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.

Understanding the molecular mechanisms of odorant binding and activation of the human OR52 family.

Structural and mechanistic studies on human odorant receptors (ORs), key in olfactory signaling, are challenging because of their low surface expression in heterologous cells. The recent structure of OR51E2 bound to propionate provided molecular insight into odorant recognition, but the lack of an inactive OR structure limited understanding of the activation mechanism of ORs upon odorant binding. Here, we determined the cryo-electron microscopy structures of consensus OR52 (OR52cs), a representative of the OR52 family, in the ligand-free (apo) and octanoate-bound states. The apo structure of OR52cs reveals a large opening between transmembrane helices (TMs) 5 and 6. A comparison between the apo and active structures of OR52cs demonstrates the inward and outward movements of the extracellular and intracellular segments of TM6, respectively. These results, combined with molecular dynamics simulations and signaling assays, shed light on the molecular mechanisms of odorant binding and activation of the OR52 family.

Deep learning framework for automated goblet cell density analysis in in-vivo rabbit conjunctiva.

Goblet cells (GCs) in the conjunctiva are specialized epithelial cells secreting mucins for the mucus layer of protective tear film and playing immune tolerance functions for ocular surface health. Because GC loss is observed in various ocular surface diseases, GC examination is important for precision diagnosis. Moxifloxacin-based fluorescence microscopy (MBFM) was recently developed for non-invasive high-contrast GC visualization. MBFM showed promise for GC examination by high-speed large-area imaging and a robust analysis method is needed to provide GC information. In this study, we developed a deep learning framework for GC image analysis, named dual-channel attention U-Net (DCAU-Net). Dual-channel convolution was used both to extract the overall image texture and to acquire the GC morphological characteristics. A global channel attention module was adopted by combining attention algorithms and channel-wise pooling. DCAU-Net showed 93.1% GC segmentation accuracy and 94.3% GC density estimation accuracy. Further application to both normal and ocular surface damage rabbit models revealed the spatial variations of both GC density and size in normal rabbits and the decreases of both GC density and size in damage rabbit models during recovery after acute damage. The GC analysis results were consistent with histology. Together with the non-invasive high-contrast imaging method, DCAU-Net would provide GC information for the diagnosis of ocular surface diseases.

Noninvasive Imaging of Conjunctival Goblet Cells as a Method for Diagnosing Dry Eye Disease in an Experimental Mouse Model.

Purpose: The purpose of this study was to evaluate a noninvasive conjunctival goblet cell (GC) imaging method for assessing dry eye disease (DED) in an experimental mouse model. Methods: Moxifloxacin-based fluorescence microscopy (MBFM) was used to examine GCs noninvasively in 56 mice. Forty-two (42) DED-induced mice were divided into 2 groups and treated topically for 14 days with cyclosporine (CsA) or normal saline (NS). In vivo MBFM imaging and clinical DED evaluations were performed and goblet cell density (GCD) and goblet cell area (GCA) were obtained and compared with histological GCD using periodic acid-Schiff (PAS) staining. Correlation and receiver operating characteristic (ROC) analyses showed MBFM's high diagnostic value. Results: The GCD and GCA of the DED mice obtained from in vivo MBFM imaging were highly correlated with clinical DED parameters and GCD obtained from PAS histology. The therapeutic effect of CsA, as observed by in vivo MBFM, was significant with respect to that of NS treatment. The ROC curves derived from in vivo MBFM showed high diagnostic value in assessing DED. Conclusions: The proposed noninvasive method has high diagnostic value in assessing the severity of DED and the effect of treatment for this disease. Translational Relevance: A noninvasive imaging method using moxifloxacin-based fluorescence microscopy was evaluated for assessing DED in an experimental mouse model. The method showed high diagnostic value in assessing the severity of DED and the effect of treatment, bridging the gap between basic research and clinical treatment. The study provides a promising tool for diagnosing and monitoring DED.

Simultaneous bright- and dark-field X-ray microscopy at X-ray free electron lasers.

