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Nobuaki Takeshita, Tomoya Oe, Tetsuo Kiso, Shuichiro Kakimoto Drug Discovery Research, Astellas Pharma Inc, Ibaraki, JapanCorrespondence: Nobuaki TakeshitaDrug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, JapanTel +81-29-852-1111Fax +81-29-850-5120Email nobuaki.takeshita@astellas.comPurpose: Although abnormal peripheral and central pain processing has been observed in fibromyalgia (FM) patients, the biomechanics and pathophysiology, surrounding the peripheral mechanism are not well understood. An intermediate conductance channel, KCa 3.1, is expressed in peripheral sensory nerve fibers where it maintains the resting membrane potential and controls nerve firing, making it a plausible target for peripheral therapeutic interventions. ASP0819, a KCa 3.1 channel opener, is an orally available molecular entity and is used in this investigation to elucidate the role of KCa 3.1 in signal processing of pain in FM.Methods: Human or rat KCa 3.1 channel-expressing cells were used for evaluating the main action of the compound. Effects of the compound on withdrawal behavior by mechanical stimulation were examined in reserpine-induced myalgia (RIM) and vagotomy-induced myalgia (VIM) models of rats. In addition, in vivo electrophysiological analysis was performed to examine the peripheral mechanisms of action of the compound. Other pain models were also examined.Results: ASP0819 increased the negative membrane potential in a concentration-dependent manner. Oral administration of ASP0819 significantly recovered the decrease in muscle pressure threshold in rat FM models of RIM and VIM. The in vivo electrophysiological experiments showed that Aδ- and C-fibers innervating the leg muscles in the RIM model demonstrated increased spontaneous and mechanically evoked firing compared with normal rats. Intravenous infusion of ASP0819 significantly reduced both the spontaneous activity and mechanically evoked responses in Aδ-fibers in the rat RIM model. ASP0819 significantly reduced the number of abdominal contractions as an indicator of abdominal pain behaviors in the rat visceral extension model and withdrawal responses in the osteoarthritis model, respectively.Conclusion: These findings suggest that ASP0819 may be a promising analgesic agent with the ability to modulate peripheral pain signal transmission. Its use in the treatment of several pain conditions should be explored, chief amongst these being FM pain.Keywords: potassium channel, fibromyalgia, pain

Insulin-degrading enzyme (IDE) cleaves amyloid beta (Aβ), insulin, and other bioactive peptides. Because Aβ and insulin are closely related to Alzheimer's disease (AD) and type-2 diabetes mellitus (T2DM), respectively, IDE is a candidate drug target for treating both AD and T2DM. However, the activity of IDE has opposing effects, including decreasing AD risk by degrading Aβ and increasing T2DM risk by degrading insulin. The opposed substrate specificity is associated with the exo- and active sites containing Tyr 314 and Tyr 831 residues, the plausible modification targets for controlling substrate specificity. In this study, we used a tyrosine-specific modification regent, Cookson reagent (4-phenyl-1,2,4-triazoline-3,5-dione, PTAD), for IDE and examined the degradation activities on Aβ 40 and insulin. Fifteen tyrosine residues, including Tyr 314 and Tyr 831 , were modified by PTAD. After incubation with PTAD-modified IDE for 3 days, insulin remained intact, whereas Aβ 40 was completely degraded. This favorable change of substrate specificity was also observed in the mixture of Aβ 40 and insulin, suggesting that tyrosine modification of IDE might be a therapeutic strategy for AD and T2DM., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV2) infection has forced social changes worldwide. Development of potent antiviral agents is necessary to prevent future pandemics. Titanium oxide, a photocatalyst, is a long-acting antiviral agent; however, its effects are weakened in the dark. Therefore, new antiviral substances that can be used in the dark are needed. Two types of nitroxyl radicals, 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) and 2-azaadamantane N-oxyl (AZADO), are commonly used as oxidation catalysts utilizing oxygen in the air as the terminal oxidant. Therefore, in this study, we aimed to evaluate the potential of these radicals as antiviral compounds with sustained activity even in the dark. We evaluated the antiviral effects of oxoammonium salts corresponding to TEMPO and AZADO (TEMPO-Oxo and AZADO-Oxo, respectively), which are the active forms of nitroxyl radicals in oxidation reactions. TEMPO-Oxo and AZADO-Oxo inhibited the binding of SARS-CoV2 spike protein receptor-binding domain (S-RBD) to angiotensin-converting enzyme 2. Notably, AZADO-Oxo exhibited a 10-fold stronger inhibitory effect than TEMPO-Oxo. TEMPO-Oxo and AZADO-Oxo also denatured S-RBD; however, effects of AZADO-Oxo were 10-fold stronger than those of TEMPO-Oxo and did not change in the dark. Some S-RBD peptides treated with AZADO-Oxo were cleaved at the N-terminal side of tyrosine residues. TEMPO-Oxo and AZADO-Oxo exhibited concentration-dependent antiviral effects against feline coronavirus. In conclusion, active forms of the nitroxyl radicals, TEMPO-Oxo and AZADO-Oxo, exerted antiviral effects by denaturing S-RBD, regardless of the presence or absence of light, suggesting their potential as novel antiviral agents., (© 2024. The Author(s).)

This study explores sustainable agricultural practices by examining the role of organic materials in enhancing native predatory mites for controlling spider mites in apple orchards. Developing techniques to conserve indigenous natural enemies is vital for sustainable agricultural production. Phytoseiid mites can control spider mites, which are among the most significant pests in apple production. To conserve phytoseiid mite populations, it is important to identify alternative prey and to determine their role in phytoseiid mite proliferation. We demonstrated that the concurrent use of specific organic fertilizers and coconut husks can increase prey Tyrophagus mites, thereby enhancing phytoseiid mite density. Our research was conducted using sticky traps at the Miyagi Prefectural Agriculture and Horticulture Research Center in Japan. The occurrence of Tyrophagus mites was significantly correlated with the occurrence of phytoseiid mites in 2 years. In laboratory experiments, the use of organic fertilizers increased the density of Tyrophagus mites by 83 × within 4 weeks. Several species of phytoseiid mites were able to lay between 0.25 and 1.03 eggs per day per female by preying on Tyrophagus larvae. A 2-year field survey revealed that the use of organic fertilizers more than doubled the density of phytoseiid mites on apple leaves, likely through promoting Tyrophagus mite proliferation on the ground. These results highlight the potential of organic fertilizers not only to enhance soil nutrients, but also to boost phytoseiid mite populations, thereby contributing to more sustainable apple production., (© 2024. The Author(s).)

Significance: Lower extremity traumatic wounds are associated with numerous perioperative challenges. Their etiologies determine the characteristics and extent of the injury. The timing of subsequent surgical intervention and wound healing optimization after lower extremity trauma are integral to successful perioperative lower extremity wound management. Recent Advances: Managing trauma to the lower extremities uses a multidisciplinary surgical approach. The objective of this review is to summarize lower limb trauma assessment, advancements in lower extremity trauma management, and the clinical applications of advanced wound care in lower limb traumatic wounds. The advent of lower limb reconstruction and the development of advanced wound care modalities have helped to improve the management of these complex injuries. Critical Issues: The extensive involvement of bone, soft tissues, nerves, and blood vessels of severe lower extremity trauma wounds presents a challenge for clinicians in both the acute care setting and during patient rehabilitation. If not properly managed, these injuries may be subject to a decline in limb function and may possibly result in limb loss. To reveal developing limb-threatening conditions, serial examinations should be performed. Future Directions: The majority of lower limb traumatic wound will benefit from the perioperative administration of an appropriate negative pressure wound therapy (NPWT)-based system, which can help to promote granulation tissue and remove wound exudate before definitive closure and/or reconstruction. NPWT should be included as an important adjunct in the surgical management of lower limb traumatic wounds.

