Optimization of α-amido boronic acids via cryo-electron microscopy analysis: Discovery of a novel highly selective immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual inhibitor.

The immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual blockade has been reported to suppress B cell differentiation and activation, suggesting that the dual inhibition of LMP7/LMP2 is a promising approach for treating autoimmune diseases. In contrast, the inhibition of the constitutive proteasome subunit ß5c correlates with cytotoxicity against non-immune cells. Therefore, LMP7/LMP2 dual inhibitors with high selectivity over ß5c may be desirable for treating autoimmune diseases. In this study, we present the optimization and discovery of α-amido boronic acids using cryo-electron microscopy (cryo-EM). The exploitation of structural differences between the proteasome subunits led to the identification of a highly selective LMP7/LMP2 dual inhibitor 19. Molecular dynamics simulation based on cryo-EM structures of the proteasome subunits complexed with 19 explained the inhibitory activity profile. In mice immunized with 4-hydroxy-3-nitrophenylacetyl conjugated to ovalbumin, results indicate that 19 is orally bioavailable and shows promise as potential treatment for autoimmune diseases.

Patchwork metasurface quantum well photodetectors with broadened photoresponse.

Complex lightwave manipulation such as broadband absorption has been realized with metasurfaces based on laterally arranged metal-dielectric-metal cavities with different geometries. However, application of these metasurfaces for optoelectronic devices by incorporating functional dielectrics remains challenging. Here, we integrate a quantum well infrared photodetector (QWIP) with a metasurface made of a patchwork of square cavities with different dimensions arranged in a subwavelength unit cell. Our detector realizes wideband photoresponse approaching the entire responsivity spectrum of the QWIP-single-sized square cavities can utilize only 60% of the possible bandwidth-and external quantum efficiencies of up to 78% at 6.8 µm. Our highly flexible design scheme enables integration of photodetectors and metasurfaces with arbitrary arrangements of cavities selectively responding to incidence with a specific wavefront.

Development of a chromophore-solid phase peptide reaction assay (C-SPRA) for assessing skin sensitization in vitro.

We developed an in vitro chromophore-solid phase peptide reaction assay (C-SPRA) using microbead-immobilized peptides and chromophores. Peptide-resins (microbeads) reacted with 14 representative chemicals to demonstrate the test's capacity to predict skin sensitization. C-SPRA enables accurate and high-throughput assessments of various chemicals, including poorly water-soluble sensitizers that are regarded as weakly potent by other methods.

Mass Spectrometry-Based Solid Phase Peptide Reaction Assay for Detecting Allergenicity Using an Immobilized Peptide-Conjugating Photo-Cleavable Linker.

The biological process of skin sensitization depends on the ability of a sensitizer to modify endogenous proteins. A direct peptide reactivity assay (DPRA), based on the biological process of skin sensitization, was developed as an alternative to controversial animal experiments. Although DPRA has been endorsed by industries and is internationally accepted as promising, it has several drawbacks, such as incompatibility with hydrophobic chemicals, inability to perform detailed reaction analysis, and ability to evaluate only single components. Here, we demonstrated that sensitizers and peptide adducts can be easily identified using a mass spectrometry-based solid-phase peptide reaction assay (M-SPRA). We synthesized peptides with a photo-cleavable linker immobilized on resins. We showed the potential of M-SPRA in predicting skin sensitization by measuring the peptide adducts that were selectively eluted from the resin after cleaving the linker post-reaction. M-SPRA provides more detailed information regarding chemical reactivity and accurate assessment of test samples, including mixtures. M-SPRA may be helpful for understanding the binding mechanism of sensitizers (toxicology), which may assist in further refining reactivity assays and aiding in the interpretation of reactivity data.

Stable Immune Response Induced by Intradermal DNA Vaccination by a Novel Needleless Pyro-Drive Jet Injector.