The structures, strain fields, and defect distributions in solid materials underlie the mechanical and physical properties across numerous applications. Many modern microstructural microscopy tools characterize crystal grains, domains and defects required to map lattice distortions or deformation, but are limited to studies of the (near) surface. Generally speaking, such tools cannot probe the structural dynamics in a way that is representative of bulk behavior. Synchrotron X-ray diffraction based imaging has long mapped the deeply embedded structural elements, and with enhanced resolution, dark field X-ray microscopy (DFXM) can now map those features with the requisite nm-resolution. However, these techniques still suffer from the required integration times due to limitations from the source and optics. This work extends DFXM to X-ray free electron lasers, showing how the [Formula: see text] photons per pulse available at these sources offer structural characterization down to 100 fs resolution (orders of magnitude faster than current synchrotron images). We introduce the XFEL DFXM setup with simultaneous bright field microscopy to probe density changes within the same volume. This work presents a comprehensive guide to the multi-modal ultrafast high-resolution X-ray microscope that we constructed and tested at two XFELs, and shows initial data demonstrating two timing strategies to study associated reversible or irreversible lattice dynamics.

Biophysical investigation of interactions between SARS-CoV-2 spike protein and neuropilin-1.

Recent studies have suggested that neuropilin-1 (NRP1) may serve as a potential receptor in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the biophysical characteristics of interactions between NRP1 and SARS-CoV-2 remain unclear. In this study, we examined the interactions between NRP1 and various SARS-CoV-2 spike (S) fragments, including the receptor-binding domain (RBD) and the S protein trimer in a soluble form or expressed on pseudovirions, using atomic force microscopy and structural modeling. Our measurements shows that NRP1 interacts with the RBD and trimer at a higher binding frequency (BF) compared to ACE2. This NRP1-RBD interaction has also been predicted and simulated via AlphaFold2 and molecular dynamics simulations, and the results indicate that their binding patterns are very similar to RBD-ACE2 interactions. Additionally, under similar loading rates, the most probable unbinding forces between NRP1 and S trimer (both soluble form and on pseudovirions) are larger than the forces between NRP1 and RBD and between trimer and ACE2. Further analysis indicates that NRP1 has a stronger binding affinity to the SARS-CoV-2 S trimer with a dissociation rate of 0.87 s-1 , four times lower than the dissociation rate of 3.65 s-1 between NRP1 and RBD. Moreover, additional experiments show that RBD-neutralizing antibodies can significantly reduce the BF for both ACE2 and NRP1. Together, the study suggests that NRP1 can be an alternative receptor for SARS-CoV-2 attachment to human cells, and the neutralizing antibodies targeting SARS-CoV-2 RBD can reduce the binding between SARS-CoV-2 and NRP1.

Molecular Analysis of Anti-Tuberculosis Drug Resistance of Mycobacterium tuberculosis Isolated in the Republic of Korea.

Rapid and accurate detection of tuberculosis (TB) drug resistance is critical for the successful treatment and control of TB. Here, we investigated resistance to anti-TB drugs and genetic variations in 215 drug-resistant Mycobacterium tuberculosis isolates in Korea. Genetic variations were observed in rpoB Ser531Leu, katG Ser315Thr, and gyrA Asp94Gly; however, the minimum inhibitory concentrations varied, which can be attributed to other resistance mechanisms. Examination of genetic relatedness among drug-resistant isolates revealed that the cluster size of resistant bacteria was less than six strains, suggesting no evidence of a large-scale epidemic caused by a specific strain. However, rpoC mutants of the rifampicin-resistant isolates were composed of five types of clusters, suggesting that these compensatory mutations advance propagation. In the present study, more than 90% of the resistance mechanisms to major anti-TB drugs were identified, and the effect of each mutation on drug resistance was estimated. With the clinical application of recent next-generation sequencing-based susceptibility testing, the present study is expected to improve the clinical utilization of genotype-based drug susceptibility testing for the diagnosis and treatment of patients with drug-resistant TB.

Genome sequence of Coxiella burnetii KZQ2 isolated from a clinical strain in the Republic of Korea.

The genome of Coxiella burnetii KZQ2, isolated from clinical patients in Korea, is 2.04 MB long. Multispacer types were ST77, and phylogenetic tree analysis showed that KZQ2 is closely related to the CbuK_Q154 chronic strain isolated from human endocarditis patients in the USA.

