Single-Cell RNA Sequencing Reveals an Immune Landscape of CD4+ T Cells in Coronary Culprit Plaques With Acute Coronary Syndrome in Humans-Brief Report.

BACKGROUND: Acute coronary syndrome (ACS) involves plaque-related thrombosis, causing primary ischemic cardiomyopathy or lethal arrhythmia. We previously demonstrated a unique immune landscape of myeloid cells in the culprit plaques causing ACS by using single-cell RNA sequencing. Here, we aimed to characterize T cells in a single-cell level, assess clonal expansion of T cells, and find a therapeutic target to prevent ACS. METHODS: We obtained the culprit lesion plaques from 4 patients with chronic coronary syndrome (chronic coronary syndrome plaques) and the culprit lesion plaques from 3 patients with ACS (ACS plaques) who were candidates for percutaneous coronary intervention with directional coronary atherectomy. Live CD45+ immune cells were sorted from each pooled plaque samples and applied to the 10× platform for single-cell RNA sequencing analysis. We also extracted RNA from other 3 ACS plaque samples and conducted unbiased TCR (T-cell receptor) repertoire analysis. RESULTS: CD4+ T cells were divided into 5 distinct clusters: effector, naive, cytotoxic, CCR7+ (C-C chemokine receptor type 7) central memory, and FOXP3 (forkhead box P3)+ regulatory CD4+ T cells. The proportion of central memory CD4+ T cells was higher in the ACS plaques. Correspondingly, dendritic cells also tended to express more HLAs (human leukocyte antigens) and costimulatory molecules in the ACS plaques. The velocity analysis suggested the differentiation flow from central memory CD4+ T cells into effector CD4+ T cells and that from naive CD4+ T cells into central memory CD4+ T cells in the ACS plaques, which were not observed in the chronic coronary syndrome plaques. The bulk repertoire analysis revealed clonal expansion of TCRs in each patient with ACS and suggested that several peptides in the ACS plaques work as antigens and induced clonal expansion of CD4+ T cells. CONCLUSIONS: For the first time, we revealed single cell-level characteristics of CD4+ T cells in patients with ACS. CD4+ T cells could be therapeutic targets of ACS. REGISTRATION: URL: https://upload.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000046521; Unique identifier: UMIN000040747.

Chronic stress alters lipid mediator profiles associated with immune-related gene expressions and cell compositions in mouse bone marrow and spleen.

Despite the importance of lipid mediators in stress and depression and their link to inflammation, the influence of stress on these mediators and their role in inflammation is not fully understood. This study used RNA-seq, LC-MS/MS, and flow cytometry analyses in a mouse model subjected to chronic social defeat stress to explore the effects of acute and chronic stress on lipid mediators, gene expression, and cell population in the bone marrow and spleen. In the bone marrow, chronic stress induced a sustained transition from lymphoid to myeloid cells, accompanied by corresponding changes in gene expression. This change was associated with decreased levels of 15-deoxy-d12,14-prostaglandin J2, a lipid mediator that inhibits inflammation. In the spleen, chronic stress also induced a lymphoid-to-myeloid transition, albeit transiently, alongside gene expression changes indicative of extramedullary hematopoiesis. These changes were linked to lower levels of 12-HEPE and resolvins, both critical for inhibiting and resolving inflammation. Our findings highlight the significant role of anti-inflammatory and pro-resolving lipid mediators in the immune responses induced by chronic stress in the bone marrow and spleen. This study paves the way for understanding how these lipid mediators contribute to the immune mechanisms of stress and depression.

Microglial subpopulations with distinct transcriptome signatures vary across brain regions in the resting mouse brain.

