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.

Prognostic factors for thyroid-stimulating immunoglobulin normalization in moderate-to-severe Graves' orbitopathy: a 36-month longitudinal study.

PURPOSE: The primary objective of this study was to identify predictive factors linked to the normalization of thyroid-stimulating immunoglobulin (TSI) levels in patients diagnosed with active, moderate-to-severe Graves' orbitopathy (GO). The study also tracked the longitudinal changes in TSI levels over a 36-month period following treatment. METHODS: The study population consisted of individuals who were recently diagnosed with active, moderate-to-severe GO and received a 12-week course of intravenous methylprednisolone (IVMP) treatment. A subgroup of patients who did not respond to the initial treatment received an additional 20 Gy of radiation therapy (RTx). TSI levels were monitored at the time of diagnosis, after treatment, and subsequently every 6 months for 36 months. Normalization was defined as a TSI level below 140%. Patients were divdied into two groups with success and failure group depending on whether TSI became normal or not. RESULTS: Out of 83 patients, 36 (43.4%) achieved normalized TSI levels within two years post-IVMP treatment. Lower initial TSI levels (< 425%), absence of additional RTx, and early treatment initiation were associated with a higher likelihood of TSI normalization (P = 0.035, P = 0.028, P < 0.001, respectively). Notably, significant differences in TSI level reduction were observed from 18 months post-treatment between the two groups (P = 0.031). A TSI cutoff value of 413% was identified as predictive for normalization at 24 months (P = 0.002). CONCLUSION: This study is the first to identify key factors that influence normalization of TSI levels in moderate-to-severe Graves' Orbitopathy. It highlights the importance of early treatment decisions, particularly for patients with initial TSI levels above 425%. Despite the treatment, less than half of the patients achieved TSI normalization within 24 months, underscoring the need for additional research to explore the relationship between TSI levels and the clinical manifestations of chronic GO.

Ultrafast Energy Transfer Process in Confined Gold Nanospheres Revealed by Femtosecond X-ray Imaging and Diffraction.

Femtosecond laser pulses drive nonequilibrium phase transitions via reaction paths hidden in thermal equilibrium. This stimulates interest to understand photoinduced ultrafast melting processes, which remains incomplete due to challenges in resolving accompanied kinetics at the relevant space-time resolution. Here, by newly establishing a multiplexing femtosecond X-ray probe, we have successfully revealed ultrafast energy transfer processes in confined Au nanospheres. Real-time images of electron density distributions with the corresponding lattice structures elucidate that the energy transfer begins with subpicosecond melting at the specimen boundary earlier than the lattice thermalization, and proceeds by forming voids. Two temperature molecular dynamics simulations uncovered the presence of both heterogeneous melting with the melting front propagation from surface and grain boundaries and homogeneous melting with random melting seeds and nanoscale voids. Supported by experimental and theoretical results, we provide a comprehensive atomic-scale picture that accounts for the ultrafast laser-induced melting and evaporation kinetics.

scL-2PAM: A Novel Countermeasure That Ameliorates Neuroinflammation and Neuronal Losses in Mice Exposed to an Anticholinesterase Organophosphate.

Due to their inhibition of acetylcholinesterase, organophosphates are among the most toxic of chemicals. Pralidoxime (a.k.a 2-PAM) is the only acetylcholinesterase reactivator approved in the U.S., but 2-PAM only poorly traverses the blood-brain barrier. Previously, we have demonstrated that scL-2PAM, a nanoformulation designed to enter the brain via receptor-mediated transcytosis, is superior to unencapsulated 2-PAM for reactivating brain acetylcholinesterase, ameliorating cholinergic crisis, and improving survival rates for paraoxon-exposed mice. Here, we employ histology and transcriptome analyses to assess the ability of scL-2PAM to prevent neurological sequelae including microglial activation, expression of inflammatory cytokines, and ultimately loss of neurons in mice surviving paraoxon exposures. Levels of the mRNA encoding chemokine ligand 2 (CCL2) were significantly upregulated after paraoxon exposures, with CCL2 mRNA levels in the brain correlating well with the intensity and duration of cholinergic symptoms. Our nanoformulation of 2-PAM was found to be superior to unencapsulated 2-PAM in reducing the levels of the CCL2 transcript. Moreover, brain histology revealed that scL-2PAM was more effective than unencapsulated 2-PAM in preventing microglial activation and the subsequent loss of neurons. Thus, scL-2PAM appears to be a new and improved countermeasure for reducing neuroinflammation and mitigating brain damage in survivors of organophosphate exposures.