Despite the extensive research conducted on Alzheimer's disease (AD) over the years, no effective drug for AD treatment has been found. Therefore, the development of new drugs for the treatment of AD is of the utmost importance. We recently reported the proteolytic activities of JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMA), synthetic peptides of nine amino acids each, derived from the Box A region of Tob1 and ANA/BTG3 proteins, respectively. Furthermore, two components of ANA-TA9, ANA-YA4 (YRMI) at the C-terminus end and ANA-SA5 (SKGQA) at the N-terminus end of ANA-TA9, exhibited proteolytic activity against amyloid-β (Aβ) fragment peptides. In this study, we identified the active center of ANA-SA5 using AEBSF, a serine protease inhibitor, and a peptide in which the Ser residue of ANA-SA5 was replaced with Leu. In addition, we demonstrate the proteolytic activity of ANA-SA5 against the soluble form Aβ42 ( a -Aβ42) and solid insoluble form s -Aβ42. Furthermore, ANA-SA5 was not cytotoxic to A549 cells. These results indicate that ANA-SA5 is a promising Catalytide and a potential candidate for the development of new peptide drugs targeting Aβ42 for AD treatment.

The universality of physical phenomena is a pivotal concept underlying quantum standards. In this context, the realization of a quantum current standard using silicon single-electron pumps necessitates the verification of the equivalence across multiple devices. Herein, we experimentally investigate the universality of pumped currents from two different silicon single-electron devices which are placed inside the cryogen-free dilution refrigerator whose temperature (mixing chamber plate) was ∼150 mK under the operation of the pump devices. By direct comparison using an ultrastable current amplifier as a galvanometer, we confirm that two pumped currents are consistent with ∼1 ppm uncertainty. Furthermore, we realize quantum-current multiplication with a similar uncertainty by adding the currents of two different gigahertz (GHz)-operated silicon pumps, whose generated currents are confirmed to be identical. These results pave the way for realizing a quantum current standard in the nanoampere range and a quantum metrology triangle experiment using silicon pump devices.

The Japanese apricot (Prunus mume) is a popular fruit tree in Japan. However, the genetic factors associated with fruit trait variations are poorly understood. In this study, we investigated nine fruit-associated traits, including harvesting time, fruit diameter, fruit shape, fruit weight, stone (endocarp) weight, ratio of stone weight to fruit weight, and rate of fruit gumming, using 110 Japanese apricot accessions over four years. A genome-wide association study (GWAS) was performed for these traits and strong signals were detected on chromosome 6 for harvesting time and fruit diameters. These peaks were shown to undergo strong artificial selection during the differentiation of small-fruit cultivars. The genomic region defined by the GWAS and XP-nSL analyses harbored several candidate genes associated with plant hormone regulation. Furthermore, the alleles of small-fruit cultivars in this region were shown to have genetic proximity to some Chinese cultivars of P. mume. These results indicate that the small-fruit trait originated in China; after being introduced into Japan, it was preferred and selected by the Japanese people, resulting in the differentiation of small-fruit cultivars., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Oxidative stress and the resulting lipid peroxidation are associated with various pathological states, including neurodegenerative diseases and cancer. The end products of lipid peroxidation, such as 4-oxo-2(E)-nonenal (ONE), 4-hydroxy-2(E)-nonenal (HNE), and methylglyoxal (MG), exert several biological effects through modification of various cellular components, including DNA and proteins. Glutathione peroxidase 1 (GPx1) is an intracellular antioxidant enzyme that uses glutathione (GSH) to reduce a variety of peroxides, thereby modulating cellular oxidative stress and redox-mediated responses. GPx1 contains nucleophilic amino acids at its active (one Sec) and GSH-binding (four Arg and one Lys) sites. We found that lipid peroxidation-derived reactive aldehydes (ONE, HNE, and MG) modified the GSH-binding site, resulting in the inhibition of GPx1 activity. Mass spectrometry-based proteomic analysis identified the sites modified by each aldehyde (ONE, 14 sites; HNE, 7 sites; MG, 9 sites). The GSH-binding sites modified were as follows: ONE, Arg57, 103, 184, and 185; HNE, Lys91; MG, Arg103. Upon incubation of GPx1 with each aldehyde, ONE reduced GPx1 activity more significantly than did HNE or MG in a dose- and time-dependent manner. The addition of GSH to GPx1 3 h after incubation with ONE prevented further inhibition by trapping ONE as a ONE-GSH adduct. However, the activity of GPx1 was not restored to the initial level, indicating that ONE modified GPx1 irreversibly. This study suggests that oxidative damage to lipids, resulting in the formation of reactive aldehydes, can amplify cellular oxidative stress via direct inactivation of GPx1, which increases the production of intracellular peroxides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Herein, improving the antibacterial activity of a hydrogel system of sodium alginate (SA) and basic chitosan (CS) using sodium hydrogen carbonate by adding AgNPs was investigated. SA-coated AgNPs produced by ascorbic acid or microwave heating were evaluated for their antimicrobial activity. Unlike ascorbic acid, the microwave-assisted method produced uniform and stable SA-AgNPs with an optimal reaction time of 8 min. Transmission electron microscopy (TEM) confirmed the formation of SA-AgNPs with an average particle size of 9 ± 2 nm. Moreover, UV-vis spectroscopy confirmed the optimal conditions for SA-AgNP synthesis (0.5% SA, 50 mM AgNO 3 , and pH 9 at 80 °C). Fourier transform infrared (FTIR) spectroscopy confirmed that the -COO - group of SA electrostatically interacted with either the Ag + or -NH 3 + of CS. Adding glucono-δ-lactone (GDL) to the mixture of SA-AgNPs/CS resulted in a low pH (below the p K a of CS). An SA-AgNPs/CS gel was formed successfully and retained its shape. This hydrogel exhibited 25 ± 2 mm and 21 ± 1 mm inhibition zones against E. coli and B. subtilis and showed low cytotoxicity. Additionally, the SA-AgNP/CS gel showed higher mechanical strength than SA/CS gels, possibly due to the higher crosslink density. In this work, a novel antibacterial hydrogel system was synthesized via 8 min of microwave heating.

Purpose: To develop a safe and precise method for intraprostatic injection, and to establish correlation between the volume of ethanol injectate and the volume of subsequent infiltrated prostate tissue., Materials and Methods: We performed intraprostatic injection of 96% ethanol using a needle which has a segment of its wall made of capillary membrane with hundreds of pores in an acute and chronic canine experiment, in heart-beating cadaveric organ donors, and in a xenograft model of human prostate cancer. Whole mount tissue sections were used for three-dimensional reconstruction of the necrotic lesions and calculation of their volumes., Results: The ethanol injection resulted in oval shaped lesions of well-delineated coagulative necrosis. In both healthy human and canine prostates, the prostatic pseudocapsule and neurovascular bundle remained intact without evidence of disruption. There was a linear correlation between administered volume of ethanol and the volume of necrotic lesion. Regression analysis showed strong correlation in the acute canine experiments and in experiments performed on xenografts of human prostate cancer. A formula was calculated for each experiment to estimate the relationship between the injected volume and the volume of infiltrated prostate tissue area., Conclusions: Intraprostatic injection using a porous needle allows for effective and predictable tissue distribution of the injectate in the prostate. Through varying the volume of the agent injected and use of needles with a different length of the porous segment, the volume of infiltrated tissue could be adjusted allowing for targeted focal treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Eubank, Švihra, DiBona, Sommers, Oe, Strnádel, Miklušica, Szépe, Marcinek, King, Plante, Ľupták, Poulsen, Kida, Baco, Švihra and Zvara.)

This study aimed to investigate the linguistic factors involved in stuttering among Japanese-speaking preschool children. The participants included 10 Japanese children who stutter, with a mean age of 5 years and 9 months. Speech samples comprised spontaneous conversations of the participants with their parents for about 20 minutes. We compared the percentages of the occurrence of stuttering-like disfluencies (SLDs) at the word and sentence levels, using the Wilcoxon signed-rank test. The results showed no significant differences in SLDs based on syllable structure when comparing light and heavy syllables and comparing consonants and vowels in the initial position of each content word. SLDs occurred more frequently in the initial than non-initial position of words and in longer rather than shorter words. Additionally, SLDs occurred more frequently in sentences that contained more 'bunsetsu' (a kind of linguistic unit in Japanese). Our study is the first to show that both word and sentence-level factors could contribute to SLDs in preschool children who stutter in agglutinating languages, such as Japanese. This aspect is rarely reported in psycholinguistic studies based on stuttering occurrence in inflecting languages, such as English.