DNA vaccination can be applied to the treatment of various infectious diseases and cancers; however, technical difficulties have hindered the development of an effective delivery method. The efficacy of a DNA vaccine depends on optimal antigen expression by the injected plasmid DNA. The pyro-drive jet injector (PJI) is a novel system that allows for adjustment of injection depth and may, thus, provide a targeted delivery approach for various therapeutic or preventative compounds. Herein, we investigated its potential for use in delivering DNA vaccines. This study evaluated the optimal ignition powder dosage, as well as its delivery effectiveness in both rat and mouse models, while comparing the results of the PJI with that of a needle syringe delivery system. We found that the PJI effectively delivered plasmid DNA to intradermal regions in both rats and mice. Further, it efficiently transfected plasmid DNA directly into the nuclei, resulting in higher protein expression than that achieved via needle syringe injection. Moreover, results from animal ovalbumin (OVA) antigen induction models revealed that animals receiving OVA expression plasmids (pOVA) via PJI exhibited dose-dependent (10 µg, 60 µg, and 120 µg) production of anti-OVA antibodies; while only low titers (< 1/100) of OVA antibodies were detected when 120 µg of pOVA was injected via needle syringe. Thus, PJI is an effective, novel method for delivery of plasmid DNA into epidermal and dermal cells suggesting its promise as a tool for DNA vaccination.

Discovery of evocalcet, a next-generation calcium-sensing receptor agonist for the treatment of hyperparathyroidism.

The calcium-sensing receptor (CaSR) plays an important role in sensing extracellular calcium ions and regulating parathyroid hormone secretion by parathyroid gland cells, and the receptor is a suitable target for the treatment of hyperparathyroidism. Cinacalcet hydrochloride is a representative CaSR agonist which widely used for the hyperparathyroidism. However, it has several issues to clinical use, such as nausea/vomiting and strong inhibition of CYP2D6. We tried to improve these issues of cinacalcet for a new pharmaceutical agent as a preferable CaSR agonist. Optimization from cinacalcet resulted in the identification of pyrrolidine compounds and successfully led to the discovery of evocalcet as an oral allosteric CaSR agonist. Evocalcet, which exhibited highly favorable profiles such as CaSR agonistic activity and good DMPK profiles, will provide a novel therapeutic option for secondary hyperparathyroidism.

Altered Expression of Retinol Metabolism-Related Genes in an ANIT-Induced Cholestasis Rat Model.

Cholestasis is defined as a reduction of bile secretion caused by a dysfunction of bile formation. Insufficient bile secretion into the intestine undermines the formation of micelles, which may result in the reduced absorption of lipids and fat-soluble vitamins. Here, we investigated the retinol homeostasis and the alterations of retinol metabolism-related genes, including ß-carotene 15,15' monooxygenase (BCMO), lecithin:retinol acyltransferase (LRAT), aldehyde dehydrogenase (ALDH), cytochrome P450 26A1 (CYP26A1), and retinoic acid receptors (RAR) ß, in a α-naphthyl isothiocyanate (ANIT)-induced cholestasis rat model. Moreover, we examined the expression of the farnesoid X receptor (FXR) target genes. Our results showed that plasma retinol levels were decreased in ANIT rats compared to control rats. On the contrary, hepatic retinol levels were not different between the two groups. The expression of FXR target genes in the liver and intestine of cholestasis model rats was repressed. The BCMO expression was decreased in the liver and increased in the intestine of ANIT rats compared to control rats. Finally, the hepatic expression of LRAT, RARß, and ALDH1A1 in cholestatic rats was decreased compared to the control rats, while the CYP26A1 expression of the liver was not altered. The increased expression of intestinal BCMO in cholestasis model rats might compensate for decreased circulatory retinol levels. The BCMO expression might be regulated in a tissue-specific manner to maintain the homeostasis of retinol.

Design of efficient molecular organic light-emitting diodes by a high-throughput virtual screening and experimental approach.

Virtual screening is becoming a ground-breaking tool for molecular discovery due to the exponential growth of available computer time and constant improvement of simulation and machine learning techniques. We report an integrated organic functional material design process that incorporates theoretical insight, quantum chemistry, cheminformatics, machine learning, industrial expertise, organic synthesis, molecular characterization, device fabrication and optoelectronic testing. After exploring a search space of 1.6 million molecules and screening over 400,000 of them using time-dependent density functional theory, we identified thousands of promising novel organic light-emitting diode molecules across the visible spectrum. Our team collaboratively selected the best candidates from this set. The experimentally determined external quantum efficiencies for these synthesized candidates were as large as 22%.

