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Aims To examine the effects of a 24-month treatment with ipragliflozin on carotid intima-media thickness (IMT) in type 2 diabetes patients. Methods and results In this multicenter, prospective, randomized, open-label, and blinded-endpoint investigator-initiated clinical trial, adults with type 2 diabetes and haemoglobin A1C (HbA1c) of 6.0–10.0% (42–86 mmol/mol) were randomized equally to ipragliflozin (50 mg daily) and non-sodium-glucose cotransporter-2 (SGLT2) inhibitor use of standard-care (control group) for type 2 diabetes and were followed-up to 24 months. The primary endpoint was the change in mean common carotid artery IMT (CCA-IMT) from baseline to 24 months. A total of 482 patients were equally allocated to the ipragliflozin (N = 241) and control (N = 241) groups, and 464 patients (median age 68 years, female 31.7%, median type 2 diabetes duration 8 years, median HbA1c 7.3%) were included in the analyses. For the primary endpoint, the changes in the mean CCA-IMT from baseline to 24 months were 0.0013 [95% confidence interval (CI), −0.0155–0.0182] mm and 0.0015 (95% CI, −0.0155–0.0184) mm in the ipragliflozin and control groups, respectively, with an estimated group difference (ipragliflozin-control) of −0.0001 mm (95% CI, −0.0191–0.0189; P = 0.989). A group difference in HbA1c change at 24 months was also non-significant between the treatment groups [−0.1% (95% CI, −0.2–0.1); P = 0.359]. Conclusion Twenty-four months of ipragliflozin treatment did not affect carotid IMT status in patients with type 2 diabetes recruited in the PROTECT study, relative to the non-SGLT2 inhibitor-use standard care for type 2 diabetes.

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.

Melanin is an organic material biosynthesized from tyrosine in pigment-producing cells. The present study reports a simple method to generate tailored functional materials in mammalian cells by chemically fabricating intracellular melanin. Our approach exploits synthetic tyrosine derivatives to hijack the melanin biosynthesis pathway in pigment-producing cells. Its application was exemplified by synthesizing and using a paramagnetic tyrosine derivative, m-YR, which endowed melanoma cells with responsiveness to external magnetic fields. The mechanical force generated by the magnet-responsive melanin forced the cells to elongate and align parallel to the magnetic power lines. Critically, even non-pigment cells were similarly remote-controlled by external magnetic fields once engineered to express tyrosinase and treated with m-YR, suggesting the versatility of the approach. The present methodology may potentially provide a new avenue for mechanobiology and magnetogenetic studies and a framework for magnetic control of specific cells.

The patient was a 34-year-old woman. Surgical resection and chemotherapy had been performed on diagnosis of malignant melanoma in year X-9. Chronic thyroiditis was diagnosed in year X-8, but her thyroid function was normal. In November of the year X-1, the patient, who had metastasis to the left lung and the left main bronchus and radically unresectable metastases with distant metastases, was treated with the anti-PD-1 antibody nivolumab. In December X-1, we initiated levothyroxine sodium for hypothyroidism after the patient suffered indolent thyroiditis due to nivolumab. In March X, the nivolumab treatment was stopped because it proved to be ineffective, then in April, anorexia, fever, and general malaise were noted. Cortisol 5.0 and ACTH 17.5 were confirmed by blood test, and the patient was diagnosed with adrenal insufficiency and was admitted to the hospital. Head MRI showed no organic lesions, and a stress test showed abnormalities only in a CRH test (low response to both ACTH and cortisol). The patient was diagnosed with isolated ACTH deficiency due to nivolumab. Side effects of thyroid dysfunction due to nivolumab are frequently observed in Japan at a rate of 14.3%, and overseas at 5.9%. However, secondary adrenocortical dysfunction is observed in overseas clinical trials at a frequency of only about 0.3%. There are few reports of such complications, and we report this as a rare case.

