Structural analyses of the GI.4 norovirus by cryo-electron microscopy and X-ray crystallography revealing binding sites for human monoclonal antibodies.

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.

Elucidating the structural changes of cellulose molecules and dynamics of Na ions during the crystal transition from cellulose I to II in low temperature and low concentration NaOH solution.

Low-concentration alkali treatments at low temperatures facilitate the crystal transition of cellulose I to II. However, the transition mechanism remains unclear. Hence, in this study, we traced the transition using in situ solid-state 13C CP/MAS NMR, WAXS, and 23Na NMR relaxation measurements. In situ solid-state 13C CP/MAS NMR and WAXS measurements revealed that soaking cellulose in NaOH at low temperatures disrupts the intramolecular hydrogen bonds and lowers the crystallinity of cellulose. The dynamics of Na ions (NaOH) play a crucial role in causing these phenomena. 23Na NMR relaxation measurements indicated that the Na-ion correlation time becomes longer during the crystal transition. This transition requires the penetration of Na ions (NaOH) into the cellulose crystal and a reduction in Na-ion mobility, which occurs at low temperatures or high NaOH concentrations. The interactions between cellulose and NaOH disrupt intramolecular hydrogen bonds, inducing a conformational change in the cellulose molecules into a more stable arrangement. This weakens the hydrophobic interactions of cellulose, and facilitates the penetration of NaOH and water into the crystal, leading to the formation of alkali cellulose. Our findings suggest that a strategy to control NaOH dynamics could lead to the discovery of a novel preparation method for cellulose II.

Thermoresponsive Property of Poly(N,N-bis(2-methoxyethyl)acrylamide) and Its Copolymers with Water-Soluble Poly(N,N-disubstituted acrylamide) Prepared Using Hydrosilylation-Promoted Group Transfer Polymerization.

The group-transfer polymerization (GTP) of N,N-bis(2-methoxyethyl)acrylamide (MOEAm) initiated by Me2EtSiH in the hydrosilylation-promoted method and by silylketene acetal (SKA) in the conventional method proceeded in a controlled/living manner to provide poly(N,N-bis(2-methoxyethyl)acrylamide) (PMOEAm) and PMOEAm with the SKA residue at the α-chain end (MCIP-PMOEAm), respectively. PMOEAm-b-poly(N,N-dimethylacrylamide) (PDMAm) and PMOEAm-s-PDMAm and PMOEAm-b-poly(N,N-bis(2-ethoxyethyl)acrylamide) (PEOEAm) and PMOEAm-s-PEOEAm were synthesized by the block and random group-transfer copolymerization of MOEAm and N,N-dimethylacrylamide or N,N-bis(2-ethoxyethyl)acrylamide. The homo- and copolymer structures affected the thermoresponsive properties; the cloud point temperature (Tcp) increasing by decreasing the degree of polymerization (x). The chain-end group in PMOEAm affected the Tcp with PMOEAmx > MCIP-PMOEAmx. The Tcp of statistical copolymers was higher than that of block copolymers, with PMOEAmx-s-PDMAmy > PMOEAmx-b-PDMAmy and PMOEAmx-s-PEOEAmy > PMOEAmx-b-PEOEAmy.

Epigenetic mechanisms to propagate histone acetylation by p300/CBP.

Histone acetylation is important for the activation of gene transcription but little is known about its direct read/write mechanisms. Here, we report cryogenic electron microscopy structures in which a p300/CREB-binding protein (CBP) multidomain monomer recognizes histone H4 N-terminal tail (NT) acetylation (ac) in a nucleosome and acetylates non-H4 histone NTs within the same nucleosome. p300/CBP not only recognized H4NTac via the bromodomain pocket responsible for reading, but also interacted with the DNA minor grooves via the outside of that pocket. This directed the catalytic center of p300/CBP to one of the non-H4 histone NTs. The primary target that p300 writes by reading H4NTac was H2BNT, and H2BNTac promoted H2A-H2B dissociation from the nucleosome. We propose a model in which p300/CBP replicates histone N-terminal tail acetylation within the H3-H4 tetramer to inherit epigenetic storage, and transcribes it from the H3-H4 tetramer to the H2B-H2A dimers to activate context-dependent gene transcription through local nucleosome destabilization.

Discovery of Novel Substrate-Competitive Lysine Methyltransferase G9a Inhibitors as Anticancer Agents.

