Development of the multiplex imaging chamber at PAL-XFEL.

Various X-ray techniques are employed to investigate specimens in diverse fields. Generally, scattering and absorption/emission processes occur due to the interaction of X-rays with matter. The output signals from these processes contain structural information and the electronic structure of specimens, respectively. The combination of complementary X-ray techniques improves the understanding of complex systems holistically. In this context, we introduce a multiplex imaging instrument that can collect small-/wide-angle X-ray diffraction and X-ray emission spectra simultaneously to investigate morphological information with nanoscale resolution, crystal arrangement at the atomic scale and the electronic structure of specimens.

Binding of human ACE2 and RBD of Omicron enhanced by unique interaction patterns among SARS-CoV-2 variants of concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, has continued to mutate and spread worldwide despite global vaccination efforts. In particular, the Omicron variant, first identified in South Africa in late November 2021, has become the dominant strain worldwide. Compared to the original strain identified in Wuhan, Omicron features 50 genetic mutations, with 15 mutations in the receptor-binding domain (RBD) of the spike protein, which binds to the human angiotensin-converting enzyme 2 (ACE2) receptor for viral entry. However, it is not completely understood how these mutations alter the interaction and binding strength between the Omicron RBD and ACE2. In this study, we used a combined steered molecular dynamics (SMD) simulation and experimental microscale thermophoresis (MST) approach to quantify the interaction between Omicron RBD and ACE2. We report that the Omicron brings an enhanced RBD-ACE2 interface through N501Y, Q498R, and T478K mutations; the changes further lead to unique interaction patterns, reminiscing the features of previously dominated variants, Alpha (N501Y) and Delta (L452R and T478K). Among the Q493K and Q493R, we report that Q493R shows stronger binding to ACE2 than Q493K due to increased interactions. Our MST data confirmed that the Omicron mutations in RBD are associated with a five-fold higher binding affinity to ACE2 compared to the RBD of the original strain. In conclusion, our results could help explain the Omicron variant's prevalence in human populations, as higher interaction forces or affinity for ACE2 likely promote greater viral binding and internalization, leading to increased infectivity.

Structure and Reactivity of Nonporphyrinic Terminal Manganese(IV)-Hydroxide Complexes in the Oxidative Electrophilic Reaction.

High-valent transition metal-hydroxide complexes have been proposed as essential intermediates in biological and synthetic catalytic reactions. In this work, we report the single-crystal X-ray structure and spectroscopic characteristics of a mononuclear nonporphyrinic MnIV-(OH) complex, [MnIV(Me3-TPADP)(OH)(OCH2CH3)]2+ (2), using various physicochemical methods. Likewise, [MnIV(Me3-TPADP)(OH)(OCH2CF3)]2+ (3), which is thermally stable at room temperature, was also synthesized and characterized spectroscopically. The MnIV-(OH) adducts are capable of performing oxidation reactions with external organic substrates such as C-H bond activation, sulfoxidation, and epoxidation. Kinetic studies, involving the Hammett correlation and kinetic isotope effect, and product analyses indicate that 2 and 3 exhibit electrophilic oxidative reactivity toward hydrocarbons. Density functional theory calculations support the assigned electronic structure and show that direct C-H bond activation of the MnIV-(OH) species is indeed possible.

Biomechanical characterization of SARS-CoV-2 spike RBD and human ACE2 protein-protein interaction.

The current COVID-19 pandemic has led to a devastating impact across the world. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus causing COVID-19) is known to use the receptor-binding domain (RBD) at viral surface spike (S) protein to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD-ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has a higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002-2004 SARS outbreak. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approaches to quantify the specific interactions between SARS-CoV-2 or SARS-CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between SARS-CoV-2 RBD and ACE2 range from 70 to 105 pN and are 30-40% higher than those of SARS-CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that SARS-CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the SARS-CoV-1 RBD-ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After removing N-linked glycans on ACE2, its mechanical binding strength with SARS-CoV-2 RBD decreases to a similar level of the SARS-CoV-1 RBD-ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1 and could help develop new strategies to block SARS-CoV-2 entry.

Characterization of the Percival detector with soft X-rays.

In this paper the back-side-illuminated Percival 2-Megapixel (P2M) detector is presented, along with its characterization by means of optical and X-ray photons. For the first time, the response of the system to soft X-rays (250 eV to 1 keV) is presented. The main performance parameters of the first detector are measured, assessing the capabilities in terms of noise, dynamic range and single-photon discrimination capability. Present limitations and coming improvements are discussed.

Creation of Fano resonances and bound states in the continuum in metallic metasurface superlattices.