Microglia are crucial for tissue homeostasis and its disturbance. However, microglial heterogeneity and its relationship with microglial activation in physiological conditions remain elusive. Using single-cell RNA sequencing, we identified microglial subpopulations with distinct transcriptome signatures in the resting brain. The distribution of two major, continuous subpopulations varied across brain regions, especially between cerebral cortices and the hypothalamus. Lipopolysaccharide and chronic social defeat stress, both of which involve the innate immune receptor TLR4, upregulate the marker genes of selective microglial subpopulations. These findings suggest that microglial subpopulations contribute to the heterogeneity of microglial transcriptome and responsiveness within and across brain regions.

N-Acetylcysteine Suppresses Microglial Inflammation and Induces Mortality Dose-Dependently via Tumor Necrosis Factor-α Signaling.

N-acetylcysteine (NAC) is an antioxidant that prevents tumor necrosis factor (TNF)-α-induced cell death, but it also acts as a pro-oxidant, promoting reactive oxygen species independent apoptosis. Although there is plausible preclinical evidence for the use of NAC in the treatment of psychiatric disorders, deleterious side effects are still of concern. Microglia, key innate immune cells in the brain, play an important role in inflammation in psychiatric disorders. This study aimed to investigate the beneficial and deleterious effects of NAC on microglia and stress-induced behavior abnormalities in mice, and its association with microglial TNF-α and nitric oxide (NO) production. The microglial cell line MG6 was stimulated by Escherichia coli lipopolysaccharide (LPS) using NAC at varying concentrations for 24 h. NAC inhibited LPS-induced TNF-α and NO synthesis, whereas high concentrations (≥30 mM) caused MG6 mortality. Intraperitoneal injections of NAC did not ameliorate stress-induced behavioral abnormalities in mice, but high-doses induced microglial mortality. Furthermore, NAC-induced mortality was alleviated in microglial TNF-α-deficient mice and human primary M2 microglia. Our findings provide ample evidence for the use of NAC as a modulating agent of inflammation in the brain. The risk of side effects from NAC on TNF-α remains unclear and merits further mechanistic investigations.

[Two-staged Operation for Bilateral Giant Bullae Successfully Performed with Veno-venous Extracorporeal Membrane Oxygenation( V-V ECMO) for First Surgery].

57-year-old man with bilateral giant lung bullae was admitted to our hospital for dyspnea on exertion as his chief complaint. The size and shape of right lung bullae were bigger and more complicated than those of left lung bullae. He underwent 2-staged bullectomy by video-assisted thoracoscopic surgery (VATS). Left lung bullectomy was expected to be more safely done, and was performed with veno-venous extracorporeal membrane oxygenation( V-V ECMO) prior to right side without any complications. The right lung bullectomy was performed 6 months after 1st operation without V-V ECMO. These reoperations were necessary due to postoperative air leak, however, his respiratory condition was well maintained by his left lung which had already been remarkably improved.

Identification and characterization of multifunctional cationic and amphipathic peptides from soybean proteins.

In this study, we identified and chemically synthesized three cationic and amphipathic peptides (Glycinin-17, BCAS-16, and BCBS-11) from soybean proteins. These peptides had high isoelectric points, high positive net charges, and included multiple hydrophobic amino acids. Subsequently, we identified multiple functions of these peptides, including antimicrobial, lipopolysaccharide-neutralizing, and angiogenic activities, and examined their cytotoxic activities against mammalian red blood cells. Glycinin-17, BCAS-16, and BCBS-11 exhibited antimicrobial activity against Porphyromonas gingivalis and Candida albicans whereas Glycinin-17 did not possess antimicrobial effects on Propionibacterium acnes and Streptococcus mutans. Membrane-depolarization assays and flow cytometric analyses showed that the antimicrobial properties of Glycinin-17, BCAS-16, and BCBS-11 against P. gingivalis, P. acnes, and S. mutans were dependent on membrane-disrupting potential. In contrast, major antimicrobial activities of these peptides against C. albicans were dependent on interactions with targets other than cell membranes. Furthermore, chromogenic Limulus amebocyte lysate assays showed that 50% effective concentrations (EC50 , 0.12-0.31 µM) of these three peptides neutralize LPS with similar potency (EC50 : 0.11 µM) to that of polymyxin B. Moreover, tube-formation assays in human umbilical vein endothelial cells showed similar angiogenic activities of the three peptides as that following treatment with LL-37. Although BCAS-16 exhibited hemolytic activity, the rate of hemolysis for Glycinin-17 and BCBS-11 in the presence of 500-µM Glycinin-17 and BCBS-11 was less than 2%. These results demonstrate that cationic and amphipathic peptides from soybean proteins, particularly Glycinin-17 and BCBS-11, have potential as multifunctional ingredients for healthcare applications.