Oxidative stress is strongly associated with the onset and/or progression of diabetes. Under conditions of oxidative stress, lipid hydroperoxides are decomposed to reactive aldehydes that have been reported to induce insulin resistance by modifying proteins involved in insulin signaling. Pyridoxamine (PM) can inhibit the formation of advanced glycation/lipoxidation end products by scavenging reactive carbonyl species. Thus, PM has emerged as a promising drug candidate for various chronic conditions, including diabetic complications. In this study, L6 skeletal muscle cells were treated with 4-oxo-2( E )-nonenal (ONE), one of the most abundant and reactive lipid-derived aldehydes. Cellular insulin resistance was assessed by measuring insulin-stimulated glucose uptake using 2-deoxyglucose. ONE induced a time- and dose-dependent decrease in glucose uptake. Liquid chromatography/electrospray ionization-mass spectrometry analysis of the reaction between ONE and insulin receptor substrate 1 (IRS1) lysate identified multiple modifications that could disturb the interaction between IRS1 and activated IR, leading to insulin resistance. Pretreatment of the cells with PM restored the ONE-induced decrease in glucose uptake. Concomitantly, the formation of PM-ONE adducts in cell culture medium was increased in a PM-dose dependent manner. PM can therefore prevent lipid hydroperoxide-derived insulin resistance by quenching ONE., Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-022-00155-z., Competing Interests: Conflict of interestThe authors do not have any conflicts of interest to declare., (© The Author(s) under exclusive licence to Korean Society of Toxicology 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Amyloid beta (Aβ) proteins are produced from amyloid precursor protein cleaved by β- and γ-secretases, and are the main components of senile plaques pathologically found in Alzheimer's disease (AD) patient brains. Therefore, the relationship between AD and Aβs has been well studied for both therapeutic and diagnostic purposes. Several enzymes have been reported to degrade Aβs in vivo , with neprilysin (NEP) and insulysin (insulin-degrading enzyme, IDE) being the most prominent. In this article, we describe the mass spectrometric characterization of peptide fragments generated using NEP and IDE, and clarify the differences in digestion specificities between these two enzymes for non-aggregated Aβ 40 , aggregated Aβ 40 , and Aβ 40 peptide fragments, including Aβ 16 . Our results allowed identification of all the peptide fragments from non-aggregated Aβ 40 : NEP, 23 peptide fragments consisting of 2-11 amino-acid residues, 17 cleavage sites; IDE, 23 peptide fragments consisting of 6-33 amino-acid residues, 15 cleavage sites. Also, we confirmed that IDE can digest only whole Aβ 40 , whereas NEP can digest both Aβ 40 and partial structures such as Aβ 16 and peptide fragments generated by the digestion of Aβ 40 by IDE. Furthermore, we confirmed that IDE and NEP are unable to digest aggregated Aβ 40 ., Competing Interests: The authors declare that there is no conflict of interest., (© 2022 The Author(s).)

Purpose: This systematic review identified and synthesized published research articles, written in Japanese, on the clinical effectiveness of a broad range of nonpharmacological interventions for school-age children who stutter., Method: A systematic review of Japanese literature published between January 1, 1980, and July 7, 2020, reporting interventions for school-age children who stutter, was carried out through a search of two databases (CiNii Article database and Japan Medical Abstract Society database) using the key words "stuttering" and "school-age" or "child" or "primary school students" or "children" or "school child" in Japanese. To be included in the review, the articles must report studies where data were subjectively reported by clinicians, where school-age participants were treated for developmental stuttering, where participants received interventions conducted by clinicians, and where quantitative outcomes (pre- and/or posttreatment) were measured; and they must be published in Japanese., Results: Forty articles met all the inclusion criteria. Most articles adopted a case series or single-case study design. A total of 179 intervention programs were identified from all the articles and broadly classified into speech therapy, psychological therapy, interventions for modifying the child's environment, and others., Conclusions: Our systematic review provided a broad overview of the treatments used for school-age children who stutter in Japan. Future research should focus on gathering more reliable, systematic, and rigorous evidence to establish the effectiveness of stuttering treatments for school-age children and thereby develop evidence-based practices.

Aims: In vivo experiments were performed to establish and validate a rat model of urethral sphincter injury and to develop a method for leak point pressure (LPP) measurements performed repeatedly in the same animal., Methods: Twenty-four Sprague-Dawley female rats underwent bladder and epidural catheter implantation. Five days later, cystometry was performed using continuous infusion. Anesthesia with isoflurane, ketamine-xylazine (KX) or fentanyl-fluanisone-midazolam (FFM) was used. After three micturition cycles, intrathecal bupivacaine was administered leading to the suppression of reflex bladder contractions. LPP measurements were performed using vertical tilt. After the initial LPP measurement, animals underwent partial resection of the striated urethral sphincter. The effect was evaluated 6 weeks after surgery, by repeating the LPP measurement in the same animal., Results: Ten out of 19 animals showed full micturition cycles under isoflurane, and all 9 animals under KX anesthesia. No significant difference in micturition pressures (Mean ± SEM; 30.1 ± 2.3 vs. 26.8 ± 1.6 mmHg) and LPP (31.0 ± 2.4 vs. 28.0 ± 0.9 mmHg) was observed between isoflurane and KX groups, respectively. Reflex micturition was suppressed with FFM. Bupivacaine led to overflow incontinence in all cases. Sphincter injury caused fibrotic changes and a significant increase in LPP (26.4 ± 2.3 before vs. 46.9 ± 4.6 mmHg after injury, p  < 0.05)., Conclusions: KX anesthesia preserves bladder contractions. Intrathecal bupivacaine eliminates reflex micturition, allowing for repeated LPP measurements in the same animal. Resection of striated sphincter resulted in increased LPP 6 weeks post injury. The site of urethral sphincter resection healed with fibrosis.

Angiotensin (Ang) peptides are the main effectors of the renin-angiotensin system (RAS) regulating diverse physiological conditions and are involved in renal and vascular diseases. Currently, quantitative analyses of Ang peptides in human plasma mainly rely on radioimmunoassay-based methods whose reported levels are quite divergent. Analyses are further complicated by the potential of Ang peptides to bind to solid surfaces, to be enzymatically decomposed during sample preparation, and to undergo post-translational modifications. A column switching-parallel LC/ESI-SRM/MS method has been developed for seven Ang peptides (Ang I, Ang II, Ang III, Ang IV, Ang 1-9, Ang 1-7, and Ang A) in human plasma. Aqueous acetonitrile (5%) containing 50 mM arginine (Arg) as a dissolving solution and a combination of protease inhibitors with formic acid were used to prevent adsorption and enzymatic degradation, respectively. Plasma samples were simply deproteinized with acetonitrile followed by clean-up with an on-line trap column via column-switching. Stable isotope dilution with [ 13 C 5 , 15 N 1 -Val]-Ang peptides as internal standards was employed for quantitative analysis. The current methodology has been successfully applied to determine the plasma levels of Ang peptides in healthy participants, suggesting future applicability to studies of various diseases related to RAS., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: Although abnormal peripheral and central pain processing has been observed in fibromyalgia (FM) patients, the biomechanics and pathophysiology, surrounding the peripheral mechanism are not well understood. An intermediate conductance channel, K Ca 3.1, is expressed in peripheral sensory nerve fibers where it maintains the resting membrane potential and controls nerve firing, making it a plausible target for peripheral therapeutic interventions. ASP0819, a K Ca 3.1 channel opener, is an orally available molecular entity and is used in this investigation to elucidate the role of K Ca 3.1 in signal processing of pain in FM., Methods: Human or rat K Ca 3.1 channel-expressing cells were used for evaluating the main action of the compound. Effects of the compound on withdrawal behavior by mechanical stimulation were examined in reserpine-induced myalgia (RIM) and vagotomy-induced myalgia (VIM) models of rats. In addition, in vivo electrophysiological analysis was performed to examine the peripheral mechanisms of action of the compound. Other pain models were also examined., Results: ASP0819 increased the negative membrane potential in a concentration-dependent manner. Oral administration of ASP0819 significantly recovered the decrease in muscle pressure threshold in rat FM models of RIM and VIM. The in vivo electrophysiological experiments showed that Aδ- and C-fibers innervating the leg muscles in the RIM model demonstrated increased spontaneous and mechanically evoked firing compared with normal rats. Intravenous infusion of ASP0819 significantly reduced both the spontaneous activity and mechanically evoked responses in Aδ-fibers in the rat RIM model. ASP0819 significantly reduced the number of abdominal contractions as an indicator of abdominal pain behaviors in the rat visceral extension model and withdrawal responses in the osteoarthritis model, respectively., Conclusion: These findings suggest that ASP0819 may be a promising analgesic agent with the ability to modulate peripheral pain signal transmission. Its use in the treatment of several pain conditions should be explored, chief amongst these being FM pain., Competing Interests: All authors are employees of Astellas Pharma Inc, during the conduct of the study. Tomoya Oe has patents issued: JP4853844, US8795632, and EP2327299. The authors report no other potential conflicts of interest for this work., (© 2021 Takeshita et al.)