Identification of structurally novel inhibitors of lysine methyltransferase G9a has been a subject of intense research in cancer epigenetics. Starting with the high-throughput screening (HTS) hit rac-10a obtained from the chemical library of the University of Tokyo Drug Discovery Initiative, the structure-activity relationship of the unique substrate-competitive inhibitors was established with the help of X-ray crystallography and fragment molecular orbital (FMO) calculations for the ligand-protein interaction. Further optimization of the in vitro characteristics and drug metabolism and pharmacokinetics (DMPK) properties led to the identification of 26j (RK-701), which is a structurally distinct potent inhibitor of G9a/GLP (IC50 = 27/53 nM). Compound 26j exhibited remarkable selectivity against other related methyltransferases, dose-dependent attenuation of cellular H3K9me2 levels, and tumor growth inhibition in MOLT-4 cells in vitro. Moreover, compound 26j showed inhibition of tumor initiation and growth in a carcinogen-induced hepatocellular carcinoma (HCC) in vivo mouse model without overt acute toxicity.

Curcumin intake during lactation suppresses oxidative stress through upregulation of nuclear factor erythroid 2-related factor 2 in the kidneys of fructose-loaded female rat offspring exposed to maternal protein restriction.

BACKGROUND: A high-fructose diet causes the progression of chronic kidney disease. Maternal malnutrition during pregnancy and lactation increases oxidative stress, leading to chronic renal diseases later in life. We investigated whether curcumin intake during lactation could suppress oxidative stress and regulate NF-E2-related factor 2 (Nrf2) expression in the kidneys of fructose-loaded female rat offspring exposed to maternal protein restriction. METHODS: Pregnant Wistar rats received diets containing 20% (NP) or 8% (LP) casein and 0 or 2.5 g "highly absorptive curcumin" /kg diet containing-LP diets (LP/LP or LP/Cur) during lactation. At weaning, female offspring received either distilled water (W) or 10% fructose solution (Fr) and were divided into four groups: NP/NP/W, LP/LP/W, LP/LP/Fr, and LP/Cur/Fr. At week 13, glucose (Glc), triacylglycerol (Tg), and malondialdehyde (MDA) levels in the plasma, macrophages number, fibrotic area, glutathione (GSH) levels, glutathione peroxidase (GPx) activity, protein expression levels of Nrf2, heme oxygenase-1 (HO-1), and superoxide dismutase 1 (SOD1) in the kidneys were examined. RESULTS: The plasma levels of Glc, TG, and MDA, the number of macrophages, and the percentage of fibrotic area in the kidneys of the LP/Cur/Fr group were significantly lower than those of the LP/LP/Fr group. The expression of Nrf2 and its downstream molecules HO-1 and SOD1, GSH levels, and GPx activity in the kidneys of the LP/Cur/Fr group were significantly higher than those of the LP/LP/Fr group. CONCLUSIONS: Maternal curcumin intake during lactation may suppress oxidative stress by upregulating Nrf2 expression in the kidneys of fructose-loaded female offspring exposed to maternal protein restriction.

Molecular Weight-Dependent Oxidation and Optoelectronic Properties of Defect-Free Macrocyclic Poly(3-hexylthiophene).

The redox behaviors of macrocyclic molecules with an entirely π-conjugated system are of interest due to their unique optical, electronic, and magnetic properties. In this study, defect-free cyclic P3HT with a degree of polymerization (DPn) from 14 to 43 was synthesized based on our previously established method, and its unique redox behaviors arising from the cyclic topology were investigated. Cyclic voltammetry (CV) showed that the HOMO level of cyclic P3HT decreases from -4.86 eV (14 mer) to -4.89 eV (43 mer), in contrast to the linear counterparts increasing from -4.94 eV (14 mer) to -4.91 eV (43 mer). During the CV measurement, linear P3HT suffered from electro-oxidation at the chain ends, while cyclic P3HT was stable. ESR and UV-Vis-NIR spectroscopy suggested that cyclic P3HT has stronger dicationic properties due to the interactions between the polarons. On the other hand, linear P3HT showed characteristics of polaron pairs with multiple isolated polarons. Moreover, the dicationic properties of cyclic P3HT were more pronounced for the smaller macrocycles.

A specific G9a inhibitor unveils BGLT3 lncRNA as a universal mediator of chemically induced fetal globin gene expression.

