscopy and a tribometer. In the case of n-dodecane with stearic acid, under dynamic conditions, a strong peak from the symmetric stretching vibrational mode of methylene, which was not observed under static conditions, appears. However, this strong methylene peak was not observed in the case of only n-dodecane. The SFG spectrum in the C-H stretching mode region of n-dodecane-d26 with stearic acid in the dynamic condition was analogous to that in the static condition. These results indicate that the interfacial structure of stearic acid does not change under sliding condition. The n-dodecane on a stearic acid adsorption film is highly aligned. Moreover, from the sliding direction dependence of the SFG measurements, the molecular orientation of n-dodecane was deduced: n-dodecane on stearic acid adsorption films orient parallel to the sliding direction. These results have shown that the stearic acid adsorption film behaves as solid-like, which has also been mentioned in previous studies. Further, our results revealed a new function of stearic acid: the stearic acid adsorption film induces the formation of a well-defined n-dodecane interfacial structure and forces the n-dodecane molecules to orient along the sliding direction at the friction interface.

Thermodynamic properties of water molecules in the presence of cosolute depend on DNA structure: a study using grid inhomogeneous solvation theory.

In conditions that mimic those of the living cell, where various biomolecules and other components are present, DNA strands can adopt many structures in addition to the canonical B-form duplex. Previous studies in the presence of cosolutes that induce molecular crowding showed that thermal stabilities of DNA structures are associated with the properties of the water molecules around the DNAs. To understand how cosolutes, such as ethylene glycol, affect the thermal stability of DNA structures, we investigated the thermodynamic properties of water molecules around a hairpin duplex and a G-quadruplex using grid inhomogeneous solvation theory (GIST) with or without cosolutes. Our analysis indicated that (i) cosolutes increased the free energy of water molecules around DNA by disrupting water-water interactions, (ii) ethylene glycol more effectively disrupted water-water interactions around Watson-Crick base pairs than those around G-quartets or non-paired bases, (iii) due to the negative electrostatic potential there was a thicker hydration shell around G-quartets than around Watson-Crick-paired bases. Our findings suggest that the thermal stability of the hydration shell around DNAs is one factor that affects the thermal stabilities of DNA structures under the crowding conditions.

Effects of Bridge Functions on Radial Distribution Functions of Liquid Water.

In this report the radial distribution functions (RDFs) of liquid water are calculated on the basis of the classical density functional theory combined with the reference interaction site model for molecular liquids. The bridge functions, which are neglected in the hypernetted-chain (HNC) approximation, are taken into account through the density expansion for the Helmholtz free energy functional up to the third order. A factorization approximation to the ternary direct correlation functions in terms of the site-site pair correlation functions is then employed in the expression of the bridge functions, thus leading to a closed set of integral equations for the determination of the RDFs. It is confirmed through numerical calculations that incorporation of the oxygen-oxygen bridge function substantially improves the poor descriptions by the HNC approximation at room temperature, e.g., for the second peak of the oxygen-oxygen RDF.

i-Motifs are more stable than G-quadruplexes in a hydrated ionic liquid.

Thermodynamic analyses and molecular dynamics calculations demonstrated that i-motifs in a hydrated ionic liquid of choline dihydrogen phosphate (choline dhp) were more stable than G-quadruplexes due to choline ion binding to loop regions in the i-motifs. Interestingly, the i-motifs formed even at physiological pH in the choline dhp-containing solution.

Effects of bridge functions on radial distribution functions of liquid water.

In this report the radial distribution functions (RDFs) of liquid water are calculated on the basis of the classical density functional theory combined with the reference interaction site model for molecular liquids. The bridge functions, which are neglected in the hypernetted-chain (HNC) approximation, are taken into account through the density expansion for the Helmholtz free energy functional up to the third order. A factorization approximation to the ternary direct correlation functions in terms of the site-site pair correlation functions is then employed in the expression of the bridge functions, thus leading to a closed set of integral equations for the determination of the RDFs. It is confirmed through numerical calculations that incorporation of the oxygen-oxygen bridge function substantially improves the poor descriptions by the HNC approximation at room temperature, e.g., for the second peak of the oxygen-oxygen RDF.