The objective of this study was to estimate variance components related to imprinting for carcass traits and physiochemical characteristics in Japanese Black cattle. The carcass records obtained from 4,220 Japanese Black feedlot cattle included carcass weight (CW), rib eye area (REA), rib thickness, subcutaneous fat thickness, and beef marbling score (BMS), and the physiochemical characteristics were fat, moisture, glycogen per proportion of moisture content, oleic acid, and monounsaturated fatty acids (MUFA). To detect gametic effects, an imprinting model was fitted. High additive heritabilities were estimated for all traits (from 0.516 for glycogen to 0.853 for fat) and were reduced in Mendelian heritability. The range of the differences was from 0.002 (CW) to 0.331 (fat and moisture), and the reductions were due to their imprinting variances. The ratio of the imprinting variance to the total additive genetic variance for REA (0.374), BMS (0.291), fat (0.387), moisture (0.388), and MUFA (0.337) were large (p < 0.05). These imprinting variances were due to the maternal contribution and suggested the existence of maternally expressed genomic imprinting effects on the traits in Japanese Black cattle. Therefore, maternal gametic effects should be considered in breeding programs for Japanese Black cattle., (© 2021 Japanese Society of Animal Science.)

Acrylamide (AA) is a toxic industrial chemical but is also found in heated potato foods such as French fries due to the Maillard reaction between amino acids and reducing sugars. However, high-temperature cooking is often required for flavoring, browning, and sterilizing of raw ingredients. Imidazole dipeptides, such as carnosine (β-alanyl-l-histidine, CAR) and anserine (β-alanyl-N π -methyl-l-histidine, ANS), are present in high concentrations in meat and are known to scavenge radical species and toxic aldehydes. Here, we investigated the reaction between CAR/ANS and AA under several conditions expected to detoxify AA by cooking with meat. The reaction products were characterized by LC-ESI-MS/MS as CAR/ANS-AA adducts at the N-terminus, and His-N τ /N π . The reactivity of CAR sites toward AA were in the order N-terminus > N τ  > N π . A selective LC-ESI-SRM/MS method was also developed and confirmed the formation of CAR/ANS-AA adducts during pan frying of minced potato and chicken breast., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Proteins are continuously exposed to diverse chemical stresses, and the resulting chemical modifications can provide significant information on biological events. Keratins are the main constituent of human skin and are the major target proteins of various chemical modifications. We have previously developed a mass spectrometry-based noninvasive proteomic methodology to screen oxidative modifications in human skin keratins. We have improved this methodology in terms of sample preparation time and amino acid sequence coverage using an on-tape digestion method. After sampling by tape stripping, skin proteins on the tape were subjected to reduction/alkylation, followed by trypsin digestion without a presolubilization step using detergents. To screen chemical modifications in keratins, target modifications and tryptic target peptides carrying the modification sites were determined from in vitro experiments with major reactive chemical species (4-hydroxy-2( E )-nonenal (HNE), 4-oxo-2( E )-nonenal, glucose, methylglyoxal, peroxynitrite, and hydrogen peroxide). The developed method was used to screen target modifications in controls and patients with a swollen red rash. Basal levels of lipid-derived modification, oxidation, nitration, and glycation in keratins were detected in controls. Principal component analysis based on the relative chemical modification resulted in a clear classification of both groups within a 95% confidence interval. Lipid-derived HNE modification increased most significantly in the patient group. This methodology can be easily applied to patients with other diseases, and the target modifications can be used as biomarkers of certain physiological conditions.