In this study, we propose a method for extracting the characteristics of body motions that contribute to reducing the takt time in a cooperative task between a dishwashing robot and a human operator. The proposed method collects the takt time and motion data from novice operators until they become experienced using an inexpensive acceleration sensor. The operation data is classified into experienced and novice periods using the variance value of the takt time. In addition, the Hidden Markov Model is generated to classify the motion data into multiple motion phases. The motion features of the operator are extracted for each phase from the generated model. The proposed method finds the motion features whose difference between the experienced and novice periods are similar to the takt time transition. It uses them as important variables. We verified the effectiveness of the proposed method by conducting experiments that simulate actual work at a restaurant. The Hidden Markov Model classified the operation phases into three categories with the AUC of 0.9. In all samples, we were able to extract the motion characteristics of the experienced operators. This study showed the potential to improve the speed of novice's progress by the extracted motion characteristics to improve education guidelines and to show operators how they should physically move.

Polymer electrolyte membrane fuel cells have recently attracted considerable attention as sustainable and eco-friendly electricity generation devices from the viewpoint of carbon neutrality. This study focuses on new discoveries related to the application of eggshell membranes to polymer electrolytes in the development of cheaper, more eco-friendly fuel cells. We observed the electricity generation of the fuel cells using an eggshell membrane as a proton-conductive material and a general carbonic acid aqueous solution. This new fuel cell will contribute to the continued improvement of available fuel cells at lower costs.

Anti‐N‐methyl‐D‐aspartate receptor (NMDA‐R) encephalitis is an autoimmune disorder in which autoantibodies in the limbic system bind to GluN1 subunits of NMDA‐Rs in the brain. We report a rare case of autoimmune polyendocrine syndrome type 3 complicated by anti‐NMDA‐R encephalitis. After hospitalization for type 1 diabetes, the 39‐year‐old patient developed various schizophreniform symptoms and seizures after cold‐like symptoms. These findings are consistent with the diagnosis of anti‐NMDA‐R encephalitis. Immune‐related encephalitis was suspected at the early phase of the disease, and cerebrospinal fluid was positive for anti‐NMDA‐R antibody. Early steroid pulse therapy was initiated during the disease course. The condition improved gradually to full recovery. Early detection and treatment of anti‐NMDA‐R encephalitis should enhance a positive outcome, considering that besides thyroid diseases and type 1 diabetes, various autoimmune diseases are associated with autoimmune polyendocrine syndrome type 3.

An enantioselective Strecker-type reaction of aldimines and ketimines was developed by using a chiral dilithium(I) binaphthyldisulfonate as a chiral acid–base cooperative catalyst. The present cata...

The ten-eleven translocation (TET) protein family, consisting of three isoforms (TET1/2/3), have been found in mammalian cells and have a crucial role in 5-methylcytosine demethylation in genomic DNA through the catalysis of oxidation reactions assisted by 2-oxoglutarate (2OG). DNA methylation/demethylation contributes to the regulation of gene expression at the transcriptional level, and recent studies have revealed that TET1 is highly elevated in malignant cells of various diseases and related to malignant alteration. TET1 inhibitors based on a scaffold of thioether macrocyclic peptides, which have been discovered by the random nonstandard peptide integrated discovery (RaPID) system, are reported. The affinity-based selection was performed against the TET1 compact catalytic domain (TET1CCD) to yield thioether macrocyclic peptides. These peptides exhibited inhibitory activity of the TET1 catalytic domain (TET1CD), with an IC50 value as low as 1.1 μm. One of the peptides, TiP1, was also able to inhibit TET1CD over TET2CD with tenfold selectivity, although it was likely to target the 2OG binding site; this provides a good starting point to develop more selective inhibitors.

Membrane proteins allow a cell to communicate with its environment by relaying a signal or transporting a molecule through the cell membrane. Elucidation of the three-dimensional structure of a membrane protein provides a greater understanding of its function and mechanisms. Ultimately, this knowledge will enlighten researchers on how these proteins can be regulated to elicit a desired cellular response, which could lead to novel therapeutic medicine. Unfortunately, the determination of the high-resolution crystal structures of transmembrane proteins remains a challenge due to their poor solubility and high conformational flexibility. Additives and cocrystallization ligands are being used to address these problems. In vitro selected macrocyclic peptides have recently been successfully employed as cocrystallization ligands. Although originally intended as inhibitors and drug lead molecules, in vitro selected macrocyclic peptides are now showing that their pharmacodynamic properties also allow them to serve as excellent cocrystallization ligands. Structures for macrocyclic peptide-bound transporters, the multidrug and toxic compound extrusion family transporter from Pyrococcus furiosus (PfMATE) and the ABC transporter subfamily B member 1 from Cyanidioschyzon meraloe (CmABCB1), have been elucidated using X-ray crystallography. The cocrystal structures reveal that the macrocyclic peptides improve crystallization by binding in a similar manner as a small molecule or a biologic. The PfMATE-binding macrocyclic peptides MaD3S and MaD5 bind to the surfaces buried in the center channel of the transporters. Although both transporters possess a center channel and substrate-binding pocket, the CmABCB1-binding macrocyclic peptide, aCAP, binds to the outer surface of the transporter in a similar manner to a biologic.