Effects of arginine and leucine substitutions on anti-endotoxic activities and mechanisms of action of cationic and amphipathic antimicrobial octadecapeptide from rice α-amylase.

Previously, we showed that the antimicrobial cationic and amphipathic octadecapeptide AmyI-1-18 from rice α-amylase (AmyI-1) inhibited the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli. In addition, we demonstrated that several AmyI-1-18 analogs containing arginine or leucine substitutions, which were designed on the basis of the helical wheel projection of AmyI-1-18, exhibited higher antimicrobial activity against human pathogenic microorganisms than AmyI-1-18. In the present study, anti-inflammatory (anti-endotoxic) activities of five AmyI-1-18 analogs containing arginine or leucine substitutions were investigated. Two single arginine-substituted and two single leucine-substituted AmyI-1-18 analogs inhibited the production of LPS-induced nitric oxide in mouse macrophages (RAW264) more effectively than AmyI-1-18. These data indicate that enhanced cationic and hydrophobic properties of AmyI-1-18 are associated with improved anti-endotoxic activity. In subsequent chromogenic Limulus amebocyte lysate assays, 50% inhibitory concentrations (IC50 ) of the three AmyI-1-18 analogs (G12R, D15R, and E9L) were 0.11-0.13 µm, indicating higher anti-endotoxic activity than that of AmyI-1-18 (IC50, 0.22 µm), and specific LPS binding activity. In agreement, surface plasmon resonance analyses confirmed direct LPS binding of three AmyI-1-18 analogs. In addition, AmyI-1-18 analogs exhibited little or no cytotoxic activity against RAW264 cells, indicating that enhancements of anti-inflammatory and LPS-neutralizing activities following replacement of arginine or leucine did not result in significant increases in cytotoxicity. This study shows that the arginine-substituted and leucine-substituted AmyI-1-18 analogs with improved anti-endotoxic and antimicrobial activities have clinical potential as dual-function host defense agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Characterization and production of multifunctional cationic peptides derived from rice proteins.

Food proteins have been identified as a source of bioactive peptides. These peptides are inactive within the sequence of the parent protein and must be released during gastrointestinal digestion, fermentation, or food processing. Of bioactive peptides, multifunctional cationic peptides are more useful than other peptides that have specific activity in promotion of health and/or the treatment of diseases. We have identified and characterized cationic peptides from rice enzymes and proteins that possess multiple functions, including antimicrobial, endotoxin-neutralizing, arginine gingipain-inhibitory, and/or angiogenic activities. In particular, we have elucidated the contribution of cationic amino acids (arginine and lysine) in the peptides to their bioactivities. Further, we have discussed the critical parameters, particularly proteinase preparations and fractionation or purification, in the enzymatic hydrolysis process for producing bioactive peptides from food proteins. Using an ampholyte-free isoelectric focusing (autofocusing) technique as a tool for fractionation, we successfully prepared fractions containing cationic peptides with multiple functions.

Inhibitory effects of a novel cationic dodecapeptide [CL(14-25)] derived from cyanate lyase of rice on endotoxic activities of LPSs from Escherichia coli and periodontopathic Aggregatibacter actinomycetemcomitans.

