Therapeutic manipulation of IKBKAP mis-splicing with a small molecule to cure familial dysautonomia.

Approximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP-familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP-FD exon 20 has a suboptimal 5' splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases.

Community-acquired fulminant Clostridioides (Clostridium) difficile infection by ribotype 027 isolate in Japan: a case report.

BACKGROUND: Clostridioides (Clostridium) difficile infection (CDI) has become an increasingly significant disease not only as healthcare-associated infection, but also as community-acquired (CA) infection worldwide. CDI caused by the NAP1/BI/027 strain is reported to be more severe, difficult to cure, and frequently associated with recurrences in North America and Europe. CASE PRESENTATION: A 68-year-old woman was referred to our hospital for continuous lower abdominal pain 4 weeks after eradication therapy against Helicobacter pylori. While she was treated with fasting on the suspicion of ischemic colitis, she experienced septic shock. Emergent subtotal proctocolectomy revealed fulminant pseudomembranous C. difficile colitis. The C. difficile isolate recovered from the patient was identified as ribotype 027, which has been reported to be uncommon in Japan. CONCLUSION: We report a rare case of CA fulminant pseudomembranous colitis caused by ribotype 027 C. difficile after H. pylori eradication therapy.

Endogenous agonist-bound S1PR3 structure reveals determinants of G protein-subtype bias.

Sphingosine-1-phosphate (S1P) regulates numerous important physiological functions, including immune response and vascular integrity, via its cognate receptors (S1PR1 to S1PR5); however, it remains unclear how S1P activates S1PRs upon binding. Here, we determined the crystal structure of the active human S1PR3 in complex with its natural agonist S1P at 3.2-Å resolution. S1P exhibits an unbent conformation in the long tunnel, which penetrates through the receptor obliquely. Compared with the inactive S1PR1 structure, four residues surrounding the alkyl tail of S1P (the "quartet core") exhibit orchestrating rotamer changes that accommodate the moiety, thereby inducing an active conformation. In addition, we reveal that the quartet core determines G protein selectivity of S1PR3. These results offer insight into the structural basis of activation and biased signaling in G protein-coupled receptors and will help the design of biased ligands for optimized therapeutics.

Intensity Interference in a Coherent Spin-Polarized Electron Beam.

We investigate the intensity interference between pairs of electrons using a spin-polarized electron beam having a high polarization and a narrow energy width. We observe spin-dependent antibunching on the basis of coincident counts of electron pairs performed with a spin-polarized transmission electron microscope, which could control the spin-polarization without any changes in the electron optics. The experimental results show that the time correlation was only affected by the spin polarization, demonstrating that the antibunching is associated with fermionic statistics. The coherent spin-polarized electron beam facilitates the extraction of intrinsic quantum interference.

Reduction of the extra-column band dispersion by a slow transport and splitting of a sample band in isocratic reversed-phase liquid chromatography.

Sample introduction method was studied to reduce the extra-column effect in reversed-phase HPLC. Slow transport of a sample band (SToSB) in the pre-column space followed by the introduction of the band into the column at a near-optimum flow rate resulted in larger plate counts for a 1.0 mmID, 5 cm long column as much as 1.4-1.6 times for solutes with a retention factor (k) of 0.5-1.8 compared to a conventional elution method. Further reduction of the extra-column effect was possible by orthogonally splitting the sample band (SplSB) by flow switching during its slow transport followed by the introduction of the leading part of the band into the column. In this case, increased plate counts of up to 2-3 times for solutes with k of 0.5-1.8 were observed for a 1.0 mmID, 5 cm column. The sample introduction method, SToSB in the injector and the pre-column tube of a few µL, was found to reduce the extra-column band variance by 0.4-0.5 µL2 for an UHPLC system with the extra-column volume (Vextra) of ca. 4.6 µL and the system variance (σextra2) of 1.1 µL2 at flow rate of 100 µL/min, while SToSB and subsequent SplSB were found to be more effective, reducing σextra2 by about 0.8 µL2. With an UHPLC instrument with Vextra of about 10 µL and σextra2 of ca. 3.6 µL2 at flow rate of 300 µL/min, 1.4-2.1 times as many plate counts were observed with SToSB and SplSB compared to the normal elution method for early-eluting solutes with k=0.25-1.7 for a column, 2.1 mmID, 5 cm long. With this UHPLC instrument, SToSB and/or SplSB resulted in the reduction of σextra2 by 1.2-2.2 µL2.

Progress in environmental high-voltage transmission electron microscopy for nanomaterials.