Membrane proteins allow a cell to communicate with its environment by relaying a signal or transporting a molecule through the cell membrane. Elucidation of the three-dimensional structure of a membrane protein provides a greater understanding of its function and mechanisms. Ultimately, this knowledge will enlighten researchers on how these proteins can be regulated to elicit a desired cellular response, which could lead to novel therapeutic medicine. Unfortunately, the determination of the high-resolution crystal structures of transmembrane proteins remains a challenge due to their poor solubility and high conformational flexibility. Additives and cocrystallization ligands are being used to address these problems. In vitro selected macrocyclic peptides have recently been successfully employed as cocrystallization ligands. Although originally intended as inhibitors and drug lead molecules, in vitro selected macrocyclic peptides are now showing that their pharmacodynamic properties also allow them to serve as excellent cocrystallization ligands. Structures for macrocyclic peptide-bound transporters, the multidrug and toxic compound extrusion family transporter from Pyrococcus furiosus (PfMATE) and the ABC transporter subfamily B member 1 from Cyanidioschyzon meraloe (CmABCB1), have been elucidated using X-ray crystallography. The cocrystal structures reveal that the macrocyclic peptides improve crystallization by binding in a similar manner as a small molecule or a biologic. The PfMATE-binding macrocyclic peptides MaD3S and MaD5 bind to the surfaces buried in the center channel of the transporters. Although both transporters possess a center channel and substrate-binding pocket, the CmABCB1-binding macrocyclic peptide, aCAP, binds to the outer surface of the transporter in a similar manner to a biologic.

The high-temperature thermolysis of fatty acid–iron complexes generates magnetic nanoparticles (MNPs) of a precisely controlled size coated with fatty acids and dispersed in oil. Because they are water-immiscible, MNPs are unsuitable for water-based biomedical applications. Ligand exchange reactions that transform oil- into water-dispersed MNPs have attracted considerable attention, but are difficult to perform. In this paper, we report the successful preparation of size-controlled and highly water-dispersed MNPs, which have 4, 8 and 20 nm diameter by a unique two-step ligand exchange reaction. As temporary ligands, we selected thiomalic acid (TMA), which possesses moderate affinity toward MNPs and is soluble in both oil and water to remove fatty acids by XANES analyses. Next we selected the citric acids as secondary ligands for TMA-exchanged MNPs to be highly dispersed in water to remove TMA from the surface of MNPs. And the resulting highly water-dispersed MNPs are expected to be available as MRI contrast agents and hyperthermia carriers.

In this study, FG beads (ferrite nanoparticles in the core covered with poly-(styrene-co-glycidyl methacrylate)) were made into fluorescent magnetic carriers (FMCs) containing the fluorescent substance, europium ion (Eu3+) complex. The developed FMCs showed several notable features such as high fluorescence intensity and high dispersibility in water. More importantly, FMCs did not leak Eu3+ complex. It is expected that the FMCs will be a useful tool for biomolecular recognition and imaging and contribute to advancement of a wide range of research fields, including cell biology and molecular imaging.