OBJECTIVE: CL(14-25), a dodecapeptide of cyanate lyase from rice, is a novel cationic α-helical antimicrobial peptide. In this study, we examined inhibitory ability of CL(14-25) against endotoxic activities of lipopolysaccharides (LPSs) from Escherichia coli and periodontal pathogenic Aggregatibacter actinomycetemcomitans. METHODS: Endotoxin-neutralizing activity of CL(14-25) was evaluated by inhibition to induction of cytokine and nitric oxide in human aortic endothelial cells (HAECs) and RAW264 mouse macrophage cells, respectively. Protective effect of CL(14-25) was determined in mice against lethal toxicity of LPS. RESULTS: IL-6 in HAECs was induced by stimulation with LPS preparations of A. actinomycetemcomitans and E. coli tested in this study, and addition of CL(14-25) to the medium caused inhibition of their induction in a dose-dependent manner. CL(14-25) inhibited NO induction in RAW264 cells by a smooth type LPS of E. coli O55:B5 and an Rc type LPS of E. coli J5 as well as lipid A of E. coli R515 in a dose-dependent manner. Simultaneous injection of E. coli O55:B5 LPS and CL(14-25) in BALB/c mice resulted in prevention of lethal toxicity of the former. The results of a Limulus amebocyte lysate assay and surface plasmon resonance analysis of interaction between CL(14-25) and E. coli LPS or lipid A showed that CL(14-25) specifically binds to a lipid A moiety of LPS. CONCLUSION: The results of present study suggest that CL(14-25) has a potential to be used as a nutraceutical agent for periodontal therapy.

Effect of alanine, leucine, and arginine substitution on antimicrobial activity against candida albicans and action mechanism of a cationic octadecapeptide derived from α-amylase of rice.

AmyI-1-18, an antimicrobial peptide, is a cationic α-helical octadecapeptide derived from α-amylase of rice (Oryza sativa L. japonica) that contains four cationic amino acid residues (two arginines and two lysines). To enhance the antifungal activity of AmyI-1-18 against Candida albicans, 11 analogs bearing substitutions with alanine, leucine, and/or arginine, which were designed on the basis of the helical wheel projection of AmyI-1-18, were synthesized, and their antifungal activity was investigated. The antifungal activities of four analogs obtained by replacing arginine or lysine with alanine were significantly reduced. The results suggested that the cationic arginine and lysine residues in AmyI-1-18 are important for its antifungal activity. The antifungal activities of two single leucine-substituted analogs were not improved, but among three single arginine-substituted analogs, AmyI-1-18(D15R) had approximately a twofold higher antifungal activity [50% growth-inhibitory concentration (IC50 ): 31 µM] than AmyI-1-18 (IC50 : 64 µM) and exhibited low hemolytic activity (4% at 100 µM). Flow cytometric analysis using propidium iodide revealed that the antifungal activity of AmyI-1-18(D15R) was dependent on its membrane-disrupting activity in a manner different from that of AmyI-1-18. Further enhancement of the cationicity and hydrophobicity of AmyI-1-18(D15R) resulted in no improvement in antifungal activity and a significant increase in hemolytic activity. In this study, the results demonstrated that the antifungal activity of AmyI-1-18 against C. albicans was enhanced through increasing its membrane-disrupting activity by replacing aspartic acid at position 15 with arginine without a significant increase in hemolytic activity. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 219-229, 2016.

Rice Bran Protein as a Potent Source of Antimelanogenic Peptides with Tyrosinase Inhibitory Activity.