T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice.

Evaluation of the therapeutic potential of RNAi for HIV infection has been hampered by the challenges of siRNA delivery and lack of suitable animal models. Using a delivery method for T cells, we show that siRNA treatment can dramatically suppress HIV infection. A CD7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2rgamma-/- mice reconstituted with human lymphocytes (Hu-PBL) or CD34+ hematopoietic stem cells (Hu-HSC). In HIV-infected Hu-PBL mice, treatment with anti-CCR5 (viral coreceptor) and antiviral siRNAs complexed to scFvCD7-9R controlled viral replication and prevented the disease-associated CD4 T cell loss. This treatment also suppressed endogenous virus and restored CD4 T cell counts in mice reconstituted with HIV+ peripheral blood mononuclear cells. Moreover, scFvCD7-9R could deliver antiviral siRNAs to naive T cells in Hu-HSC mice and effectively suppress viremia in infected mice. Thus, siRNA therapy for HIV infection appears to be feasible in a preclinical animal model.

Enrichment of Activated Fibroblasts as a Potential Biomarker for a Non-Durable Response to Anti-Tumor Necrosis Factor Therapy in Patients with Crohn's Disease.

We investigated whether the response to anti-tumor necrosis factor (anti-TNF) treatment varied according to inflammatory tissue characteristics in Crohn's disease (CD). Bulk RNA sequencing (RNA-seq) data were obtained from inflamed and non-inflamed tissues from 170 patients with CD. The samples were clustered based on gene expression profiles using principal coordinate analysis (PCA). Cellular heterogeneity was inferred using CiberSortx, with bulk RNA-seq data. The PCA results displayed two clusters of CD-inflamed samples: one close to (Inflamed_1) and the other far away (Inflamed_2) from the non-inflamed samples. Inflamed_1 was rich in anti-TNF durable responders (DRs), and Inflamed_2 was enriched in non-durable responders (NDRs). The CiberSortx results showed that the cell fraction of activated fibroblasts was six times higher in Inflamed_2 than in Inflamed_1. Validation with public gene expression datasets (GSE16879) revealed that the activated fibroblasts were enriched in NDRs over Next, we used DRs by 1.9 times pre-treatment and 7.5 times after treatment. Fibroblast activation protein (FAP) was overexpressed in the Inflamed_2 and was also overexpressed in the NDRs in both the RISK and GSE16879 datasets. The activation of fibroblasts may play a role in resistance to anti-TNF therapy. Characterizing fibroblasts in inflamed tissues at diagnosis may help to identify patients who are likely to respond to anti-TNF therapy.

Development of an experimental apparatus to observe ultrafast phenomena by tender X-ray absorption spectroscopy at PAL-XFEL.

Understanding the ultrafast dynamics of molecules is of fundamental importance. Time-resolved X-ray absorption spectroscopy (TR-XAS) is a powerful spectroscopic technique for unveiling the time-dependent structural and electronic information of molecules that has been widely applied in various fields. Herein, the design and technical achievement of a newly developed experimental apparatus for TR-XAS measurements in the tender X-ray range with X-ray free-electron lasers (XFELs) at the Pohang Accelerator Laboratory XFEL (PAL-XFEL) are described. Femtosecond TR-XAS measurements were conducted at the Ru L3-edge of well known photosensitizer tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)3]2+) in water. The results indicate ultrafast photoinduced electron transfer from the Ru center to the ligand, which demonstrates that the newly designed setup is applicable for monitoring ultrafast reactions in the femtosecond domain.

Statistical analysis of hard X-ray radiation at the PAL-XFEL facility performed by Hanbury Brown and Twiss interferometry.

A Hanbury Brown and Twiss interferometry experiment based on second-order correlations was performed at the PAL-XFEL facility. The statistical properties of the X-ray radiation were studied within this experiment. Measurements were performed at the NCI beamline at 10 keV photon energy under various operation conditions: self-amplified spontaneous emission (SASE), SASE with a monochromator, and self-seeding regimes at 120 pC, 180 pC and 200 pC electron bunch charge. Statistical analysis showed short average pulse duration from 6 fs to 9 fs depending on the operational conditions. A high spatial degree of coherence of about 70-80% was determined in the spatial domain for the SASE beams with the monochromator and self-seeding regime of operation. The obtained values describe the statistical properties of the beams generated at the PAL-XFEL facility.