生体分子のアフィニティ精製は生命現象の分離レベルでの解明に不可欠であり，長年にわたりゲルカラム法が用いられてきた．しかしこの方法は高純度のものを得るのが非常に困難であり，純度を高めるために他の方法の併用が不可欠であること，精製で得られるタンパク質濃度が低いこと，精製にかなりの時間を有するなどの問題点が挙げられていた．そこで筆者らは孔をもたず，比表面積が非常に大きいサブミクロンサイズの微粒子を設計し，DNA やペプチド，タンパク質の他，薬剤などの生体にはない物質を固定化して，これらに結合するタンパク質の同定を長年にわたって研究してきた．本報ではこれらのアフィニティ精製に関する特筆すべき結果に関して述べる．また精製操作をさらに簡便化するために，磁石で回収可能な芯に強磁性体のナノフェライト微粒子を含有した磁性体含有高分子微粒子を作製し，それを用いたアフィニティ精製についても報告する．

For affinity purification of drug target protein we have developed magnetic carriers, narrow in size distribution (184±9 nm), which exhibit minimal non-specific binding of unwanted proteins. The carriers were highly dispersed in aqueous solutions and highly resistant to organic solvents, which enabled immobilization of various hydrophobic chemicals as probes on the carrier surfaces. Utilizing the carriers we have automated the process of separation and purification of the target proteins that had been done by manual operation previously.

We developed novel magnetic nano-carriers around 180 nm in diameter for affinity purification. Prepared magnetic nano-carriers possessed uniform core/shell/shell nano-structure composed of 40 nm magnetite particles/poly(styrene-co-glycidyl methacrylate (GMA))/polyGMA, which was constructed by admicellar polymerization. By utilizing relatively large 40 nm magnetite particles with large magnetization, the magnetic nano-carriers could show good response to permanent magnet. Thanks to uniform polymer shell with high physical/chemical stability, the magnetic nano-carriers could disperse in a wide range of organic solvent without disruption of core/shell structure and could immobilize various kinds of drugs. We examined affinity purification using our prepared magnetic nano-carriers with anti-cancer agent methotrexate (MTX) as ligand. Our magnetic nano-carriers showed higher performance compared to commercially available magnetic beads in terms of purification efficiency of target including extent of non-specific binding protein.

Despite the wide utility of ferrite nanoparticles (FP), a methodology to conjugate heterologous molecules to FP is still limited and characterization of small molecule-conjugated FP is not well known. Here, we describe what kinds of proteins and amino acids are selectively immobilized onto FP when FP is synthesized in the presence of these molecules. Two-dimentional gel electrophoresis (2D SDS-PAGE) showed that proteins with low pI value were selectively bound to FP. Quantitative analyses using HPLC suggested that L-aspartic acid (Asp) and L-cysteine (Cys) were bound to FP selectively among natural amino acids examined. Additional analysis of compounds-conjugated FP revealed that selective binding of Asp to FP was attributed with its molecular structure. It was found that the substructure of amino acid-bound to FP specifically was composed of a defined chelation of two carboxyl groups separated by two carbon atoms as deduced from FT-IR measurement. Thus, we concluded that molecules possessing two carboxyl groups separated by two carbons were bound to FP spontaneously and selectively, which might enable the attachment of free functional groups onto the FP surface if their molecules have functional groups other than carboxyl groups. The resulting complex might be applicable as a chemical tag to immobilize various molecules onto FP.

Under neutral conditions (pH=7-9) and temperature as low as 4°C we successfully synthesized ferrite nanoparticles with an intermediate structure between Fe3O4 and γ -Fe2O3 from an aqueous solution. These experimental conditions enabled us to immobilize even very unstable bioactive molecules onto the surfaces of the ferrite nanoparticles during their synthesis. The bioactive molecules were strongly fixed onto the ferrite surface intermediated by specific amino-acids or related-structure molecules each having pairs of carboxyl (COOH) groups. The COOH group pair was found to make a chemical bond with the ferrite particles. Utilizing the strong bond between such molecules and ferrites, we successfully prepared bioactive ferrite nanobeads where the ferrite nanoparticles were encapsulated in a polymer that exhibited negligible non-specific absorption of proteins. We also succeeded in fixing bioactive molecules onto the surfaces of the polymer coating of the ferrite nanobeads. We describe promising applications of our novel bioactive ferrite nanobeads such as high performance magnetic carriers for bio-screening, hypothermia, MRI contrast enhancement and magnetic drug delivery.

Direct observation of sulfur sites for sulfur-containing compounds (cysteine and its oxidative derivatives) on ferrite nanoparticles (FP) was performed by X-ray adsorption near edge structure (XANE...