Rice (Oryza sativa) is consumed as a staple food globally, and rice bran, the byproduct, is an unused biomass that is ultimately discarded as waste. Thus, in the present study, a technique for producing tyrosinase inhibitory peptides from rice bran protein (RBP) was developed. Simultaneous treatment of RBP with chymotrypsin and trypsin produced numerous peptides. Subsequently, six tyrosinase inhibitory peptides were isolated from the hydrolysate fractions in a multistep purification protocol, and their amino acid sequences were determined. Three of these peptides had a C-terminal tyrosine residue and exhibited significant inhibitory effects against tyrosinase-mediated monophenolase reactions. Furthermore, peptide CT-2 (Leu-Gln-Pro-Ser-His-Tyr) potently inhibited melanogenesis in mouse B16 melanoma cells without causing cytotoxicity, suggesting the potential of CT-2 as an agent for melanin-related skin disorder treatment. The present data indicate that RBP is a potent source of tyrosinase inhibitory peptides and that simultaneous treatment of RBP with chymotrypsin and trypsin efficiently produces these peptides.

Antimicrobial activity and mechanism of action of a novel cationic α-helical octadecapeptide derived from α-amylase of rice.

AmyI-1-18, an octadecapeptide derived from α-amylase (AmyI-1) of rice (Oryza sativa L. japonica), is a novel cationic α-helical antimicrobial peptide (AMP) that contains two lysine and two arginine residues. The antimicrobial activity of AmyI-1-18 against human pathogens was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. Of the ten kinds of human pathogens, AmyI-1-18 exhibited antimicrobial activity against nine. Its 50% growth-inhibitory concentrations (ICs50 ) against Porphyromonas gingivalis, Propionibacterium acnes, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans were 13, 19, 50, 64, and 77 µM, respectively. AmyI-1-18 had little or no hemolytic activity even at 500 µM, and showed negligible cytotoxicity up to 1200 µM. The degree of 3,3'-dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of AmyI-1-18 was significantly lower than that induced by the addition of melittin. Flow cytometric analysis showed that the percentages of P. aeruginosa, S. mutans, and C. albicans cells stained with propidium iodide (PI), a DNA-intercalating dye, were 89%, 43%, and 3%, respectively, when AmyI-1-18 was added at a concentration equal to its 4×IC50 . Therefore, the antimicrobial activity of AmyI-1-18 against P. aeruginosa and S. mutans appears to be mainly attributable to its membrane-disrupting activity. In contrast, its antimicrobial activity against P. gingivalis and C. albicans most likely depends upon interactions with intracellular targets other than their cell membranes. Collectively, these results indicate that AmyI-1-18 is useful as a safe and potent AMP against the pathogens described above in many fields of healthcare.

Contribution of cationic amino acids toward the inhibition of Arg-specific cysteine proteinase (Arg-gingipain) by the antimicrobial dodecapeptide, CL(14-25), from rice protein.

CL(14-25), a dodecapeptide, exhibits antimicrobial activity against Porphyromonas gingivalis with the 50% growth-inhibitory concentration (IC50 ) value of 145 µM, and arginine-specific gingipain (Rgp)-inhibitory activity. Kinetic analysis revealed that CL(14-25) is a mixed-type inhibitor, with inhibition constants (Ki and Ki ' values) of 1.4 × 10(-6) M and 4.3 × 10(-6) M, respectively. To elucidate the contributions of four cationic amino acid residues at the N- and C-termini of CL(14-25) toward Rgp-inhibitory activity, we investigated the Rgp-inhibitory activities of truncated and alanine-substituted analogs of CL(14-25). Rgp-inhibitory activities significantly decreased by truncated analogs, CL(15-25) and CL(16-25), whereas those of CL(14-24) and CL(14-23) were almost as high as that of CL(14-25). Rgp-inhibitory activities of alanine-substituted analogs, CL(R14A) and CL(R14A, R15A) also significantly decreased, whereas those of CL(K25A) and CL(R24A, K25A) were higher than that of CL(14-25). These results suggest that the arginine residue at position 15 substantially contributes to the Rgp-inhibitory activity and that the arginine residue at position 14 plays important roles in exerting Rgp-inhibitory activity. In this study, we demonstrated that CL(K25A) was a potent, dual function, peptide inhibitor candidate, exhibiting Rgp-inhibitory activity with Ki and Ki ' of 9.6 × 10(-7) M and 1.9 × 10(-6) M, respectively, and antimicrobial activity against P. gingivalis with an IC50 value of 51 µM.