Background: Distraction osteogenesis has been broadly used to treat various structural bone deformities and defects. However, prolonged healing time remains a major problem. Various approaches including the use of low-intensity pulsed ultrasound, parathyroid hormone, and bone morphogenetic proteins (BMPs) have been studied to shorten the treatment period with limited success. Our previous studies of rats have reported that the transcutaneous application of CO2 accelerates fracture repair and bone-defect healing in rats by promoting angiogenesis, blood flow, and endochondral ossification. This therapy may also accelerate bone generation during distraction osteogenesis, but, to our knowledge, no study investigating CO2 therapy on distraction osteogenesis has been reported., Questions/purposes: We aimed to investigate the effect of transcutaneous CO2 during distraction osteogenesis in rabbits, which are the most suitable animal as a distraction osteogenesis model for a lengthener in terms of limb size. We asked: Does transcutaneous CO2 during distraction osteogenesis alter (1) radiographic bone density in the distraction gap during healing; (2) callus parameters, including callus bone mineral content, volumetric bone mineral density, and bone volume fraction; (3) the newly formed bone area, cartilage area, and angiogenesis, as well as the expression of interleukin-6 (IL-6), BMP-2, BMP-7, hypoxia-inducible factor (HIF) -1α, and vascular endothelial growth factor (VEGF); and (4) three-point bend biomechanical strength, stiffness, and energy?, Methods: Forty 24-week-old female New Zealand white rabbits were used according to a research protocol approved by our institutional ethical committee. A distraction osteogenesis rabbit tibia model was created as previously described. Briefly, an external lengthener was applied to the right tibia, and a transverse osteotomy was performed at the mid-shaft. The osteotomy stumps were connected by adjusting the fixator to make no gap. After a 7-day latency phase, distraction was continued at 1 mm per day for 10 days. Beginning the day after the osteotomy, a 20-minute transcutaneous application of CO2 on the operated leg using a CO2 absorption-enhancing hydrogel was performed five times per week in the CO2 group (n = 20). Sham treatment with air was administered in the control group (n = 20). Animals were euthanized immediately after the distraction period (n = 10), 2 weeks (n = 10), and 4 weeks (n = 20) after completion of distraction. We performed bone density quantification on the plain radiographs to evaluate consolidation in the distraction gap with image analyzing software. Callus parameters were measured with micro-CT to assess callus microstructure. The newly formed bone area and cartilage area were measured histologically with safranin O/fast green staining to assess the progress of ossification. We also performed immunohistochemical staining of endothelial cells with fluorescein-labeled isolectin B4 and examined capillary density to evaluate angiogenesis. Gene expressions in newly generated callus were analyzed by real-time polymerase chain reaction. Biomechanical strength, stiffness, and energy were determined from a three-point bend test to assess the mechanical strength of the callus., Results: Radiographs showed higher pixel values in the distracted area in the CO2 group than the control group at Week 4 of the consolidation phase (0.98 ± 0.11 [95% confidence interval 0.89 to 1.06] versus 1.19 ± 0.23 [95% CI 1.05 to 1.34]; p = 0.013). Micro-CT demonstrated that bone volume fraction in the CO2 group was higher than that in the control group at Week 4 (5.56 ± 3.21 % [95% CI 4.32 to 6.12 %] versus 11.90 ± 3.33 % [95% CI 9.63 to 14.25 %]; p = 0.035). There were no differences in any other parameters (that is, callus bone mineral content at Weeks 2 and 4; volumetric bone mineral density at Weeks 2 and 4; bone volume fraction at Week 2). At Week 2, rabbits in the CO2 group had a larger cartilage area compared with those in the control group (2.09 ± 1.34 mm [95% CI 1.26 to 2.92 mm] versus 5.10 ± 3.91 mm [95% CI 2.68 to 7.52 mm]; p = 0.011). More newly formed bone was observed in the CO2 group than the control group at Week 4 (68.31 ± 16.32 mm [95% CI 58.19 to 78.44 mm] versus 96.26 ± 19.37 mm [95% CI 84.25 to 108.26 mm]; p < 0.001). There were no differences in any other parameters (cartilage area at Weeks 0 and 4; newly formed bone area at Weeks 0 and 2). Immunohistochemical isolectin B4 staining showed greater capillary densities in rabbits in the CO2 group than the control group in the distraction area at Week 0 and surrounding tissue at Weeks 0 and 2 (distraction area at Week 0, 286.54 ± 61.55 /mm [95% CI 232.58 to 340.49] versus 410.24 ± 55.29 /mm [95% CI 361.78 to 458.71]; p < 0.001; surrounding tissue at Week 0 395.09 ± 68.16/mm [95% CI 335.34 to 454.83] versus 589.75 ± 174.42/mm [95% CI 436.86 to 742.64]; p = 0.003; at Week 2 271.22 ± 169.42 /mm [95% CI 122.71 to 419.73] versus 508.46 ± 49.06/mm [95% CI 465.45 to 551.47]; p < 0.001 respectively). There was no difference in the distraction area at Week 2. The expressions of BMP -2 at Week 2, HIF1-α at Week 2 and VEGF at Week 0 and 2 were greater in the CO2 group than in the control group (BMP -2 at Week 2 3.84 ± 0.83 fold [95% CI 3.11 to 4.58] versus 7.32 ± 1.63 fold [95% CI 5.88 to 8.75]; p < 0.001; HIF1-α at Week 2, 10.49 ± 2.93 fold [95% CI 7.91 to 13.06] versus 20.74 ± 11.01 fold [95% CI 11.09 to 30.40]; p < 0.001; VEGF at Week 0 4.80 ± 1.56 fold [95% CI 3.43 to 6.18] versus 11.36 ± 4.82 fold [95% CI 7.13 to 15.59]; p < 0.001; at Week 2 31.52 ± 8.26 fold [95% CI 24.27 to 38.76] versus 51.05 ± 15.52 fold [95% CI 37.44 to 64.66]; p = 0.034, respectively). There were no differences in any other parameters (BMP-2 at Week 0 and 4; BMP -7 at Weeks 0, 2 and 4; HIF-1α at Weeks 0 and 4; IL-6 at Weeks 0, 2 and 4; VEGF at Week 4). In the biomechanical assessment, ultimate stress and failure energy were greater in the CO2 group than in the control group at Week 4 (ultimate stress 259.96 ± 74.33 N [95% CI 167.66 to 352.25] versus 422.45 ± 99.32 N [95% CI 299.13 to 545.77]; p < 0.001, failure energy 311.32 ± 99.01 Nmm [95% CI 188.37 to 434.25] versus 954.97 ± 484.39 Nmm [95% CI 353.51 to 1556.42]; p = 0.003, respectively). There was no difference in stiffness (216.77 ± 143.39 N/mm [95% CI 38.73 to 394.81] versus 223.68 ± 122.17 N/mm [95% CI 71.99 to 375.37]; p = 0.92)., Conclusion: Transcutaneous application of CO2 accelerated bone generation in a distraction osteogenesis model of rabbit tibias. As demonstrated in previous studies, CO2 treatment might affect bone regeneration in distraction osteogenesis by promoting angiogenesis, blood flow, and endochondral ossification., Clinical Relevance: The use of the transcutaneous application of CO2 may open new possibilities for shortening healing time in patients with distraction osteogenesis. However, a deeper insight into the mechanism of CO2 in the local tissue is required before it can be used in future clinical practice.

Background: Long-term monitoring data is helpful to understand the fluctuation of susceptibility and pattern of cross resistance in insecticide resistance management. After the occurrence of imidacloprid resistance, the brown planthopper (BPH) has gradually developed resistance to thiamethoxam and clothianidin since 2010, but not to dinotefuran and nitenpyram. Here, we analyzed susceptibilities data of five neonicotinoids during 2005-2017 in East Asia and Vietnam to conduct cross-resistance patterns among neonicotinoids. To determine the factors of development of cross resistance in laboratory bioassays, we used the imidacloprid resistant and control strains that were selected from filed populations in the Philippines and Vietnam., Results: The Linear Mixed Models (LMM) analyses of insecticide susceptibility data showed that the slope values of imidacloprid resistance effects were 0.68 and 1.09 for resistance to thiamethoxam and clothianidin, respectively. Laboratory bioassay results showed that the LD 50 values for thiamethoxam and clothianidin in resistant strains (1.4-5.5 μg g -1 ) were 3.2-16.4 times higher than those in the control strains (0.28-1.5 μg g -1 ). However, the increase in the LD 50 values for imidacloprid was not related to that for dinotefuran and nitenpyram based on the results of the LMM analysis and laboratory bioassay., Conclusion: Our results demonstrate that the development of imidacloprid resistance result in strong-cross resistance to some neonicotinoids, thiamethoxam and clothianidin, but not to others, dinotefuran and nitenpyram. We anticipate that our findings will be a starting point for understanding mechanism of the different trend of cross resistance by analyzing long-term susceptibility data and laboratory bioassays in insect pests. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Mycobacterium tuberculosis ( M. tuberculosis ) produces a diverse range of antigenic proteins in its dormant phase. The cytokine profiles of CD4 + T cell responses, especially subsets other than Th1 type (non-Th1 type), against these latency-associated M. tuberculosis antigens such as α-crystallin (Acr), heparin-binding hemagglutinin (HBHA), and mycobacterial DNA-binding protein 1 (MDP-1) remain elusive in relation to the clinical stage of M. tuberculosis infection. In the present study, peripheral blood mononuclear cells (PBMCs) collected from different stages of M. tuberculosis -infected cases and control PBMCs were stimulated with these antigens and ESAT-6/CFP-10. Cytokine profiles of CD4 + T cells were evaluated by intracellular cytokine staining using multicolor flow cytometry. Our results demonstrate that Th1 cytokine responses were predominant after TB onset independent of the type of antigen stimulation. On the contrary, non-Th1 cytokine responses were preferentially induced by latency-associated M. tuberculosis antigens, specifically IL-10 response against Acr in latent M. tuberculosis infection. From these results, we surmise a shift in the CD4 + T cell response from mixed non-Th1 to Th1 dominant type during TB progression., (Copyright © 2019 Yamashita, Oe, Kawakami, Osada-Oka, Ozeki, Terahara, Yasuda, Edwards, Tanaka, Tsunetsugu-Yokota, Matsumoto and Ariyoshi.)

Angiotensin (Ang) II is a major bioactive peptide of the renin/angiotensin system and is involved in various cardiovascular functions and diseases. Ang II type 1 (AT 1 ) receptor mediates most of the physiological effects of Ang II. Previous studies have revealed that the lipid peroxidation products 4-oxo-2( E )-nonenal (ONE) and 4-hydroxy-2( E )-nonenal (HNE) readily modify the N-terminus and Asp 1 , Arg 2 , and His 6 residues of Ang II, and these modifications alter the biological activities of Ang II. Ang II is known to stimulate the formation of reactive oxygen species (ROS) that mediate cardiovascular remodeling. Another major consequence of ROS-derived damage is lipid peroxidation, which generates genotoxic aldehydes such as ONE and HNE. This study demonstrated that Ang II induced lipid peroxidation-derived modifications of cellular molecules in EA.hy926 cells, a human vascular endothelial cell line. Ang P (ONE- and ROS-derived N-terminal pyruvamide Ang II) and [His 6 (HNE)]-Ang II were detected in the medium of EA.hy926 cells incubated with Ang II, and their concentrations increased dose-dependently upon the addition of ascorbic acid (AscA) and CuSO 4 . Cells were then subjected to metabolic labeling using SILFAC ( s table i sotope l abeling by f atty a cids in c ell culture) with [ 13 C 18 ]-linoleic acid. Analysis of cellular phospholipids indicated over 90% labeling. [ 13 C 9 ]-Thiadiazabicyclo-ONE-glutathione adduct as well as Ang P and [His 6 ([ 13 C 9 ]-HNE)]-Ang II was detected in the labeled cells upon treatment with Ang II and their concentrations increased in an Ang II dose-dependent manner. Incubation of the labeled cells with losartan, an AT 1 receptor blocker, inhibited the formation of modified Ang IIs in a dose-dependent manner. These results indicate that Ang II induces lipid peroxidation and modification of various cellular molecules and these reactions are mediated by the activation of AT 1 receptor. Therefore, lipid peroxidation could be one mechanism by which Ang II contributes to cardiovascular dysfunction.