Interstitial Fluid Sphingosine-1-Phosphate in Murine Mammary Gland and Cancer and Human Breast Tissue and Cancer Determined by Novel Methods.

The tumor microenvironment is a determining factor for cancer biology and progression. Sphingosine-1-phosphate (S1P), produced by sphingosine kinases (SphKs), is a bioactive lipid mediator that regulates processes important for cancer progression. Despite its critical roles, the levels of S1P in interstitial fluid (IF), an important component of the tumor microenvironment, have never previously been measured due to a lack of efficient methods for collecting and quantifying IF. The purpose of this study is to clarify the levels of S1P in the IF from murine mammary glands and its tumors utilizing our novel methods. We developed an improved centrifugation method to collect IF. Sphingolipids in IF, blood, and tissue samples were measured by mass spectrometry. In mice with a deletion of SphK1, but not SphK2, levels of S1P in IF from the mammary glands were greatly attenuated. Levels of S1P in IF from mammary tumors were reduced when tumor growth was suppressed by oral administration of FTY720/fingolimod. Importantly, sphingosine, dihydro-sphingosine, and S1P levels, but not dihydro-S1P, were significantly higher in human breast tumor tissue IF than in the normal breast tissue IF. To our knowledge, this is the first reported S1P IF measurement in murine normal mammary glands and mammary tumors, as well as in human patients with breast cancer. S1P tumor IF measurement illuminates new aspects of the role of S1P in the tumor microenvironment.

Highly efficient blue electroluminescence based on thermally activated delayed fluorescence.

Organic compounds that exhibit highly efficient, stable blue emission are required to realize inexpensive organic light-emitting diodes for future displays and lighting applications. Here, we define the design rules for increasing the electroluminescence efficiency of blue-emitting organic molecules that exhibit thermally activated delayed fluorescence. We show that a large delocalization of the highest occupied molecular orbital and lowest unoccupied molecular orbital in these charge-transfer compounds enhances the rate of radiative decay considerably by inducing a large oscillator strength even when there is a small overlap between the two wavefunctions. A compound based on our design principles exhibited a high rate of fluorescence decay and efficient up-conversion of triplet excitons into singlet excited states, leading to both photoluminescence and internal electroluminescence quantum yields of nearly 100%.

Increase of ß2-integrin on adhesion of THP-1 cells to collagen vitrigel membrane.

When human monocyte-derived leukemia (THP-1) cells, which are floating cells, are stimulated with lipid peroxides, or Streptococcus suis, these cells adhere to a plastic plate or endothelial cells. However, it is unclear whether or not non-stimulated THP-1 cells adhere to collagen vitrigel membrane (CVM). In this study, firstly, we investigated the rate of adhesion of THP-1 cells to CVM. When THP-1 cells were not stimulated, the rate of adhesion to CVM was high. Then, to identify adhesion molecules involved in adhesion of THP-1 cells to CVM, expressions of various cell adhesion molecules on the surface of THP-1 cells adhering to CVM were measured. ß-actin, ß-catenin, and ß1-integrin expressions did not change in non-stimulated THP-1 cells cultured on CVM compared with those in cells cultured in a flask, but ß2-integrin expression markedly increased.

Dehydroepiandrosterone alters vitamin E status and prevents lipid peroxidation in vitamin E-deficient rats.

In humans, dehydroepiandrosterone and its sulfate ester metabolite DHEA-S are secreted predominantly from the adrenal cortex, and dehydroepiandrosterone is converted to steroid hormones, including androgens and estrogens, and neurosteroid. Dehydroepiandrosterone exerts protective effects against several pathological conditions. Although there are reports on the association between dehydroepiandrosterone and vitamins, the exact relationship between dehydroepiandrosterone and vitamin E remains to be determined. Therefore, we attempted to elucidate the effect of dehydroepiandrosterone on vitamin E status and the expression of various vitamin E-related proteins, including binding proteins, transporters, and cytochrome P450, in vitamin E-deficient rats. Plasma α-tocopherol levels in vitamin E-deficient rats increased in response to dehydroepiandrosterone administration. The expression of hepatic α-tocopherol transfer protein was repressed in vitamin E-deficient rats compared to that in control rats; however, dehydroepiandrosterone administration significantly upregulated this expression. Hepatic expression of CYP4F2, an α-tocopherol metabolizing enzyme, in vitamin E-deficient rats was decreased by dehydroepiandrosterone administration, whereas hepatic expression of ATP-binding cassette transporter A1, an α-tocopherol transporter, was not altered following dehydroepiandrosterone administration. Dehydroepiandrosterone repressed lipid peroxidation in the liver of vitamin E-deficient rats. Therefore, adequate dehydroepiandrosterone supplementation may improve lipid peroxidation under several pathological conditions, and dehydroepiandrosterone may modulate α-tocopherol levels through altered expression of vitamin E-related proteins.