A perfect metal film with a periodic arrangement of cut-through slits, an anisotropic metallic metamaterial film, mimics a dielectric slab and supports guided electromagnetic waves in the direction perpendicular to the slits. Since the guided Bloch modes exist only below the light line, conventional metallic metamaterial films do not exhibit interesting leaky-wave effects, such as bound states in the continuum and Fano resonances. Here, we introduce metallic metasurface superlattices that include multiple slits in a period and demonstrate that the superlattices support the Fano resonances and bound states in the continuum. We show that the number of Fano resonances and bound states depend on the number of slits in a period of superlattices through rigorous finite element method simulations. Experimental results in microwave region also support the creation of Fano resonance and bound states in the continuum by the increment of the number of slits in a period of superlattices.

Metasurfaces with Bound States in the Continuum Enabled by Eliminating First Fourier Harmonic Component in Lattice Parameters.

Conventional photonic lattices, such as metamaterials and photonic crystals, exhibit various interesting physical properties that are attributed to periodic modulations in lattice parameters. In this study, we introduce novel types of photonic lattices, namely Fourier-component-engineered metasurfaces, that do not possess the first Fourier harmonic component in the lattice parameters. We demonstrate that these metasurfaces support the continuous high-Q bound states near second stop bands. The concept of engineering Fourier harmonic components in periodic modulations provides a new method to manipulate electromagnetic waves in artificial periodic structures.

Proton Switch in the Secondary Coordination Sphere to Control Catalytic Events at the Metal Center: Biomimetic Oxo Transfer Chemistry of Nickel Amidate Complex.

High-valent metal-oxo species are key intermediates for the oxygen atom transfer step in the catalytic cycles of many metalloenzymes. While the redox-active metal centers of such enzymes are typically supported by anionic amino acid side chains or porphyrin rings, peptide backbones might function as strong electron-donating ligands to stabilize high oxidation states. To test the feasibility of this idea in synthetic settings, we have prepared a nickel(II) complex of new amido multidentate ligand. The mononuclear nickel complex of this N5 ligand catalyzes epoxidation reactions of a wide range of olefins by using mCPBA as a terminal oxidant. Notably, a remarkably high catalytic efficiency and selectivity were observed for terminal olefin substrates. We found that protonation of the secondary coordination sphere serves as the entry point to the catalytic cycle, in which high-valent nickel species is subsequently formed to carry out oxo-transfer reactions. A conceptually parallel process might allow metalloenzymes to control the catalytic cycle in the primary coordination sphere by using proton switch in the secondary coordination sphere.

Longitudinal Label-Free Two-Photon Microscopy of Cellular Healing Processes in Non-Ablative Fractional Laser Wounds.

BACKGROUND AND OBJECTIVES: Wound healing is an important biomedical problem with various associated complications. Although cutaneous wound healing has been studied in vivo extensively using various optical imaging methods, early-stage cellular healing processes were difficult to study due to scab formation. The objective of this study is to demonstrate that minimal laser wounds and optical microscopy can access the detailed cellular healing processes of cutaneous wounds from the early stage. STUDY DESIGN/MATERIALS AND METHODS: A non-ablative fractional laser (NAFL) and label-free two-photon microscopy (TPM) were used to induce minimal cutaneous wounds and to image the wounds in three-dimension. Sixteen hairless mice and a single human volunteer were used. NAFL wounds were induced in the hindlimb skin of the mice and in the forearm skin of the human subject. The NAFL wounds were longitudinally imaged during the healing period, starting from an hour post wound induction in the earliest and until 21 days. Cells in the wound and surrounding normal skin were visualized based on two-photon excited auto-fluorescence (TPAF), and cellular changes were tracked by analyzing longitudinal TPM images both qualitatively and quantitatively. Damage and recovery in the skin dermis were tracked by using the second harmonic generation (SHG) signal of collagen. Immunofluorescence and hematoxylin and eosin histology analysis were conducted to validate the TPM results of the murine skin. RESULTS: Cellular healing processes in NAFL wounds and surroundings could be observed by longitudinal TPM. In the case of murine skin, various healing phases including inflammation, re-epithelization, granulation tissue formation, and late remodeling phase including collagen regeneration were observed in the same wounds owing to minimal or no scab formation. The re-epithelization process was analyzed quantitatively by measuring cell density and thickness of the epithelium in the wound surroundings. In the case of the human skin, the access inside the wound was blocked for a few days post wound induction due to scabs but the cellular changes in the wound surroundings were observed from the early stage. Cellular healing processes in the NAFL wound of the human skin were similar to those in murine skin. CONCLUSIONS: The minimal NAFL wound model and label-free TPM demonstrated the cell level assessment of wound healing processes with applicability to human subjects. © 2021 Wiley Periodicals LLC.