Effect of substituting arginine and lysine with alanine on antimicrobial activity and the mechanism of action of a cationic dodecapeptide (CL(14-25)), a partial sequence of cyanate lyase from rice.

The antimicrobial activity of analogs obtained by substituting arginine and lysine in CL(14-25), a cationic α-helical dodecapeptide, with alanine against Porphyromonas gingivalis, a periodontal pathogen, varied significantly depending on the number and position of cationic amino acids. The alanine-substituted analogs had no hemolytic activity, even at a concentration of 1 mM. The antimicrobial activities of CL(K20A) and CL(K20A, K25A) were 3.8-fold and 9.1-fold higher, respectively, than that of CL(14-25). The antimicrobial activity of CL(R15A) was slightly lower than that of CL(14-25), suggesting that arginine at position 15 is not essential but is important for the antimicrobial activity. The experiments in which the alanine-substituted analogs bearing the replacement of arginine at position 24 and/or lysine at position 25 were used showed that arginine at position 24 was crucial for the antimicrobial activity whenever lysine at position 25 was substituted with alanine. Helical wheel projections of the alanine-substituted analogs indicate that the hydrophobicity in the vicinity of leucine at position 16 and alanines at positions 18 and/or 21 increased by substituting lysine at positions 20 and 25 with alanine, respectively. The degrees of diSC3 -5 release from P. gingivalis cells and disruption of GUVs induced by the alanine-substituted analogs with different positive charges were not closely related to their antimicrobial activities. The enhanced antimicrobial activities of the alanine-substituted analogs appear to be mainly attributable to the changes in properties such as hydrophobicity and amphipathic propensity due to alanine substitution and not to their extents of positive charge (cationicity).

Crystal structure of α-amylase from Oryza sativa: molecular insights into enzyme activity and thermostability.

AmyI-1 is an α-amylase from Oryza sativa (rice) and plays a crucial role in degrading starch in various tissues and at various growth stages. This enzyme is a glycoprotein with an N-glycosylated carbohydrate chain, a unique characteristic among plant α-amylases. In this study, we report the first crystal structure of AmyI-1 at 2.2-Å resolution. The structure consists of a typical (ß/α)8-barrel, which is well-conserved among most α-amylases in the glycoside hydrolase family-13. Structural superimposition indicated small variations in the catalytic domain and carbohydrate-binding sites between AmyI-1 and barley α-amylases. By contrast, regions around the N-linked glycosylation sites displayed lower conservation of amino acid residues, including Asn-263, Asn-265, Thr-307, Asn-342, Pro-373, and Ala-374 in AmyI-1, which are not conserved in barley α-amylases, suggesting that these residues may contribute to the construction of the structure of glycosylated AmyI-1. These results increase the depths of our understanding of the biological functions of AmyI-1.

Intense impact of IL-1ß expressing inflammatory macrophages in acute aortic dissection.

There is no treatment for acute aortic dissection (AAD) targeting inflammatory cells. We aimed to identify the new therapeutic targets associated with inflammatory cells. We characterized the specific distribution of myeloid cells of both human type A AAD samples and a murine AAD model generated using angiotensin II (ANGII) and ß-aminopropionitrile (BAPN) by single-cell RNA sequencing (scRNA-seq). We also examined the effect of an anti-interleukin-1ß (IL-1ß) antibody in the murine AAD model. IL1B+ inflammatory macrophages and classical monocytes were increased in human AAD samples. Trajectory analysis demonstrated that IL1B+ inflammatory macrophages differentiated from S100A8/9/12+ classical monocytes uniquely observed in the aorta of AAD. We found increased infiltration of neutrophils and monocytes with the expression of inflammatory cytokines in the aorta and accumulation of inflammatory macrophages before the onset of macroscopic AAD in the murine AAD model. In blocking experiments using an anti-IL-1ß antibody, it improved survival of murine AAD model by preventing elastin degradation. We observed the accumulation of inflammatory macrophages expressing IL-1ß in both human AAD samples and in a murine AAD model. Anti-IL-1ß antibody could improve the mortality rate in mice, suggesting that it may be a treatment option for AAD.