Altered retinol status and expression of retinol-related proteins in streptozotocin-induced type 1 diabetic model rats.

Diabetes is a metabolic disorder characterized by chronic hyperglycemia. Advanced diabetes is associated with severe complications and impaired nutritional status. Here, we assessed the expression of retinol-associated proteins, including ß-carotene 15,15'-monooxygenase (BCMO), lecithin:retinol acyltransferase (LRAT), aldehyde dehydrogenase (ALDH), and cytochrome P450 26A1 (CYP26A1), and measured retinol levels in the plasma and liver of streptozotocin (STZ)-induced type 1 diabetic model rats. Compared to the levels in the control rats, retinol levels in the plasma and liver of STZ rats were decreased and increased, respectively. Hepatic expression of the LRAT gene in STZ rats was lower than that in the controls. In the liver of STZ rats, the expression of ALDH1A1, a retinal metabolizing enzyme was higher, whereas ALDH1A2 expression was lower than in the controls. Hepatic CYP26A1 expression in STZ rats was significantly higher than in the control rats. BCMO expression levels in the liver and intestine of STZ rats were much lower than those of the controls. Altered BCMO expression might affect retinol status. It is considered that the metabolic availability of retinol was lessened despite the accelerated catabolism of retinol; therefore, retinol mobilization may be unbalanced in the liver of rats in the type 1 diabetic state.

Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

Crossover from capacitive to inductive electromagnetic responses in near self-complementary metallic checkerboard patterns.

Transmission spectra of near-self-complementary metallic checkerboard patterns (MCPs) exhibit a drastic change when the metal squares are brought into contact with each other from a noncontact state. Transmission spectra of near-self-complementary samples, which are fabricated by printing technology, show rather gradual systematic change with changing the nominal metal square size while keeping the period because of randomness naturally introduced by the limited accuracy of the printer. The spectra have transmission-invariant frequencies, which means that the spectra are a superposition of two types of spectra, the ratio of which depends on the nominal square size. The correlation seen in the real and imaginary parts of the complex amplitude spectra can be interpreted based on the Kramers-Kronig relation. As an application of the sensitiveness of the transmission spectrum of the MCPs to connectivity of the metal squares, the revealing of an optically hidden pattern by a terahertz beam is demonstrated.

The α-tocopherol status and expression of α-tocopherol-related proteins in methionine-choline deficient rats treated with vitamin E.

Non-alcoholic fatty liver disease is the most common liver disorder in developed countries, and its incidence is increasing in all population groups. As an antioxidant, vitamin E is effective in the treatment of non-alcoholic fatty liver disease, although the mechanism is still unclear. Methionine-choline deficient Wistar rats (n = 5) used as an experimental model of non-alcoholic fatty liver disease were fed a vitamin E-enriched diet (500 mg/kg) for 4 weeks. The effects were assessed by measuring lipid peroxidation, α-tocopherol levels, and the expression of α-tocopherol-related proteins in the liver. In vitamin E-treated methionine-choline deficient rats, lipid peroxidation was reduced, but liver histopathological changes were not improved. Hepatic α-tocopherol levels in these rats were significantly elevated compared to normal rats treated with vitamin E. Expression of liver α-tocopherol transfer protein in vitamin E-treated methionine-choline deficient rats was significantly repressed compared to methionine-choline deficient rats. The expression of liver cytochrome P450 4F2 and ATP-binding cassette transporter protein 1, involved in metabolism and transport of α-tocopherol, respectively, was significantly repressed in vitamin E-treated methionine-choline deficient rats. In methionine-choline deficient rats, vitamin E treatment altered the hepatic α-tocopherol-related protein expression, which may affect α-tocopherol status in the liver, leading to reduced lipid peroxidation.