Multi-Focus Image Fusion Using Focal Area Extraction in a Large Quantity of Microscopic Images.

The non-invasive examination of conjunctival goblet cells using a microscope is a novel procedure for the diagnosis of ocular surface diseases. However, it is difficult to generate an all-in-focus image due to the curvature of the eyes and the limited focal depth of the microscope. The microscope acquires multiple images with the axial translation of focus, and the image stack must be processed. Thus, we propose a multi-focus image fusion method to generate an all-in-focus image from multiple microscopic images. First, a bandpass filter is applied to the source images and the focus areas are extracted using Laplacian transformation and thresholding with a morphological operation. Next, a self-adjusting guided filter is applied for the natural connections between local focus images. A window-size-updating method is adopted in the guided filter to reduce the number of parameters. This paper presents a novel algorithm that can operate for a large quantity of images (10 or more) and obtain an all-in-focus image. To quantitatively evaluate the proposed method, two different types of evaluation metrics are used: "full-reference" and "no-reference". The experimental results demonstrate that this algorithm is robust to noise and capable of preserving local focus information through focal area extraction. Additionally, the proposed method outperforms state-of-the-art approaches in terms of both visual effects and image quality assessments.

Polarization-differentiated band dynamics of resonant leaky modes at the lattice Γ point.

In the physical description of photonic lattices, leaky-mode resonance and bound states in the continuum are central concepts. Understanding of their existence conditions and dependence on lattice parameters is of fundamental interest. Primary leaky-wave effects are associated with the second stop band at the photonic lattice Γ point. The pertinent band gap is defined by the frequency difference between the leaky-mode band edge and the bound-state edge. This paper address the polarization properties of the band gaps resident in laterally periodic one-dimensional photonic lattices. We show that the band gaps pertinent to TM and TE leaky modes exhibit significantly differentiated evolution as the lattice parameters vary. This is because the TM band gap is governed by a surface effect due to the discontinuity of the dielectric constant at the interfaces of the photonic lattice as well as by a Bragg effect due to the periodic in-plane dielectric constant modulation. We find that when the lattice is thin (thick), the surface (Bragg) effect dominates the Bragg (surface) effect in the formation of the TM band. This leads to complex TM band dynamics with multiple band closures possible under parametric variation. In complete contrast, the TE band gap is governed only by the Bragg effect thus exhibiting simpler band dynamics. This research elucidates the important effect of polarization on resonant leaky-mode band dynamics whose explanation has heretofore not been available.

Redox-Inactive Metal Ions That Enhance the Nucleophilic Reactivity of an Alkylperoxocopper(II) Complex.

The importance of redox-inactive metal ions in modulating the reactivity of redox-active biological systems is a subject of great current interest. In this work, the effect of redox-inactive metal ions (M3+ = Sc3+, Y3+, Yb3+, La3+) on the nucleophilic reactivity of a mononuclear ligand-based alkylperoxocopper(II) complex, [Cu(iPr2-tren-C(CH3)2O2)]+ (1), was examined. 1 was prepared by the addition of hydrogen peroxide and triethylamine to the solution of [Cu(iPr3-tren)(CH3CN)]+ (iPr3-tren = tris[2-(isopropylamino)ethyl]amine) via the formation of [Cu(iPr3-tren)(O2H)]+ (2) in methanol (CH3OH) at 30 °C. 1 was characterized using density functional theory (DFT) calculations and spectroscopic methods such as UV-vis, resonance Raman (rR), and electron paramagnetic resonance (EPR). DFT calculations support the electronic structure of 1 with an intermediate geometry between the trigonal-bipyramidal and square-pyramidal geometries, which is consistent with the observed EPR signal exhibiting a signal with g⊥ = 2.03 (A⊥ = 16 G) and g|| = 2.19 (A|| = 158 G). The Cu-O bond stretching frequency of 1 was observed at 507 cm-1 for 16O2 species (486 cm-1 for 18O2 species), and its O-O vibrational energy was determined to be 799 cm-1 for 16O2 species (759 cm-1 for 18O2 species) by rR spectroscopy. The reactivity of 1 was investigated in oxidative nucleophilic reactions. The positive slope of the Hammett plot (ρ = 2.3(1)) with para-substituted benzaldehydes and the reactivity order with 1°-, 2°-, and 3°-CHO demonstrate well the nucleophilic character of this copper(II) ligand-based alkylperoxo complex. The Lewis acidity of M3+ improves the oxidizing ability of 1. The modulated reactivity of 1 with M3+ was revealed to be an opposite trend of the Lewis acidity of M3+ in aldehyde deformylation.