Highly efficient graphene terahertz modulator with tunable electromagnetically induced transparency-like transmission.

Graphene-based optical modulators have been extensively studied owing to the high mobility and tunable permittivity of graphene. However, weak graphene-light interactions make it difficult to achieve a high modulation depth with low energy consumption. Here, we propose a high-performance graphene-based optical modulator consisting of a photonic crystal structure and a waveguide with graphene that exhibits an electromagnetically-induced-transparency-like (EIT-like) transmission spectrum at terahertz frequency. The high quality-factor guiding mode to generate the EIT-like transmission enhances light-graphene interaction, and the designed modulator achieves a high modulation depth of 98% with a significantly small Fermi level shift of 0.05 eV. The proposed scheme can be utilized in active optical devices that require low power consumption.

Whole-Genome Sequencing of Mycobacterium tuberculosis Isolated from Three Hospitals in South Korea.

Mycobacterium tuberculosis clinical isolates CN118, CN272, CB131, IP004, and IP016 were isolated from three hospitals in South Korea. Here, we report the whole-genome sequences of the five M. tuberculosis clinical isolates to aid the understanding of their genetic diversity.

Whole-Genome Sequencing of Clinical Isolates of Mycobacterium tuberculosis Isolated before and after Treatment.

Mycobacterium tuberculosis clinical isolates CN177_0W, CN177_2W, CB060_0W, and CB060_2W were isolated from two tuberculosis patients in South Korea. Here, we report the whole-genome sequences of clinical isolates of M. tuberculosis isolated before and after tuberculosis treatment.

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.

CHARMM-GUI Enhanced Sampler for various collective variables and enhanced sampling methods.

Enhanced sampling methodologies modifying underlying Hamiltonians can be used for the systems with a rugged potential energy surface that makes it hard to observe convergence using conventional unbiased molecular dynamics (MD) simulations. We present CHARMM-GUI Enhanced Sampler, a web-based tool to prepare various enhanced sampling simulations inputs with user-selected collective variables (CVs). Enhanced Sampler provides inputs for the following nine methods: accelerated MD, Gaussian accelerated MD, conformational flooding, metadynamics, adaptive biasing force, steered MD, temperature replica exchange MD, replica exchange solute tempering 2, and replica exchange umbrella sampling for the method-implemented MD packages including AMBER, CHARMM, GENESIS, GROMACS, NAMD, and OpenMM. Users only need to select a group of atoms via intuitive web-implementation in order to define commonly used nine CVs of interest: center of mass based distance, angle, dihedral, root-mean-square-distance, radius of gyration, distance projected on axis, two types of angles projected on axis, and coordination numbers. The enhanced sampling methods are tested with several biological systems to illustrate their efficiency over conventional MD. Enhanced Sampler with carefully optimized system-dependent parameters will help users to get meaningful results from their enhanced sampling simulations.

Transcriptomic analysis in the striatum reveals the involvement of Nurr1 in the social behavior of prenatally valproic acid-exposed male mice.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that exhibits neurobehavioral deficits characterized by abnormalities in social interactions, deficits in communication as well as restricted interests, and repetitive behaviors. The basal ganglia is one of the brain regions implicated as dysfunctional in ASD. In particular, the defects in corticostriatal function have been reported to be involved in the pathogenesis of ASD. Surface deformation of the striatum in the brains of patients with ASD and their correlation with behavioral symptoms was reported in magnetic resonance imaging (MRI) studies. We demonstrated that prenatal valproic acid (VPA) exposure induced synaptic and molecular changes and decreased neuronal activity in the striatum. Using RNA sequencing (RNA-Seq), we analyzed transcriptome alterations in striatal tissues from 10-week-old prenatally VPA-exposed BALB/c male mice. Among the upregulated genes, Nurr1 was significantly upregulated in striatal tissues from prenatally VPA-exposed mice. Viral knockdown of Nurr1 by shRNA significantly rescued the reduction in dendritic spine density and the number of mature dendritic spines in the striatum and markedly improved social deficits in prenatally VPA-exposed mice. In addition, treatment with amodiaquine, which is a known ligand for Nurr1, mimicked the social deficits and synaptic abnormalities in saline-exposed mice as observed in prenatally VPA-exposed mice. Furthermore, PatDp+/- mice, a commonly used ASD genetic mouse model, also showed increased levels of Nurr1 in the striatum. Taken together, these results suggest that the increase in Nurr1 expression in the striatum is a mechanism related to the changes in synaptic deficits and behavioral phenotypes of the VPA-induced ASD mouse model.