A 50-kilodalton Cry2A peptide is lethal to Bombyx mori and Lymantria dispar.

The Cry2Aa3 gene was introduced into asporogenic Bacillus thuringiensis, and the synthesized protoxin killed Bombyx mori and Lymantria dispar larvae. Chymotrypsin hydrolyzed the linkages between 49Tyr/Val50 and 145Lys/Ser146 in the protoxin, and 50- and 58-kDa fragments were generated, respectively. Both peptides killed the larvae of both insects.

Selective concentration of antimicrobial peptides to heat-treated porous silica gel using adsorption/desorption.

Heat-treated porous silica gel (HT silica gel) previously developed by our group has selectively adsorbed cationic peptides at a pH of 7. Therefore, we focused on the use of antimicrobial peptides (AMPs) as bioactive peptides (BPs). First, 32 AMPs and 32 randomly designed peptides were generated using Fmoc solid synthesis, and their adsorption ratio to HT-silica gel was investigated. Thirty two AMPs showed a relatively higher adsorption ratio of 58.8% compared to that of randomly designed peptides, which was 35.3%. Desorption conditions were investigated using Amyl-1-18 antimicrobial peptides. Next, pepsin hydrolysate from rice endosperm protein (REP) powder was prepared by ourselves. The REP hydrolysate containing dry matter (7.5 mg) was applied to the adsorption/desorption (AD) procedure using HT silica gel to obtain 1.6 mg of AD hydrolysate. When the two hydrolysates were subjected to mass spectrometry, 305 concentrated peptides were obtained. In total, 26 peptides with high content and high enrichment ratios were listed and synthesized. When the antimicrobial activity of these 26 peptides was evaluated using Cutibacterium acnes, five peptides consisting of 12-27 amino acids were identified as novel AMPs. Two of these peptides, which were derived from rice glutelin, showed antimicrobial activity against all four microbes, including Porphyromonas gingivalis, Escherichia coli, and Streptococcus mutans. In the present study, we showed that AMPs could be easily enriched from protein hydrolysate using HT silica gel. The adsorption/desorption procedure using HT silica gel was confirmed to be a useful tool for convenient BP separation.

Memantine ameliorates the impairment of social behaviors induced by a single social defeat stress as juveniles.

Clinically, juveniles are more sensitive to stress than adults, and exposure to stress as juveniles prolongs psychiatric symptoms and causes treatment resistance. However, the efficacy of antidepressants for juveniles with psychiatric disorders is unknown. In the present study, we investigated whether the expression or development of impaired social behavior was attenuated by memantine, a non-competitive NMDA receptor antagonist. In addition, we clarified the molecular mechanisms related to intracellular signal transduction through NMDA receptors and the ameliorating effect of memantine in mice with impaired social behavior. Acute administration of memantine before the social interaction test, but not before exposure to social defeat stress, attenuated social behavioral impairment. A single social defeat stress increased the phosphorylation of NMDA receptor subunit GluN2A and extracellular-signal-related kinase 1/2 (ERK1/2). Memantine inhibited the increase of phosphorylated GluN2A and ERK1/2 resulting from social interaction behavior. In both GluN2A deficient and pharmacological blockaded mice, social behavioral impairment was not observed in the social interaction test through regulation of ERK1/2 phosphorylation. These findings suggest that memantine ameliorates social behavioral impairment in mice exposed to a single social defeat stress as juveniles by regulating the NMDA receptor and subsequent ERK1/2 signaling activation. Memantine may constitute a novel therapeutic drug for stress-related psychiatric disorders in juveniles with adverse juvenile experiences.