Characterizing the intrinsic properties of individual XFEL pulses via single-particle diffraction.

With each single X-ray pulse having its own characteristics, understanding the individual property of each X-ray free-electron laser (XFEL) pulse is essential for its applications in probing and manipulating specimens as well as in diagnosing the lasing performance. Intensive research using XFEL radiation over the last several years has introduced techniques to characterize the femtosecond XFEL pulses, but a simple characterization scheme, while not requiring ad hoc assumptions, to address multiple aspects of XFEL radiation via a single data collection process is scant. Here, it is shown that single-particle diffraction patterns collected using single XFEL pulses can provide information about the incident photon flux and coherence property simultaneously, and the X-ray beam profile is inferred. The proposed scheme is highly adaptable to most experimental configurations, and will become an essential approach to understanding single X-ray pulses.

Targeted nanocomplex carrying siRNA against MALAT1 sensitizes glioblastoma to temozolomide.

Intrinsic therapeutic resistance especially in cancer stem cells (CSCs) together with extensive tumor cell infiltration and restricted permeation of the blood-brain barrier (BBB) by drugs may all contribute to the treatment failure in patients with glioblastoma multiforme (GBM). Accumulating evidence suggests that long non-coding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a role in tumor cell infiltration and therapeutic resistance of GBM. Using our tumor-targeted nanocomplex, we have modulated the expression of MALAT1 and investigated its impact on GBM cells. Importantly, our nanocomplex is able to target CSCs that are considered to be the prime culprits in therapeutic resistance and recurrence of GBM. Attenuation of MALAT1 by RNA interference significantly lowered the growth, motility and stemness of GBM cells. In addition, silencing of MALAT1 clearly improved the sensitivity of GBM cells to chemotherapeutic agents including the current first-line therapy of GBM [temozolomide (TMZ)]. In animal models of GBM, tumor involution with a modest but statistically significant survival benefit was achieved with concurrent treatment of TMZ and nanocomplex-mediated silencing of MALAT1. These results suggest that combining standard TMZ treatment with lncRNA-targeting therapies using our nanocomplex could substantially enhance the very poor prognosis for GBM patients.

Efficient pathway enrichment and network analysis of GWAS summary data using GSA-SNP2.

Pathway-based analysis in genome-wide association study (GWAS) is being widely used to uncover novel multi-genic functional associations. Many of these pathway-based methods have been used to test the enrichment of the associated genes in the pathways, but exhibited low powers and were highly affected by free parameters. We present the novel method and software GSA-SNP2 for pathway enrichment analysis of GWAS P-value data. GSA-SNP2 provides high power, decent type I error control and fast computation by incorporating the random set model and SNP-count adjusted gene score. In a comparative study using simulated and real GWAS data, GSA-SNP2 exhibited high power and best prioritized gold standard positive pathways compared with six existing enrichment-based methods and two self-contained methods (alternative pathway analysis approach). Based on these results, the difference between pathway analysis approaches was investigated and the effects of the gene correlation structures on the pathway enrichment analysis were also discussed. In addition, GSA-SNP2 is able to visualize protein interaction networks within and across the significant pathways so that the user can prioritize the core subnetworks for further studies. GSA-SNP2 is freely available at https://sourceforge.net/projects/gsasnp2.

A tumor-targeting nanomedicine carrying the p53 gene crosses the blood-brain barrier and enhances anti-PD-1 immunotherapy in mouse models of glioblastoma.

Despite its anticipated clinical potential, anti-PD-1 immunotherapy has only yielded poor outcomes in recent clinical trials for glioblastoma patients. Strategies combining anti-PD-1 antibody with other treatment modalities are being explored to alter the immunosuppressive microenvironment that appears to characterize these anti-PD-1-insensitive tumors. Here, we evaluated whether introducing wild-type p53 gene via a tumor-targeting nanomedicine (termed SGT-53) could provide immune stimulation and augment anti-PD-1 therapy in mouse syngeneic GL261 tumor models (either subcutaneous or intracranial). In both models, anti-PD-1 monotherapy had no demonstrable therapeutic effect. However, combining anti-PD-1 with our investigational nanomedicine SGT-53 was very effective in inhibiting tumor growth, inducing tumor cell apoptosis and increasing intratumoral T-cell infiltration. A significant survival benefit was observed in mice bearing intracranial glioblastoma receiving combination treatment. Importantly, SGT-53 upregulated PD-L1 expression both in vitro and in vivo. Transcriptome analysis revealed modulation of genes linked to either cancer progression or immune activation after combination treatment. Our data suggest that SGT-53 can boost antitumor immunity and sensitize glioblastoma to anti-PD-1 therapy by converting immunologically "cold" tumors into "hot" tumors. Combining SGT-53 with anti-PD-1 might benefit more patients from anti-PD-1 immunotherapy and our data support evaluation of this combination in patients with glioblastoma.