Neuromyelitis optica spectrum disorders (NMOSD) constitute rare autoimmune disorders of the CNS that are primarily characterized by severe inflammation of the spinal cord and optic nerve. Approximately 75% of NMOSD patients harbour circulating pathogenic autoantibodies targeting the aquaporin-4 water channel (AQP4). The source of these autoantibodies remains unclear, but parallels between NMOSD and other autoantibody-mediated diseases posit compromised B cell tolerance checkpoints as common underlying and contributing factors. Using a well established assay, we assessed tolerance fidelity by creating recombinant antibodies from B cell populations directly downstream of each checkpoint and testing them for polyreactivity and autoreactivity. We examined a total of 863 recombinant antibodies. Those derived from three anti-AQP4-IgG seropositive NMOSD patients (n = 130) were compared to 733 antibodies from 15 healthy donors. We found significantly higher frequencies of poly- and autoreactive new emigrant/transitional and mature naïve B cells in NMOSD patients compared to healthy donors (P-values < 0.003), thereby identifying defects in both central and peripheral B cell tolerance checkpoints in these patients. We next explored whether pathogenic NMOSD anti-AQP4 autoantibodies can originate from the pool of poly- and autoreactive clones that populate the naïve B cell compartment of NMOSD patients. Six human anti-AQP4 autoantibodies that acquired somatic mutations were reverted back to their unmutated germline precursors, which were tested for both binding to AQP4 and poly- or autoreactivity. While the affinity of mature autoantibodies against AQP4 ranged from modest to strong (Kd 15.2-559 nM), none of the germline revertants displayed any detectable binding to AQP4, revealing that somatic hypermutation is required for the generation of anti-AQP4 autoantibodies. However, two (33.3%) germline autoantibody revertants were polyreactive and four (66.7%) were autoreactive, suggesting that pathogenic anti-AQP4 autoantibodies can originate from the pool of autoreactive naïve B cells, which develops as a consequence of impaired early B cell tolerance checkpoints in NMOSD patients., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The carbon stable isotopic composition (δ 13 C) is often analyzed to quantify the addition of acidulants to Japanese apricot liqueur, but little is known about the variation in the δ 13 C values of the main organic acids arising from differences in the ripeness of Japanese apricots. We show that in Japanese apricot liqueur prepared using fruits at different stages of ripeness, the δ 13 C values of citric acid and malic acid ranged from -25.1‰ to -23.7‰ and from -22.3‰ to -19.7‰, respectively, and the δ 13 C values decreased as the fruit ripened. The average δ 13 C value of citric acid from liqueurs was 0.7‰ higher than that from fresh fruits, whereas the δ 13 C values of malic acid showed no isotope discrimination. The variation in δ 13 C values of the main organic acids in Japanese apricot liqueurs will help detect acidulant addition and control authenticity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Acetaminophen (Paracetamol, APAP) has been widely used for many decades as an analgesic and antipyretic agent but APAP overdose often causes acute adverse reactions, particularly liver damage. The metabolically oxidized form of APAP, N-acetyl-p-benzoquinone imine (NAPQI), is chemically reactive and binds covalently to proteins. Therefore, NAPQI is believed to be the key metabolite that causes hepatotoxicity, especially under conditions of glutathione depletion. Other APAP-induced adverse reactions, such as skin damage, are rare and remain poorly studied. Here, we report a case study of a male patient who presented with an acute swelling skin rash (without hepatotoxicity) caused by therapeutic doses of APAP. Plasma samples were collected at 17 hr after dosing (during the manifestation of symptoms) and at one month (after recovery) and were subjected to LC-MS analysis of NAPQI-adducts. A significant concentration of NAPQI-cysteine adduct (33 pmol/mL) was found together with low concentrations of NAPQI-N-acetylcysteine adduct (2.0 pmol/mL) and NAPQI-glutathione adduct (0.13 pmol/mL). However, the NAPQI-albumin adduct was below the detection limit (below 0.001% modification on albumin) despite a previous report of high concentrations of NAPQI-albumin adduct following acute liver injury. Therefore, the observed APAP-induced skin damage may have had a different cause from APAP-induced liver injury.

Several graphene quantized Hall resistance (QHR) devices manufactured at the National Institute of Standards and Technology (NIST) were compared to GaAs QHR devices and a 100 Ω standard resistor at the National Institute for Advanced Industrial Science and Technology (AIST). Measurements of the 100 Ω resistor with the graphene QHR devices agreed within 5 nΩ/Ω of the values for the 100 Ω resistor obtained through GaAs measurements. The electron density of the graphene devices was adjusted at AIST to restore device properties such that operation was possible at low magnetic flux densities of 4 T to 6 T. This adjustment was accomplished with a functionalization method utilized at NIST, allowing for consistent tunability of the graphene QHR devices with simple annealing. Such a method replaces older and less predictable methods for adjusting graphene for metrological suitability. The milestone results demonstrate the ease with which graphene can be used to make resistance comparison measurements among many National Metrology Institutes.

Imidazole dipeptides, such as carnosine (β-alanyl-l-histidine) and anserine (β-alanyl-N π -methyl-l-histidine), are highly localized in excitable tissues, including skeletal muscle and nervous tissue, and play important roles such as scavenging reactive oxygen species and quenching reactive aldehydes. We have demonstrated several reactions between imidazole dipeptides (namely, carnosine, and anserine) and a lipid peroxide-derived reactive aldehyde 4-oxo-2(E)-nonenal. Seven carnosine adducts and two anserine adducts were characterized using liquid chromatography/electrospray ionization-multiple-stage mass spectrometry. Adduct formation occurred between imidazole dipeptides and 4-oxo-2(E)-nonenal mainly through Michael addition, Schiff base formation, and/or Paal-Knorr reaction. The reactions were much more complicated than the reaction with a similar lipid peroxide-derived reactive aldehyde, 4-hydroxy-2(E)-nonenal., (© 2018 European Peptide Society and John Wiley & Sons, Ltd.)

Candidate drugs that can be metabolically transformed into reactive electrophilic products, such as epoxides, quinones, and nitroso compounds, are of special concern because subsequent covalent binding to bio-macromolecules can cause adverse drug reactions, such as allergic reactions, hepatotoxicity, and genotoxicity. Several strategies have been reported for screening reactive metabolites, such as a covalent binding assay with radioisotope-labeled drugs and a trapping method followed by LC-MS/MS analyses. Of these, a trapping method using glutathione is the most common, especially at the early stage of drug development. However, the cysteine of glutathione is not the only nucleophilic site in vivo; lysine, histidine, arginine, and DNA bases are also nucleophilic. Indeed, the glutathione trapping method tends to overlook several types of reactive metabolites, such as aldehydes, acylglucuronides, and nitroso compounds. Here, we introduce an alternate way for screening reactive metabolites as follows: A mixture of the light and heavy isotopes of simplified amino acid motifs and a DNA motif is used as a biomimetic trapping cocktail. This mixture consists of [ 2 H 0 ]/[ 2 H 3 ]-1-methylguanidine (arginine motif, Δ 3 Da), [ 2 H 0 ]/[ 2 H 4 ]-2-mercaptoethanol (cysteine motif, Δ 4 Da), [ 2 H 0 ]/[ 2 H 5 ]-4-methylimidazole (histidine motif, Δ 5 Da), [ 2 H 0 ]/[ 2 H 9 ]-n-butylamine (lysine motif, Δ 9 Da), and [ 13 C 0 , 15 N 0 ]/[ 13 C 1 , 15 N 2 ]-2'-deoxyguanosine (DNA motif, Δ 3 Da). Mass tag triggered data-dependent acquisition is used to find the characteristic doublet peaks, followed by specific identification of the light isotope peak using MS/MS. Forty-two model drugs were examined using an in vitro microsome experiment to validate the strategy. Graphical abstract Biomimetic trapping cocktail to screen reactive metabolites.