A new environmental high-voltage transmission electron microscope (E-HVEM) was developed by Nagoya University in collaboration with JEOL Ltd. An open-type environmental cell was employed to enable in-situ observations of chemical reactions on catalyst particles as well as mechanical deformation in gaseous conditions. One of the reasons for success was the application of high-voltage transmission electron microscopy to environmental (in-situ) observations in the gas atmosphere because of high transmission of electrons through gas layers and thick samples. Knock-on damages to samples by high-energy electrons were carefully considered. In this paper, we describe the detailed design of the E-HVEM, recent developments and various applications. This article is part of a discussion meeting issue 'Dynamic in situ microscopy relating structure and function'.

Fast enantiomeric separation of amino acids using liquid chromatography/mass spectrometry on a chiral crown ether stationary phase.

Fast enantiomeric separation of amino acids was studied by liquid chromatography/mass spectrometry (LC/MS) on a chiral crown ether stationary phase. A chiral crown ether bonded silica column (3 mm internal diameter (i.d.), 5 cm long) packed with 3 µm particles was employed instead of a 15 cm column packed with 5 µm particles used in our previous study. In addition, the extra-column variance, becoming more serious for smaller columns, was reduced by replacing 0.127 mm i.d. post-column tubes with shorter, smaller-diameter (0.0635 mm i.d.) tubes. The results demonstrated the benefits of using shorter columns packed with smaller particles and the reduction of the extra-column band broadening for fast enantiomeric separation. Finally, the enantiomeric separation of 18 pairs of proteinogenic amino acids was achieved within 2 min with a resolution (Rs) > 1.5 for each pair using an isocratic mobile phase of acetonitrile/water/trifluoroacetic acid (ACN/W/TFA) = 96/4/0.5, and a flow rate 1.2 mL/min at 30°C. This is the highest throughput method for simultaneous chiral separation of all proteinogenic amino acids except proline to date.

Tunable Liquid Chromatographic Separation of H/D Isotopologues Enabled by Aromatic π Interactions.

We report hydrogen/deuterium (H/D) isotope effects based on weak intermolecular interactions with polar functional groups and aromatic rings in liquid chromatography (LC). Various LC experiments with different aromatic analytes, separation media, and nonpolar mobile phases were conducted under normal phase LC conditions, where the hydrophobic interaction was completely suppressed. The separation media that had polar functional groups, such as silanol groups, allowed for higher separation efficiencies for the pairs of aromatic H/D isotopologues. In comparing the 13C NMR spectra of protiated and deuterated aromatic analytes, the electron density of the deuterated analyte was found to be slightly higher than that of the protiated analytes. In the case of silanol functional groups, aromatic rings of the analyte acted as donors through the OH-π interaction to hydrogen atoms in the silanol groups. Thus, the deuterated analytes were able to be greatly retained by the stronger OH-π interactions. Furthermore, a C70-fullerene bonded monolithic column (C70 column), which effectively provides CH-π interactions, allowed the opposite isotope effect. Briefly, an electrostatic attraction based on the dipole-(induced) dipole interaction dominated in the CH-π interactions, according to a van't Hoff analysis. Hence, the bonding lengths of the C-H or D bonds were sensitively affected, such that we were able to conclude that the CH-π interaction depended on the geometric effect. Applying these opposing H/D isotope effects, we were able to finally demonstrate effective H/D isotopologue separations by utilizing the complementary action of the OH-π and CH-π interactions.

Quantum de-trapping and transport of heavy defects in tungsten.

The diffusion of defects in crystalline materials1 controls macroscopic behaviour of a wide range of processes, including alloying, precipitation, phase transformation and creep2. In real materials, intrinsic defects are unavoidably bound to static trapping centres such as impurity atoms, meaning that their diffusion is dominated by de-trapping processes. It is generally believed that de-trapping occurs only by thermal activation. Here, we report the direct observation of the quantum de-trapping of defects below around one-third of the Debye temperature. We successfully monitored the de-trapping and migration of self-interstitial atom clusters, strongly trapped by impurity atoms in tungsten, by triggering de-trapping out of equilibrium at cryogenic temperatures, using high-energy electron irradiation and in situ transmission electron microscopy. The quantum-assisted de-trapping leads to low-temperature diffusion rates orders of magnitude higher than a naive classical estimate suggests. Our analysis shows that this phenomenon is generic to any crystalline material.

3D Vessel Image Reconstruction by MDCT for Surgical Indication and Timing of Strangulating Small Bowel Obstructions.