Comparing the spatial coherence of the natural and focused X-rays from a free electron laser.

The degree of spatial coherence, as basic characteristics of the radiation, becomes an important guide to evaluate the performance of X-rays from newly introduced advanced light sources including the X-ray free electron laser (XFEL). Often the modification of the X-ray wavefronts to fulfill various applications is necessary, but also there is the need to preserve its coherence property. However, experimental investigation directly comparing the coherence property of focused X-ray radiations with the unmodified ones has not been available. We have performed Young's double-slit experiments by recording diffraction patterns both from slit apertures for unfocused XFEL radiation and from pairs of Au nanoparticles for one-micron focused XFEL radiations. The results confirm that the degree of spatial coherence is preserved for well-built K-B focusing mirrors.

Nanotherapeutics for Gene Modulation that Prevents Apoptosis in the Brain and Fatal Neuroinflammation.

The failure of therapeutic agents to cross the blood-brain barrier (BBB) has been a major impediment in the treatment of neurological disorders and brain tumors. We have addressed this issue using an immunoliposome nanocomplex (designated scL) that delivers therapeutic nucleic acids across the BBB into the deep brain via transcytosis mediated by transferrin receptors. We validated brain delivery of payloads after systemic administration by monitoring uptake of fluorescently labeled payloads and by confirming up- or down-modulation of specific target gene expression in the brain, mainly in neuronal cells. As proof of concept for the therapeutic potential of our delivery system, we employed scL delivering an siRNA targeting tumor necrosis factor alpha to suppress neuroinflammation and neuronal apoptosis and to protect mice in lethal endotoxemia triggered by bacterial lipopolysaccharide. Brain delivery of therapeutic payloads via scL has major implications for the development of treatments for neurological disorders and brain tumors.

Genetic risk score raises the risk of incidence of chronic kidney disease in Korean general population-based cohort.

BACKGROUND: Chronic kidney disease (CKD) is a common disease, affecting about 10% of the general population. The genetic component about CKD incidence in Asian population is not well known. The aim of the study is to find the genetic loci associated with incident CKD and to figure out the effect of genetic variation on the development of CKD. METHODS: We conducted a genome-wide association (GWA) study regarding the development of CKD based on two population-based cohorts of Korean Genome Epidemiology Study. 3617 Koreans from two different cohorts, aged 40-49 years without CKD at initial visit, were included in our analysis. We used 2510 individuals in Ansan as the discovery set and another 1107 individuals from Ansung as the replication set. At baseline, members of both cohorts provided information on creatinine, and DNA samples were collected for genotyping. Single nucleotide polymorphisms that surpassed a significance threshold of P < 5 × 10-3 were selected. RESULTS: A total of 281 among 3617 developed CKD during the follow-up period. Incident CKD group was older (P < 0.001), included more female (P < 0.001), and had more hypertension and diabetes (P < 0.001). We identified 12 SNPs that are associated with incident CKD in the GWA study and made genetic risk score using these SNPs. In multiple Cox regression analysis, genetic risk score was still a significant associated factor (HR 1.311, CI 1.201, 1.431, P < 0.001). CONCLUSIONS: We identified several loci highly associated with incident CKD. The findings suggest the need for further investigations on the genetic propensity for incident CKD.

Accuracy of machine learning for differentiation between optic neuropathies and pseudopapilledema.