Sickle cell disease (SCD) is a heritable disorder caused by ß-globin gene mutations. Induction of fetal γ-globin is an established therapeutic strategy. Recently, epigenetic modulators, including G9a inhibitors, have been proposed as therapeutic agents. However, the molecular mechanisms whereby these small molecules reactivate γ-globin remain unclear. Here we report the development of a highly selective and non-genotoxic G9a inhibitor, RK-701. RK-701 treatment induces fetal globin expression both in human erythroid cells and in mice. Using RK-701, we find that BGLT3 long non-coding RNA plays an essential role in γ-globin induction. RK-701 selectively upregulates BGLT3 by inhibiting the recruitment of two major γ-globin repressors in complex with G9a onto the BGLT3 gene locus through CHD4, a component of the NuRD complex. Remarkably, BGLT3 is indispensable for γ-globin induction by not only RK-701 but also hydroxyurea and other inducers. The universal role of BGLT3 in γ-globin induction suggests its importance in SCD treatment.

Mechanical Thrombectomy for Acute Common Carotid Artery Occlusion.

Mechanical thrombectomy (MT) is the standard treatment for acute large occlusion of the cerebral artery. Evidence for the success of this procedure was based on the treatment of patients with internal carotid artery and middle cerebral artery thrombi. There are a few reports on thrombi extending to the common carotid artery (CCA). We document our endovascular procedure and the clinical outcome in seven consecutive patients who underwent MT for CCA thrombi between September 2016 and April 2021. Their mean National Institutes of Health Stroke Scale score was 20.0 (range, 9-30), and the mean diffusion-weighted imaging Alberta Stroke Program Early Computed Tomography Score on magnetic resonance images was 8.7 (range, 7-10). In six patients, MT of the CCA occlusion was successful, and the mean puncture-to-reperfusion time was 84 minutes (range, 39-211 minutes). In five patients, successful reperfusion was obtained. The mean total pass number was 4.1 (range, 2-7). Due to large thrombi, we performed balloon guide catheter (BGC) occlusion in three patients. Sheath occlusion occurred in two, and thrombus migration into the femoral artery around the sheath was observed in two patients. The mean modified Rankin Scale score 3 months post-stroke was 3.6 (range, 2-5). When the removal of a large CCA thrombus is attempted in a single step, catheter and sheath occlusion may occur, and this increases the risk for critical systemic artery occlusion. Therefore, we suggest that MT be combined with the BGC technique and propose the use of a large aspiration catheter to decrease the volume of the thrombus.

Photon extraction enhancement of praseodymium ions in gallium nitride nanopillars.

Lanthanoid-doped Gallium Nitride (GaN) integrated into nanophotonic technologies is a promising candidate for room-temperature quantum photon sources for quantum technology applications. We manufactured praseodymium (Pr)-doped GaN nanopillars of varying size, and showed significantly enhanced room-temperature photon extraction efficiency compared to unstructured Pr-doped GaN. Implanted Pr ions in GaN show two main emission peaks at 650.3 nm and 651.8 nm which are attributed to 3P0-3F2 transition in the 4f-shell. The maximum observed enhancement ratio was 23.5 for 200 nm diameter circular pillars, which can be divided into the emitted photon extraction enhancement by a factor of 4.5 and the photon collection enhancement by a factor of 5.2. The enhancement mechanism is explained by the eigenmode resonance inside the nanopillar. Our study provides a pathway for Lanthanoid-doped GaN nano/micro-scale photon emitters and quantum technology applications.

A case of pancreatic duct stones treated with electrohydraulic lithotripsy through a duodenal ulcer.

Video 1Crush the large-sized stones directly using an electrohydraulic lithotripsy device.

Size Control and Enhanced Stability of Silver Nanoparticles by Cyclic Poly(ethylene glycol).

Silver nanoparticles (AgNPs) are used in a wide range of applications, and the size control and stability of the nanoparticles are crucial aspects in their applications. In the present study, cyclized poly(ethylene glycol) (c-PEG) with various molecular weights, along with linear PEG with hydroxy chain ends (HO-PEG-OH) and methoxy chain ends (MeO-PEG-OMe) were applied for the Tollens' synthesis of AgNPs. The particle size was significantly affected by the topology and end groups of PEG. For example, the size determined by TEM was 40 ± 7 nm for HO-PEG5k-OH, 21 ± 4 nm for c-PEG5k, and 48 ± 9 nm for MeO-PEG5k-OMe when the molar ratio of PEG to AgNO3 (ω) was 44. The stability of AgNPs was also drastically improved by cyclization; the relative UV-Vis absorption intensity (A/A0 × 100%) at λmax to determine the proportion of persisting AgNPs in an aqueous NaCl solution (37.5 mM) was 58% for HO-PEG5k-OH, 80% for c-PEG5k, and 40% for MeO-PEG5k-OMe, despite the fact that AgNPs with c-PEG5k were much smaller than those with HO-PEG5k-OH and MeO-PEG5k-OMe.