BACKGROUND: Patients with strangulating small bowel obstructions (SBOs) can rapidly deteriorate, in condition; therefore, immediate and appropriate diagnosis is required. However, some cases of SBO are difficult to diagnose using axial computed tomography (CT) images alone. The impact of 3D vessel imaging for the diagnosis, surgical indication, and timing of strangulating SBOs was investigated, prospectively. METHODS: Clinical data were collected for 111 strangulating SBOs and 48 simple SBOs from patients receiving surgical interventions from January 2009 to March 2018. The accuracy of preoperative diagnoses for the type of SBO was evaluated. Among 159 patients, 27 underwent contrast-enhanced CT imaging as well as prospectively reconstructed 3D vessel imaging of the superior mesenteric artery, vein, and branches. The concordance rate of operative findings and preoperative diagnoses of the type of SBO were compared between axial CT imaging alone and combination of axial and 3D vessel imaging. RESULTS: Overall concordance rate of diagnosis for the type of SBO by axial imaging was 93.1% and that of strangulating and simple SBOs was 92.8% and 93.8%, respectively. Combined axial and 3D vessel imaging resulted in 100% accuracy of preoperative diagnoses for both types of SBO. In addition, abnormalities could be classified from 3D vessel images as central twists or peripheral twists, and deteriorated vascular flow could also be detected. CONCLUSIONS: The combination of axial imaging and 3D vessel imaging can be used to accurately diagnose SBOs, and this imaging technique may be useful for determining the surgical indication and suitable timing of strangulating SBOs.

[A Case of Rectal Cancers with Pneumonia Combined with Interesting Body Pressure Distribution Before and after Treatment].

A 74-year-old man with aspiration pneumonia was admitted in the internal medicine department. CT revealed progressive carcinoma of RS-Ra, which became the focus of intervention after the pneumonia was treated. After having been allowed temporary discharge, the patient was rehospitalized for surgical operation and rehabilitation. During the perioperative period of cancer patients, we use body distribution data for teaching and environment setting, which is a useful tool in rehabilitation. Rectal cancer was observed after hospitalization for pneumonia, presenting an interesting case. Therefore, care needs to be taken as body pressure distribution may be modified by comorbidity.

Slow injector-to-column sample transport to maximize resolution in liquid chromatography: Theory versus practice.

A simple chromatographic model including extra-column sample bandspreading is built and validated experimentally. It is used to quantify the advantage of slowly transporting under isocratic elution of the injected sample band along the pre-column volume of the chromatographic system compared to the conventional injection method at constant flow rate. For fast analyses (<30 s), the model predicts a maximum relative gain of 20% for the isocratic peak capacity of weakly retained compounds (k < 1) at the price of a 60% increase in retention time (+20 s) for short (3.0 cm long) narrow-bore (2.1 mm i.d.) columns packed with sub-2 µm particles and for low-dispersion (∼2 µL2 extra-column volume variance) vHPLC systems. These predictions were confirmed experimentally using a 2.1 mm × 30 mm column packed with 1.9 µm XBridge-C18 for the rapid separation of small molecules in 25 s at 40 °C using a mixture of methanol and water (75/25, v/v) as the eluent. For longer analyses (>30 s), the model allows for the determination of the optimum and slow injection speed that maximizes the trade-off between the analysis time and the isocratic peak capacity. For 2.1 mm × 20 mm columns packed with 2 µm particles and a highest retention factor of 10, the optimum injection speed is about 10-15% of the operating column flow rate for a maximum relative gain of 5% in overall isocratic peak capacity at constant elution time. Overall, the proposed injection method is directly applicable for enhanced resolution of weakly retained polar compounds in RPLC without the need of changing separation conditions, which may strongly increase the retention times of the late apolar eluters.

Influence of gas environment and heating on atomic structures of platinum nanoparticle catalysts for proton-exchange membrane fuel cells.

Atomic-scale relaxations of platinum nanoparticles (Pt NPs) for fuel-cell catalysts are evaluated by spherical-aberration corrected environmental transmission electron microscopy (ETEM) under reference high-vacuum and N2 atmospheres, and then under reactive H2, CO and O2 atmospheres, combined with ex situ durability test using an electrochemical half-cell. In high-vacuum, increasing roughness due to continuous relaxation of surface-adsorbed Pt atoms is quantified in real-space. Under H2 and N2 atmospheres at a critical partial pressure of 1 × 10-2 Pa the stability of the surface facets is for the first time found to be improved. The adsorption behaviour of CO molecules is investigated using experimentally measured Pt-Pt bond lengths on the topmost surface layer of Pt NPs. The deactivation of Pt NPs in the anode environment of a proton-exchange-membrane fuel-cell is demonstrated at the atomic-scale in the ETEM, and the transformation of NPs into disordered nanoclusters is systematically quantified using the partial size distribution of Pt atomic clusters under controlled heating experiments at 423, 573 and 723 K.