BACKGROUND: This study is to evaluate the accuracy of machine learning for differentiation between optic neuropathies, pseudopapilledema (PPE) and normals. METHODS: Two hundred and ninety-five images of optic neuropathies, 295 images of PPE, and 779 control images were used. Pseudopapilledema was defined as follows: cases with elevated optic nerve head and blurred disc margin, with normal visual acuity (> 0.8 Snellen visual acuity), visual field, color vision, and pupillary reflex. The optic neuropathy group included cases of ischemic optic neuropathy (177), optic neuritis (48), diabetic optic neuropathy (17), papilledema (22), and retinal disorders (31). We compared four machine learning classifiers (our model, GoogleNet Inception v3, 19-layer Very Deep Convolution Network from Visual Geometry group (VGG), and 50-layer Deep Residual Learning (ResNet)). Accuracy and area under receiver operating characteristic curve (AUROC) were analyzed. RESULTS: The accuracy of machine learning classifiers ranged from 95.89 to 98.63% (our model: 95.89%, Inception V3: 96.45%, ResNet: 98.63%, and VGG: 96.80%). A high AUROC score was noted in both ResNet and VGG (0.999). CONCLUSIONS: Machine learning techniques can be combined with fundus photography as an effective approach to distinguish between PPE and elevated optic disc associated with optic neuropathies.

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.

Focusing X-ray free-electron laser pulses using Kirkpatrick-Baez mirrors at the NCI hutch of the PAL-XFEL.

The Pohang Accelerator Laboratory X-ray Free-Electron Laser (PAL-XFEL) is a recently commissioned X-ray free-electron laser (XFEL) facility that provides intense ultrashort X-ray pulses based on the self-amplified spontaneous emission process. The nano-crystallography and coherent imaging (NCI) hutch with forward-scattering geometry is located at the hard X-ray beamline of the PAL-XFEL and provides opportunities to perform serial femtosecond crystallography and coherent X-ray diffraction imaging. To produce intense high-density XFEL pulses at the interaction positions between the X-rays and various samples, a microfocusing Kirkpatrick-Baez (KB) mirror system that includes an ultra-precision manipulator has been developed. In this paper, the design of a KB mirror system that focuses the hard XFEL beam onto a fixed sample point of the NCI hutch, which is positioned along the hard XFEL beamline, is described. The focusing system produces a two-dimensional focusing beam at approximately 2 µm scale across the 2-11 keV photon energy range. XFEL pulses of 9.7 keV energy were successfully focused onto an area of size 1.94 µm × 2.08 µm FWHM.

Apparent power-law behavior of water's isothermal compressibility and correlation length upon supercooling.

The isothermal compressibility and correlation length of supercooled water obtained from small-angle X-ray scattering (SAXS) were analyzed by fits based on an apparent power-law in the temperature range from 280 K down to the temperature of maximum compressibility at 229 K. Although the increase in thermodynamic response functions is not towards a critical point, it is still possible to obtain an apparent power law all the way to the maximum values with best-fit exponents of γ = 0.40 ± 0.01 for the isothermal compressibility and ν = 0.26 ± 0.03 for the correlation length. The ratio between these exponents is close to a value of ≈0.5, as expected for a critical point, indicating the proximity of a potential second critical point. Comparison of γ obtained from experiment with molecular dynamics simulations on the iAMOEBA water model shows that it would be located at pressures in the neighborhood of 1 kbar. The high value and sharpness of the compressibility maximum observed in the experiment are not reproduced by any of the existing classical water models, thus inviting further development of simulation models of water.

Nonstoichiometric Solution-Processed BaTiO3 Film for Gate Insulator Applications.

Solution processed barium titanate (BTO) was used to fabricate an Al/BaTiO3/p-Si metal-insulator-semiconductor (MIS) structure, which was used as a gate insulator. Changes in the electrical characteristics of the film were investigated as a function of the film thickness and post deposition annealing conditions. Our results showed that a thickness of 5 layers and an annealing temperature of 650 °C produced the highest electrical performance. BaxTi1-xO3 was altered at x = 0.10, 0.30, 0.50, 0.70, 0.90, and 1.0 to investigate changes in the electrical properties as a function of composition. The highest dielectric constant of 87 was obtained for x = 0.10, while the leakage current density was suppressed as Ba content increased. The lowest leakage current density was 1.34×10-10 A/cm2, which was observed at x = 0.90. The leakage current was related to the resistivity of the film, the interface states, and grain densification. Space charge limited current (SCLC) was the dominant leakage mechanism in BTO films based on leakage current analysis. Although a Ba content of x = 0.90 had the highest trap density, the traps were mainly composed of Ti-vacancies, which acted as strong electron traps and affected the film resistivity. A secondary phase, Ba2TiO4, which was observed in cases of excess Ba, acted as a grain refiner and provided faster densification of the film during the thermal process. The absence of a secondary phase in BaO (x = 1.0) led to the formation of many interface states and degradation in the electrical properties. Overall, the insulator properties of BTO were improved when the composition ratio was x = 0.90.