Activation of the SigE-SigB signaling pathway by inhibition of the respiratory electron transport chain and its effect on rifampicin resistance in Mycobacterium smegmatis.

Using a mutant of Mycobacterium smegmatis lacking the major aa3 cytochrome c oxidase of the electron transport chain (Δaa3), we demonstrated that inhibition of the respiratory electron transport chain led to an increase in antibiotic resistance of M. smegmatis to isoniazid, rifampicin, ethambutol, and tetracycline. The alternative sigma factors SigB and SigE were shown to be involved in an increase in rifampicin resistance of M. smegmatis induced under respiration-inhibitory conditions. As in Mycobacterium tuberculosis, SigE and SigB form a hierarchical regulatory pathway in M. smegmatis through SigE-dependent transcription of sigB. Expression of sigB and sigE was demonstrated to increase in the Δaa3 mutant, leading to upregulation of the SigB-dependent genes in the mutant. The pho U2 (MSMEG_1605) gene implicated in a phosphate-signaling pathway and the MSMEG_1097 gene encoding a putative glycosyltransferase were identified to be involved in the SigB-dependent enhancement of rifampicin resistance observed for the Δaa3 mutant of M. smegmatis. The significance of this study is that the direct link between the functionality of the respiratory electron transport chain and antibiotic resistance in mycobacteria was demonstrated for the first time using an electron transport chain mutant rather than inhibitors of electron transport chain.

Helicity-driven chiral self-sorting supramolecular polymerization with Ag+: right- and left-helical aggregates.

The study of chiral self-sorting is extremely important for understanding biological systems and for developing applications for the biomedical field. In this study, we attempted unprecedented chiral self-sorting supramolecular polymerization accompanying helical inversion with Ag+ in one enantiomeric component. Bola-type terpyridine-based ligands (R-L1 and S-L1) comprising R- or S-alanine analogs were synthesized. First, R-L1 dissolved in DMSO/H2O (1 : 1, v/v) forms right-handed helical fibers (aggregate I) via supramolecular polymerization. However, after the addition of AgNO3 (0.2-1.1 equiv.) to the R-L1 ligand, in particular, it was found that aggregate II with left-handed helicity is generated from the [R-L1(AgNO3)2] complex through the [R-L1Ag]+ complex via the dissociation of aggregate I by a multistep with an off pathway, thus demonstrating interesting self-sorting properties driven by helicity and shape discrimination. In addition, the [R-L1(AgNO3)2] complex, which acted as a building block to generate aggregate III with a spherical structure, existed as a metastable product during the formation of aggregate II in the presence of 1.2-1.5 equiv. of AgNO3. Furthermore, the AFM and CD results of two samples prepared using aggregates I and III with different volume ratios were similar to those obtained upon the addition of AgNO3 to free R-L1. These findings suggest that homochiral self-sorting in a mixture system occurred by the generation of aggregate II composed of the [R-L1Ag]+ complex via the rearrangement of both, aggregates I and III. This is a unique example of helicity- and shape-driven chiral self-sorting supramolecular polymerization induced by Ag+ starting from one enantiomeric component. This research will improve understanding of homochirality in complex biological models and contribute to the development of new chiral materials and catalysts for asymmetric synthesis.

Binding of Human ACE2 and RBD of Omicron Enhanced by Unique Interaction Patterns Among SARS-CoV-2 Variants of Concern.

The 2019 coronavirus disease (COVID-19) pandemic has had devastating impacts on our global health. 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 now overtaken the Delta variant and 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, Q493K/R, 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). 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 result 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.