Chemoproteomic Identification of Blue-Light-Damaged Proteins.

Visible light, particularly in the blue region of the spectrum, can cause cell dysfunction through the generation of singlet oxygen, contributing to cellular aging and age-related pathologies. Although photooxidation of nucleic acids, lipids, and amino acids has been extensively studied, the magnitude and span of blue-light-induced protein damages within proteome remain largely unknown. Herein we present a chemoproteomic approach to mapping blue-light-damaged proteins in live mammalian cells by exploiting a nucleophilic alkyne chemical probe. A gene ontology enrichment analysis revealed that cell surface proteins are more readily oxidized than other susceptible sets of proteins, including mitochondrial proteins. In particular, the integrin family of cell surface receptors (ITGs) was highly ranked in the mammalian cells tested, including human corneal endothelial cells. The blue-light-oxidized ITGB1 protein was functionally inactive in promoting cell adhesion and proliferation, suggesting that the photodamage of integrins contributes to the blue-light-induced cell dysfunction. Further application of our method to various cells and tissues should lead to a comprehensive analysis of light-sensitive proteins.

Development and multi-institutional validation of an artificial intelligence-based diagnostic system for gastric biopsy.

To overcome the increasing burden on pathologists in diagnosing gastric biopsies, we developed an artificial intelligence-based system for the pathological diagnosis of gastric biopsies (AI-G), which is expected to work well in daily clinical practice in multiple institutes. The multistage semantic segmentation for pathology (MSP) method utilizes the distribution of feature values extracted from patches of whole-slide images (WSI) like pathologists' "low-power view" information of microscopy. The training dataset included WSIs of 4511 gastric biopsy tissues from 984 patients. In tissue-level validation, MSP AI-G showed better accuracy (91.0%) than that of conventional patch-based AI-G (PB AI-G) (89.8%). Importantly, MSP AI-G unanimously achieved higher accuracy rates (0.946 ± 0.023) than PB AI-G (0.861 ± 0.078) in tissue-level analysis, when applied to the cohorts of 10 different institutes (3450 samples of 1772 patients in all institutes, 198-555 samples of 143-206 patients in each institute). MSP AI-G had high diagnostic accuracy and robustness in multi-institutions. When pathologists selectively review specimens in which pathologist's diagnosis and AI prediction are discordant, the requirement of a secondary review process is significantly less compared with reviewing all specimens by another pathologist.

PEGylation of silver nanoparticles by physisorption of cyclic poly(ethylene glycol) for enhanced dispersion stability, antimicrobial activity, and cytotoxicity.

Silver nanoparticles (AgNPs) are practically valuable in biological applications. However, no steady PEGylation has been established, which is essential for internal use in humans or animals. In this study, cyclic PEG (c-PEG) without any chemical inhomogeneity is physisorbed onto AgNPs to successfully PEGylate and drastically enhance the dispersion stability against physiological conditions, white light, and high temperature. In contrast, linear HO-PEG-OH and MeO-PEG-OMe do not confer stability to AgNPs, and HS-PEG-OMe, which is often used for gold nanoparticles, sulfidates the surface to considerably degrade the properties. TEM shows an essentially intact nanostructure of c-PEG-physisorbed AgNPs even after heating at 95 °C, while complete disturbance is observed for other AgNPs. Molecular weight- and concentration-dependent stabilization by c-PEG is investigated, and DLS and ζ-potential measurements prove the formation of a c-PEG layer on the surface of AgNPs. Furthermore, c-PEG-physisorbed AgNPs exhibit persistent antimicrobial activity and cytotoxicity.

Structure-Activity Relationship and In Silico Evaluation of cis- and trans-PCPA-Derived Inhibitors of LSD1 and LSD2.

trans-2-Phenylcycloproylamine (trans-PCPA) has been used as the scaffold to develop covalent-binding inhibitors against lysine-specific demethylase 1 (LSD1/KDM1A), a therapeutic target for several cancers. However, the effects of different structural moieties on the inhibitory activity, selectivity, and reactivity of these derivatives, including the cis isomers, against LSD1 and its paralogue LSD2/KDM1B are not fully understood. Here we synthesized 65 cis- and trans-PCPA derivatives and evaluated their inhibitory activity against LSD1 and LSD2. One of the derivatives, 7c (cis-4-Br-2,5-F2-PCPA; S1024), inhibited LSD1 and LSD2 with K i values of 0.094 µM and 8.4 µM, respectively, and increased the level of dimethylated histone H3 at K4 in CCRF-CEM cells. A machine learning-based regression model (Q 2 = 0.61) to predict LSD1-inhibitory activity was also constructed and showed a good prediction accuracy (R 2 = 0.81) for 12 test-set compounds, including 7c. The present methodology would be useful when designing covalent-binding inhibitors for other enzymes.