Pyridoxamine (PM) is a promising drug candidate for treating various chronic conditions/diseases in which oxidative stress and carbonyl compounds are important factors affecting pathogenicity. These abilities of PM are mainly attributed to its inhibition of advanced glycation and lipoxidation end product formation, by scavenging reactive carbonyl species. PM might therefore prevent protein damage from lipid hydroperoxide-derived aldehydes such as 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE) by trapping them. It was previously reported that PM reacts with ONE to produce pyrrolo-1,3-oxazine (PO8) through the formation of pyrido-1,3-oxazine (PO1/PO2). In this study, we found that ONE and HNE yield an identical product containing a pyrrole ring (PO7, PH2) upon reaction with PM. The structure of PO7/PH2 was shown by LC-MS and NMR analyses to be 1-(2-hydroxy-6-hydroxymethyl-3-methylpyridin-4-ylmethyl)-2-pentylpyrrole. PO1, PO7/PH2, and PO8 were the main stable PM-ONE/HNE adducts. In the incubation of human serum albumin (HSA) with ONE or HNE, Lys residues provided the most favorable modification sites for both aldehydes, and the number of HNE-modified sites was higher than that of ONE-modified sites. When HSA was allowed to react with a linoleic acid hydroperoxide in the presence of ascorbic acid, ONE modified more residues (10 Lys, 3 His, 2 Arg) than did HNE (8 His, 2 Lys), indicating the relative reactivity of aldehydes towards amino acid residues. Upon treatment with increasing concentrations of PM, the concentrations of ONE-modified HSA peptides, but not of HNE-modified peptides, were reduced significantly and dose-dependently. Concomitantly, the formation of PM-ONE adducts increased in a dose-dependent manner. The inhibition effect of PM was also confirmed in the cell system subjected to oxidative stress. Our results demonstrate that PM can inhibit lipid hydroperoxide-derived damage to proteins by trapping ONE preferentially, and the resulting PM-ONE adducts can be used as a dosimeter for ONE production to determine the levels of lipid peroxidation.

Monoclonal antibody-based therapeutic agents (antibody drugs) have attracted considerable attention as a new type of drug. Concomitantly, the use of quantitative approaches for characterizing antibody drugs, such as liquid chromatography (LC)-mass spectrometry (MS), has increased. Generally, selective quantification of antibody drugs is done using unique peptides from variable regions (V H and V L ) as surrogate peptides. Further, numerous internal standards (ISs) such as stable isotope-labeled (SIL)-intact proteins and SIL-surrogate peptides are used. However, developing LC-MS methodology for characterizing antibody drugs is time-consuming and costly. Therefore, LC-MS is difficult to apply for this purpose, particularly during the drug discovery stage when numerous candidates must be evaluated. Here, we demonstrate an efficient approach to developing a quantitative LC/electrospray ionization (ESI)-selected reaction monitoring (SRM)/MS method for characterizing antibody drugs. The approach consists of the following features: (i) standard peptides or SIL-IS are not required; (ii) a peptide from the homologous monoclonal antibody serves as an IS; (iii) method development is monitored using a spiked plasma sample and one quantitative MS analysis; and (iv) three predicted SRM assays are performed to optimize quantitative SRM conditions such as transition, collision energy, and declustering potential values. Using this strategy, we developed quantitative SRM methods for infliximab, alemtuzumab, and bevacizumab with sufficient precision (<20%)/accuracy (<±20%) for use in the drug discovery stage. We have also demonstrated that choosing a higher homologous peptide pair (from analyte mAb/IS mAb) is necessary to obtain the sufficient precision and accuracy. Graphical abstract ᅟ.

Lipid hydroperoxide-mediated modifications of proteins are receiving increasing attention because of their possible involvement in various degenerative diseases. These biological effects are attributed to the ability of lipid peroxidation-derived aldehydes to react with the nucleophilic sites of proteins. Here we describe a methodology involving metabolic labeling coupled with mass spectrometry-based proteomic analysis that enables global screening of lipid hydroperoxide-mediated protein modifications in a cell system. The lipidome of MCF-7 cells was labeled by incubating the cells with 1.4μM [ 13 C 18 ]-linoleic acid (LA) until the LA to [ 13 C 18 ]-LA ratio became 1:1. This approach was termed SILFAC (stable isotope labeling by fatty acids in cell culture). Analysis of the cellular phospholipids indicated that [ 13 C 18 ]-LA was incorporated quantitatively. The labeled cells were subjected to oxidative stress using a calcium ionophore and l-ascorbic acid, which promote the generation of reactive aldehydes from cellular LA and [ 13 C 18 ]-LA. After protein extraction and digestion with trypsin, isotope pattern dependent MS was used to analyze peptides modified by 1:1 ratios of the 12 C and 13 C aldehyde isomers. Using the current methodology, we identified the major lipid hydroperoxide-mediated modifications to proteins in MCF-7 cells without the need for chemical labeling or further affinity purification., Significance: Lipid peroxidation-derived aldehydes (LPDAs) such as 4-oxo-2(E)-nonenal and 4-hydroxy-2(E)-nonenal can readily react with proteins and peptides to produce a variety of covalent modifications and cross-linkages, resulting in protein dysfunction and altered gene regulation. Various analytical approaches have therefore been developed to detect and characterize protein modifications mediated by LPDAs. However, most of the methods are not specific for LPDA modifications or designed for proteins modified by a target aldehyde. Here we describe the coupling of stable isotope labeling by fatty acids in cell culture (SILFAC) with an isotope pattern dependent MS-based proteomic strategy to provide a global screening tool for the identification of lipid hydroperoxide-mediated protein modifications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Punishment facilitates large-scale cooperation among humans, but how punishers, who incur an extra cost of punishment, can successfully compete with non-punishers, who free-ride on the punisher's policing, poses an evolutionary puzzle. One answer is by coordinating punishment to minimise its cost. Notice, however, that in order to effectively coordinate their punishment, potential punishers must know in advance whether others would also be willing to punish a particular norm violator. Such knowledge might hinder coordination by tempting potential punishers to free-ride on other punishers. Previous research suggests that moral emotions, such as moral outrage and moral disgust, serve as a commitment device and drive people to carry out the costly act of punishment. Accordingly, we tested whether the perception of socially shared condemnation (i.e., knowledge that others also condemn a particular violator) would amplify moral outrage and moral disgust, and diminish empathy for the violator. Study 1 (scenario-based study) revealed that perceived shared condemnation was correlated positively with moral outrage and moral disgust, and negatively with empathy. Study 2 experimentally demonstrated that information indicating that others also condemn a particular norm violation amplified moral outrage. Lastly, Study 3 (autobiographical recall study) confirmed the external validity of the finding.