Assessing the Metabolic and Physical Effects of Combined DPP4 and SGLT2 Inhibitor Therapy in Patients with Type-2 Diabetes Mellitus: An Observational Prospective Pilot Study.

Introduction: This study aimed to assess the efficacy of combined administration of dipeptidyl peptide-4 (DPP4) and sodium-glucose cotransporter-2 (SGLT2) inhibitors on metabolic disorders and their preferable and complementary effects. Methods: The effectiveness of a 24-week intervention on metabolic parameters (including glucose profile), physical functions (grip strength and calf circumference), and health-related quality of life (HR-QOL) was analyzed using the International Physical Activity Questionnaire and Geriatric Depression Scale 5. A total of 39 patients with type-2 diabetes mellitus (T2DM) treated with the combination of DPP4 and SGLT2 inhibitors were included in this multicenter pilot study. Results: Combination therapy significantly reduced the HbA1c level (median [interquartile range]) after 24 weeks (pretreatment: 7.7% [7.3-8.2] vs. posttreatment: 7.1% [6.6-7.9], P < 0.001). The grip strength significantly increased after 24 weeks (1.7 ± 2.7 kg, P < 0.001), while the mean calf circumference and body mass index significantly decreased. In particular, administration of the SGLT2 inhibitor significantly increased total physical activity in participants aged ≥65 years (P = 0.003), while psychological QOL did not significantly improve. Conclusions: Combination therapy with DPP4 and SGLT2 inhibitors decreased HbA1c levels and improved physical function in patients with T2DM. This study confirmed the effectiveness of combination therapy for metabolic disorders and suggested its beneficial and complementary effects. Therefore, advances in treatment plans to achieve further improvements in glucose profiles using DPP4 and SGLT2 inhibitors are recommended to enhance the QOL of patients with T2DM. Clinical trial number: University Hospital Medical Information Network Center: UMIN000045375.

Z-Selective ethenolysis with a ruthenium metathesis catalyst: experiment and theory.

The Z-selective ethenolysis activity of chelated ruthenium metathesis catalysts was investigated with experiment and theory. A five-membered chelated catalyst that was successfully employed in Z-selective cross metathesis reactions has now been found to be highly active for Z-selective ethenolysis at low ethylene pressures, while tolerating a wide variety of functional groups. This phenomenon also affects its activity in cross metathesis reactions and prohibits crossover reactions of internal olefins via trisubstituted ruthenacyclobutane intermediates. In contrast, a related catalyst containing a six-membered chelated architecture is not active for ethenolysis and seems to react through different pathways more reminiscent of previous generations of ruthenium catalysts. Computational investigations of the effects of substitution on relevant transition states and ruthenacyclobutane intermediates revealed that the differences of activities are attributed to the steric repulsions of the anionic ligand with the chelating groups.

Diffraction-unlimited optical imaging of unstained living cells in liquid by electron beam scanning of luminescent environmental cells.

An environmental cell with a 50-nm-thick cathodoluminescent window was attached to a scanning electron microscope, and diffraction-unlimited near-field optical imaging of unstained living human lung epithelial cells in liquid was demonstrated. Electrons with energies as low as 0.8 - 1.2 kV are sufficiently blocked by the window without damaging the specimens, and form a sub-wavelength-sized illumination light source. A super-resolved optical image of the specimen adhered to the opposite window surface was acquired by a photomultiplier tube placed below. The cells after the observation were proved to stay alive. The image was formed by enhanced dipole radiation or energy transfer, and features as small as 62 nm were resolved.

Waveguide lasing from V-shaped ZnO microstructure.

A V-shaped optical resonance cavity was obtained from ZnO microstructures grown by thermal chemical vapor deposition. Strong laser emissions were observed in three regions--the tip of the two branches and the bottom facet of the V-shaped microstructures--under UV laser excitation at room temperature. In the region where the diameter of the branches was smaller than the wavelength of the laser light, light could not propagate into the tip due to the cutoff phenomenon, resulting in partial reflection. Quasi-Fabry-Perot resonance in the branch and light reflection at the bottom facet characterized the V-shaped microcavity.