Morphological features of mucous secretory organ and mucous secretion of loach Misgurnus anguillicaudatus skin for friction drag reduction.

We examined the functional morphology of loach Misgurnus anguillicaudatus skin by using synchrotron X-ray micro-computed tomography (SR-µCT) and high-contrast staining using osmium tetroxide or phosphotungstic acid (PTA), which enhances the image contrast of soft tissues. The captured high-spatial resolution images revealed that the surface ornamentations were stuck in the basement membrane of the loach scales. The ornamentations consisting of grooves (radii) and ridges (circuli) that can move freely and bend flexibly. The cross-sectional lateral microstructures of flat, concave and convex loach skins were observed from a live image of loach skin obtained through dark-field optical coherence tomography (OCT) imaging. The thickness of loach skin was changed with varying empty space between the mucous-cell layer and the scales by bending motion of loach. In addition, through direct measurement of drag reduction of loach skin, the mucous layer was found to have a strong influence on the reduction of skin friction. The present results enhance the understanding of the functional morphologies of mucous layer of loach to secrete mucus for skin friction reduction.

Spoligotype Variation of Mycobacterium tuberculosis Strains Prevailing in Korea.

Tuberculosis (TB) is an ongoing global health problem, including in South Korea. To manage TB efficiently, it is necessary to understand the epidemiology, transmission route, and characteristics of prevailing Mycobacterium tuberculosis strains. In this study, we investigated microevolutions over time in the spoligotype patterns of M. tuberculosis isolated from TB patients in Korea. We collected 1,055 clinical M. tuberculosis isolates from 16 provinces in Korea from 1994 to 2006 and analyzed them by spoligotyping. We observed 26 subfamilies, including two large predominant families: a Beijing family (72.7%) and the T family (19.1%). Specifically, the abundance of spoligotype SIT269 from the Beijing-like subfamily significantly increased in the 2000s relative to the 1990s in Korea. This study provides an overview of the M. tuberculosis genotype trends over time in Korea. These data also indicate that we should consider the influence of the newly growing SIT269 subtype identified in the Beijing family.

Multifarious injection chamber for molecular structure study (MICOSS) system: development and application for serial femtosecond crystallography at Pohang Accelerator Laboratory X-ray Free-Electron Laser.

The multifarious injection chamber for molecular structure study (MICOSS) experimental system has been developed at the Pohang Accelerator Laboratory X-ray Free-Electron Laser for conducting serial femtosecond crystallography. This system comprises several instruments such as a dedicated sample chamber, sample injectors, sample environment diagnostic system and detector stage for convenient distance manipulation. Serial femtosecond crystallography experiments of lysozyme crystals have been conducted successfully. The diffraction peaks have reached to ∼1.8 Šresolution at the photon energy of 9.785 keV.

A new fluorescence imaging technique for visualizing hepatobiliary structures using sodium fluorescein: result of a preclinical study in a rat model.

BACKGROUND: Near-infrared fluorescence imaging has been recently applied in the field of hepatobiliary surgery. Our objective was to apply blue light fluorescence imaging to cholangiography and liver mapping during laparoscopic surgery. Therefore, we designed a preclinical study to evaluate the feasibility of using blue light fluorescence for cholangiography and liver mapping in a rat model. METHODS: Sodium fluorescein solution (1 mL to each individual) were administered intravenously to 20 male Sprague-Dawley rats (6 weeks old, 200-250 g), after laparotomy. Whole abdominal organs were observed under blue light (at a wavelength of 440-490 nm) emitted from a commercialized LED curing light. RESULTS: Immediately after the tracer solution was administered into the circulatory system of the rat, it was possible to visualize the location of the kidneys and the bile duct under blue light emitted from the light source. The liver was vaguely stained green by the tracer, while the ureters were not. After establishing biliary retention via duct clamping in the left lateral segment of the liver, the green color of the segment became distinct by the tracer, which showed vague coloration following release of the clamp. CONCLUSION: We established the preclinical basis for using blue light fluorescence cholangiography and liver mapping in this study. The clinical feasibility of these techniques during laparoscopic cholecystectomy and hepatectomy remained to be demonstrated.

The Effects of Artemisinin on the Cytolytic Activity of Natural Killer (NK) Cells.