Japanese apricot liqueur manufacturers are required to control the quality and authenticity of their liqueur products. Citric acid made from corn is the main acidulant used in commercial liqueurs. In this study, we conducted spiking experiments and carbon and hydrogen stable isotope analyses to detect exogenous citric acid used as an acidulant in Japanese apricot liqueurs. Our results showed that the δ 13 C values detected exogenous citric acid originating from C 4 plants but not from C 3 plants. The δ 2 H values of citric acid decreased as the amount of citric acid added increased, whether the citric acid originated from C 3 or C 4 plants. Commercial liqueurs with declared added acidulant provided higher δ 13 C values and lower δ 2 H values than did authentic liqueurs and commercial liqueurs with no declared added acidulant. Carbon and hydrogen stable isotope analyses are suitable as routine methods for detecting exogenous citric acid in Japanese apricot liqueur., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A method for detecting the undeclared addition of acidic ingredients is required to control the authenticity of Japanese apricot liqueur. We developed an analytical procedure that minimizes carbon isotope discrimination for measurement of the δ(13)C values of citric and malic acid isolated from Japanese apricot liqueur. Our results demonstrated that freeze-drying is preferable to nitrogen spray-drying, because it does not significantly affect the δ(13)C values of citric acid and results in smaller isotope discrimination for malic acid. Both 0.1% formic acid and 0.2% phosphoric acid are acceptable HPLC mobile phases for the isolation of citric and malic acid, although the δ(13)C values of malic acid exhibited relatively large variation compared with citric acid following isolation using either mobile phase. The developed procedure allows precise δ(13)C measurements of citric and malic acid isolated from Japanese apricot liqueur., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Proteins are continuously exposed to various reactive chemical species (reactive oxygen/nitrogen species, endogenous/exogenous aldehydes/epoxides, etc.) due to physiological and chemical stresses, resulting in various chemical modifications such as oxidation, nitration, glycation/glycoxidation, lipidation/lipoxidation, and adduct formation with drugs/chemicals. Abundant proteins with a long half-life, such as hemoglobin (Hb, t 1/2 63 days, ∼150 mg/mL), are believed to be major targets of reactive chemical species that reflect biological events. Chemical modifications on Hb have been investigated mainly by mechanistic in vitro experiments or in vivo/clinical experiments focused on single target modifications. Here, we describe an optimized LC/ESI-SRM/MS method to screen oxidized, nitrated, lipidated, and glycated sites on Hb. In vivo preliminary results suggest that this method can detect simultaneously the presence of oxidation (+16 Da) of α-Met(32), α-Met(76), β-Met(55), and β-Trp(15) and adducts of malondialdehyde (+54 Da) and glycation (+162 Da) of β-Val(1) in a blood sample from a healthy volunteer. Graphical Abstract Screening chemical modifications on hemoglobin.

A mass spectrometry (MS)-based proteomic methodology was employed to monitor oxidative modifications in keratins, the main constituents of human skin ("Non-invasive proteomic analysis of human skin keratins: screening of methionine oxidation in keratins by mass spectrometry" [1], "UV irradiation-induced methionine oxidation in human skin keratins: mass spectrometry-based non-invasive proteomic analysis" [2]). Human skin proteins were obtained non-invasively by tape stripping and solubilized in sodium dodecyl sulfate (SDS) buffer, followed by purification and digestion using the filter-aided sample preparation method. The tryptic peptides were then analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-MS, tandem MS (MS/MS), and LC/ESI-selected reaction monitoring (SRM)/MS. The MS/MS data were generated to confirm amino acid sequences and oxidation sites of tryptic peptides D(290)VDGAYMTK(298) (P1) and N(258)MQDMVEDYR(267) (P2), which contain the most susceptible oxidation sites (Met(259), Met(262), and Met(296) in K1 keratin) upon UVA irradiation [2]. Subsequently, quantitative determination of the relative oxidation levels of P1 and P1 [2] was achieved by LC/ESI-SRM/MS analyses of P1 and P2 together with their oxidized forms after exposure to UVA radiation or treatment with hydrogen peroxide (H2O2).

Ultraviolet (UV) radiation is the major environmental factor that causes oxidative skin damage. Keratins are the main constituents of human skin and have been identified as oxidative target proteins. We have recently developed a mass spectrometry (MS)-based non-invasive proteomic methodology to screen oxidative modifications in human skin keratins. Using this methodology, UV effects on methionine (Met) oxidation in human skin keratins were investigated. The initial screening revealed that Met(259), Met(262), and Met(296) in K1 keratin were the most susceptible oxidation sites upon UVA (or UVB) irradiation of human tape-stripped skin. Subsequent liquid chromatography/electrospray ionization-MS and tandem MS analyses confirmed amino acid sequences and oxidation sites of tryptic peptides D(290)VDGAYMTK(298) (P1) and N(258)MQDMVEDYR(267) (P2). The relative oxidation levels of P1 and P2 increased in a time-dependent manner upon UVA irradiation. Butylated hydroxytoluene was the most effective antioxidant for artifactual oxidation of Met residues. The relative oxidation levels of P1 and P2 after UVA irradiation for 48 h corresponded to treatment with 100mM hydrogen peroxide for 15 min. In addition, Met(259) was oxidized by only UVA irradiation. The Met sites identified in conjunction with the current proteomic methodology can be used to evaluate skin damage under various conditions of oxidative stress., Biological Significance: We demonstrated that the relative Met oxidation levels in keratins directly reflected UV-induced damages to human tape-stripped skin. Human skin proteins isolated by tape stripping were analyzed by MS-based non-invasive proteomic methodology. Met(259), Met(262), and Met(296) in K1 keratin were the most susceptible oxidation sites upon UV irradiation. Met(259) was oxidized by only UVA irradiation. Quantitative LC/ESI-SRM/MS analyses confirmed a time-dependent increase in the relative oxidation of target peptides (P1 and P2) containing these Met residues, upon UVA irradiation of isolated human skin. The relative oxidation levels of P1 and P2 along with the current proteomic methodology could be applied to the assessment of oxidative stress levels in skin after exposure to sunlight., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The N-termini of peptides and proteins can be subjected to highly diverse modifications, including acetylation, myristoylation, pyroglutamylation, and epimerization. These modifications affect protein stability, localization, and activity as well as alter the chemical properties of the N-terminus. Oxidative stress is known to induce the direct oxidation of amino acid side chains and peptide backbones in proteins. Alternatively, polyunsaturated fatty acids can be oxidized to lipid hydroperoxides, which further decompose to form highly reactive aldehydes such as 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE). ONE and HNE modify various amino acid residues and induce protein cross-linking. However, there have been few studies on oxidative stress-mediated N-terminal modifications and the resulting functional changes. Our recent studies have reported several novel N-terminal modifications that result in the formation of α-ketoamide, transamination, cyclization, and epimerization. These novel N-terminal modifications are the focus of this review. We also outline recent advances in approaches for N-terminal analysis, which have been developed over the last several decades., (Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Type 1 diabetes (T1D) patients show abnormalities in early B cell tolerance checkpoints, resulting in the accumulation of large numbers of autoreactive B cells in their blood. Treatment with rituximab, an anti-CD20 mAb that depletes B cells, has been shown to preserve β cell function in T1D patients and improve other autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. However, it remains largely unknown how anti-B cell therapy thwarts autoimmunity in these pathologies. Here, we analyzed the reactivity of Abs expressed by single, mature naive B cells from 4 patients with T1D before and 52 weeks after treatment to determine whether rituximab resets early B cell tolerance checkpoints. We found that anti-B cell therapy did not alter the frequencies of autoreactive and polyreactive B cells, which remained elevated in the blood of all patients after rituximab treatment. Moreover, the limited proliferative history of autoreactive B cells after treatment revealed that these clones were newly generated B cells and not self-reactive B cells that had escaped depletion and repopulated the periphery through homeostatic expansion. We conclude that anti-B cell therapy may provide a temporary dampening of autoimmune processes through B cell depletion. However, repletion with autoreactive B cells may explain the relapse that occurs in many autoimmune patients after anti-B cell therapy.

The 1858T protein tyrosine phosphatase nonreceptor type 22 ( PTPN22 T ) allele is one of the main risk factors associated with many autoimmune diseases and correlates with a defective removal of developing autoreactive B cells in humans. To determine whether inhibiting PTPN22 favors the elimination of autoreactive B cells, we first demonstrated that the PTPN22 T allele interfered with the establishment of central B cell tolerance using NOD-scid-common γ chain knockout (NSG) mice engrafted with human hematopoietic stem cells expressing this allele. In contrast, the inhibition of either PTPN22 enzymatic activity or its expression by RNA interference restored defective central B cell tolerance in this model. Thus, PTPN22 blockade may represent a therapeutic strategy for the prevention or treatment of autoimmunity.

Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)