EEG Correlation Coefficient Change with Motor Task Activation Can Be a Predictor of Functional Recovery after Hemiparetic Stroke.

BACKGROUND: Recently, it was reported that the extent of cortico-cortical functional connections can be estimated by the correlation coefficient based on electroencephalography (EEG) monitoring. We aimed to investigate whether the EEG correlation coefficient change with motor task activation can predict the functional outcomes of hemiparetic stroke patients. METHODS: Sixteen post-stroke hemiparetic patients admitted to our rehabilitation ward were studied. On admission, EEG recording to calculate the correlation coefficient was performed at rest and during motor task activation. For the analysis of the EEG data, the program software FOCUS (NIHON KOHDEN, Japan) was used. The motor function of paretic limbs was evaluated with the Fugl-Meyer Assessment (FMA) on admission and 4 weeks after admission. RESULTS: Significant increases in the correlation coefficient with motor task activation were noted in C3-F3 or C4-F4, C3-F7 or C4-F8, and F3-F7 or F4-F8 of the lesional hemisphere. Among them, the rate of the correlation coefficient change in F3-F7 or F4-F8 in the lesional hemisphere was significantly correlated with the rate of the upper-limb FMA score change. CONCLUSION: The extent of the EEG correlation coefficient change with motor task activation in F3-F7 or F4-F8 of the lesional hemisphere may help predict the motor functional outcomes of hemiparetic upper limbs after stroke.

Changes of cortico-cortical neural connections associated with motor functional recovery after stroke.

OBJECTIVES: During functional recovery after stroke, some neural connections in the brain are augmented and new neural networks are constructed to compensate for impaired neurological functions. Recently, it was reported that the extent of cortico-cortical neural connections can be estimated by correlation analysis based on electroencephalography (EEG). The purpose of this study was to investigate changes of correlation coefficients in the cerebral cortex with motor functional recovery after stroke. MATERIALS AND METHODS: Twenty-two post-stroke hemiparetic patients admitted to our rehabilitation ward (mean age at admission: 71.4 ± 12.9 years old), were studied. For the evaluation of hemiparesis, Fugl-Meyer Assessment (FMA) was applied. All subjects underwent EEG with electrodes placed according to the international 10-20 system for correlation analysis, on admission to our ward and 4 weeks after admission. EEG data were analyzed with the program software FOCUS (NIHON KOHDEN, Japan), and squared correlation coefficients in some cortico-cortical areas of the cerebral cortex were calculated. RESULTS: The correlation coefficients in some cortico-cortical areas of the lesional hemisphere, such as C3-F3 or C4-F4, C3-F7 or C4-F8, and F3-F7 or F4-F8, significantly increased with rehabilitation training. The change of the correlation coefficient in F3-F7 or F4-F8 and F7-T3 or F8-T4 in the lesional hemisphere was significantly correlated with the change of the upper-limb FMA. CONCLUSIONS: The augmentation of cortico-cortical connections, represented by an increase of the correlation coefficient in the lesional hemisphere, may contribute to motor functional recovery, especially in hemiparetic upper limbs, after stroke.

Small molecule-based detection of non-canonical RNA G-quadruplex structures that modulate protein translation.

Tandem repeats of guanine-rich sequences in RNA often form thermodynamically stable four-stranded RNA structures. Such RNA G-quadruplexes have long been considered to be linked to essential biological processes, yet their physiological significance in cells remains unclear. Here, we report a approach that permits the detection of RNA G-quadruplex structures that modulate protein translation in mammalian cells. The approach combines antibody arrays and RGB-1, a small molecule that selectively stabilizes RNA G-quadruplex structures. Analysis of the protein and mRNA products of 84 cancer-related human genes identified Nectin-4 and CapG as G-quadruplex-controlled genes whose mRNAs harbor non-canonical G-quadruplex structures on their 5'UTR region. Further investigations revealed that the RNA G-quadruplex of CapG exhibits a structural polymorphism, suggesting a possible mechanism that ensures the translation repression in a KCl concentration range of 25-100 mM. The approach described in the present study sets the stage for further discoveries of RNA G-quadruplexes.