Artemisinin, a chemical compound used for the treatment of malaria, has been known to show anti-cancer activity. However, the effect of this chemical on natural killer (NK) cells, which are involved in tumor killing, remains unknown. Here, we demonstrate that artemisinin exerts a potent anti-cancer effect by activating NK cells. NK-92MI cells pre-treated with artemisinin were subjected to a cytotoxicity assay using K562 cells. The results showed that artemisinin significantly enhances the cytolytic activity of NK cells in a dose-dependent manner. Additionally, the artemisinin-enhanced cytotoxic effect of NK-92MI cells on tumor cells was accompanied by the stimulation of granule exocytosis, as evidenced by the detection of CD107a expression in NK cells. Moreover, this enhancement of cytotoxicity by artemisinin was also observed in human primary NK cells from peripheral blood. Our results suggest that artemisinin enhances human NK cell cytotoxicity and degranulation. This is the first evidence that artemisinin exerts antitumor activity by enhancing NK cytotoxicity. Therefore, these results provide a deeper understanding of the action of artemisinin and will contribute to the development and application of this class of compounds in cancer treatment strategies.

Delivery of miR-155 to retinal pigment epithelial cells mediated by Burkitt's lymphoma exosomes.

Exosomes are extracellularly secreted vesicles ranging from 40 to 100 nm in diameter that are thought to play important roles in intercellular communication. Exosomes contain numerous proteins, RNA, and lipids that can affect the status of recipient cells under various pathological conditions. MicroRNAs (miRNAs) are small non-coding RNAs that play a major role in post-transcriptional gene silencing by interacting with the 3'-untranslated regions of target genes. Epstein-Barr virus (EBV) has been reported to induce sustained elevation of cellular miRNAs such as miR-155. We hypothesized that miRNAs delivered by exosomes might affect the angiogenesis of retinal pigment epithelial (RPE) cells. Here, we demonstrated that co-culture of EBV-positive Burkitt's lymphoma (BL) cells (Raji) with retinal pigment epithelial (ARPE-19) cells increased the level of miR-155 in recipient cells whereas no major difference was detected for co-culture with EBV-negative BL cells (Ramos). Isolated Raji exosomes increased transcriptional and translational levels of VEGF-A in ARPE-19 cells, which was reversely correlated with von Hippel-Lindau expression. A human umbilical vein endothelial cell tube formation assay showed that delivery of ectopic miR-155 rendered ARPE-19 cells proangiogenic. Our results demonstrate that sustained accumulation of miR-155 mediated by exosomes might affect remote recipient cells such as retinal pigment epithelial cells.

Melphalan-induced apoptosis of EBV-transformed B cells through upregulation of TAp73 and XAF1 and nuclear import of XPA.

Melphalan (Mel) is widely used to treat patients with hematologic cancer, including multiple myeloma, but its mechanism of action in EBV-transformed B cells is poorly described. In this study, we demonstrate a novel mechanism by which transcriptionally active p73 (TAp73) induces translocation of X-linked inhibitor of apoptosis protein-associated factor 1 (XAF1) and xeroderma pigmentosum group A (XPA) during apoptosis caused by Mel treatment. We observed that Mel induced significant generation of reactive oxygen species (ROS) and subsequent apoptosis, as well as an early phosphorylation of p38 MAPK that preceded expression of the mitochondria membrane potential disruption-related molecules and the cleavage of caspases. In particular, Mel led to upregulation of TAp73, XAF1, and Puma and induced XPA nuclear import and translocation of Bax into mitochondria. Mel-induced apoptosis was inhibited by pretreatment with the ROS scavenger 4-amino-2,4-pyrrolidine-dicarboxylic acid (APDC) and the p38 MAPK inhibitor SB203580. We supposed that ROS generation might be the first event in Mel-induced apoptosis, because APDC blocked the increase in ROS, p38 MAPK, and TAp73, but SB203580 did not block ROS generation. Moreover, Mel elicited activation of ATR, and APDC inhibited phosphorylation of ATR but not SB203580. APDC and SB203580 completely blocked XPA and Bax translocation. We conclude that Mel promotes TAp73-mediated XAF1 and Puma expression via ROS generation and ATR/p38 MAPK pathway activation, thereby triggering apoptosis. Our results provide evidence of a novel alternate regulatory mechanism of TAp73 and reveal that Mel may be a therapeutic drug for curing EBV-related malignancies.

Propagation of spoof surface plasmon on metallic square lattice: bending and splitting of self-collimated beams.

The propagation characteristics of spoof surface plasmon modes are studied in both real and reciprocal spaces. From the metallic square lattice, we obtain constant frequency contours by directly measuring electric fields in the microwave frequency regime. The anisotropy of the measured constant frequency contour supports the presence of the negative refraction and the self-collimation which are confirmed from measured electric fields. Additionally, we demonstrate the spoof surface plasmon beam splitter in which the splitting ratio of the self-collimated beam is controlled by varying the height of rods.