LPA Induces Keratinocyte Differentiation and Promotes Skin Barrier Function through the LPAR1/LPAR5-RHO-ROCK-SRF Axis.

The skin barrier protects the body from water loss, allergens, and pathogens. Profilaggrin is produced by differentiated keratinocytes and is processed into filaggrin monomers. These monomers cross-link keratin filaments and are also decomposed to natural moisturizing factors in the stratum corneum for skin hydration and barrier function. Deficits in FLG expression impair skin barrier function and underlie skin diseases such as dry skin and atopic dermatitis. However, intrinsic factors that regulate FLG expression and their mechanisms of action remain unknown. Here, we show that lysophosphatidic acid induces FLG expression in human keratinocytes via the LPAR1 and LPAR5 receptors and the downstream RHO-ROCK-SRF pathway. Comprehensive gene profiling analysis further showed that lysophosphatidic acid not only induces FLG expression but also facilitates keratinocyte differentiation. Moreover, lysophosphatidic acid treatment significantly up-regulated FLG production in a three-dimensional culture model of human skin and promoted barrier function in mouse skin in vivo. Thus, our work shows a previously unsuspected role for lysophosphatidic acid and its downstream signaling in the maintenance of skin homeostasis, which may serve as a novel therapeutic target for skin barrier dysfunction.

Mechanistic study on the high-selectivity enantioseparation of amino acids using a chiral crown ether-bonded stationary phase and acidic, highly organic mobile phase by liquid chromatography/time-of-flight mass spectrometry.

The separation mechanism of amino acid enantiomers using a chiral crown ether-bonded stationary phase, CROWNPAK CR-I(+), and acetonitrile (ACN)-rich mobile phases (MPs) was studied. The retention factors of proteinogenic l-amino acids (except proline) formed U-shaped plots against the ACN content in the MP with a sharp increase at a high ACN content, while d-amino acids showed much smaller increases or monotonous decreases in retention within the same range. The use of an acidic, highly organic MP with trifluoroacetic acid (TFA) provided a high enantioselectivity with a short separation time from the contribution of the increased binding of the ammonium group of the analytes to the crown ether functionality of the stationary phase and electrostatic repulsion counteracting the hydrophilic partition mechanism. Optimizing the sample diluent and MP alleviated the peak distortion caused by a moving water band that accompanied the hydrophilic interaction liquid chromatography-like elution conditions. The liquid chromatography/time-of-flight mass spectrometry method with the optimized MP - ACN/ethanol/water/TFA = 80/15/5/0.5 (v/v/v/v) - enabled the determination of eighteen pairs of proteinogenic amino acid enantiomers within 10 min. The conditions also provided the following advantages: (i) fast and highly reproducible separations under isocratic conditions, (ii) high sensitivity and low backpressure using the MP with a high organic content, and (iii) highly reliable peak identification by combining two columns (CR-I(+) and CR-I(-)), reversing the elution orders of the enantiomers.

Twinned/untwinned catalytic gold nanoparticles identified by applying a convolutional neural network to their Hough transformed Z-contrast images.

In this article, we demonstrate that a convolutional neural network (CNN) can be effectively used to determine the presence of twins in the atomic resolution scanning transmission electron microscopy (STEM) images of catalytic Au nanoparticles. In particular, the CNN screening of Hough transformed images resulted in significantly higher accuracy rates as compared to those obtained by applying this technique to the raw STEM images. The proposed method can be utilized for evaluating the statistical twining fraction of Au nanoparticles that strongly affects their catalytic activity.

Reduction of the extra-column band dispersion by a slow transport of a sample band from the injector to the column in isocratic reversed-phase liquid chromatography.

Extra-column band dispersion during the transport of a sample band from the injector to the column can be reduced by a flow rate program starting with a low flow rate until the sample band has approached to, or just entered into the column, followed by an increased flow rate suitable for the solute separation in the column. Such a sample introduction method increased the plate counts of a 50 mm long column, 1.0 or 2.1 mmID, especially for early-eluting solutes by up to several times compared to a conventional elution method, when a 0.254 mmID, 15.2 cm connection tubing was used. Increase in plate counts of up to 50-70% was possible for solutes with retention factors smaller than 1.0 for the columns connected with a 0.13 mmID, 15 cm tube. The method also seems to reduce the contribution of the void space at the column inlet to the band dispersion. The elution method including a slow transport of the sample band in the pre-column space of 10 µL or less may require a little longer separation time than normal elution, but it was shown to be effective for increasing the observed efficiency of a small column for solutes with small retention factors.