Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Co-generation of palladium nanoparticles and phosphate supported on metal-organic frameworks as hydrogenation catalysts.

In this study, we demonstrated the direct synthesis of sodium dihydrogen phosphate (PA) containing palladium nanoparticles (PdNPs) supported on a metal-organic framework (MOF). The resulting composite containing PA molecules coexisting with PdNPs demonstrated improved hydrogenation catalytic performance compared to the composites without PA.

Explicit and implicit attitudes toward smoking: Dissociation of attitudes and different characteristics for an implicit attitude in smokers and nonsmokers.

Smoking is a global health risk for premature death and disease. Recently, addictive behaviors, like smoking, were considered to be guided by explicit and implicit processes. The existence of a dissociation between the two attitudes in nonsmokers and the causes of the differences in implicit attitudes toward smoking have not been fully investigated. We investigated the explicit and implicit attitudes toward smoking via a self-reported scale and the single category implicit association test (SC-IAT), respectively, among undergraduate and graduate health sciences students. In addition, we applied the drift-diffusion model (DDM) on the SC-IAT and examined the behavioral characteristics that caused differences in implicit attitude toward smoking between smokers and nonsmokers. The results showed the existence of a dissociation between explicit and implicit attitudes toward smoking among nonsmokers. In addition, nonsmokers had a higher decision threshold than smokers and a higher drift rate in the condition where negative words were associated with smoking. Nonsmokers engaged in SC-IAT with more cautious attitudes and responded more easily in a negative condition since it was consistent with their true attitudes. Conversely, smokers did not show a significant difference in the drift rate between the conditions. These results suggested that the differences in an implicit attitude between smokers and nonsmokers were caused by differences in evidence accumulation speed between the positive and negative conditions. The existence of dissociation between implicit and explicit attitudes toward smoking may indicate the difficulty of measuring true attitude in nonsmokers in a questionnaire survey. Additionally, the DDM results explained the difference of implicit attitude between smokers and nonsmokers; it may provide information on the mechanisms of addictive behaviors and a basis for therapy. However, whether these results are affected by cultural differences requires further investigation.

Semi-automated brain responses in communication: A magnetoencephalographic hyperscanning study.

Face to face communication is interactive, and involves continuous feedforward and feedback of information, thoughts, and feelings to the opposite party. To accurately assess the neural processing underlying these interactions, synchronous and simultaneous recording of the brain activity from both parties is needed, a method known as hyperscanning. Here, we investigated the neural processing underlying nonverbal face-to-face communication using a magnetoencephalographic (MEG) hyperscanning system, comprising two fiber optically connected MEGs. Eight pairs of subjects participated. Each individual in each pair viewed a combined 80 randomized 20 s trials of 40 real-time and 40 recorded (hereafter, real and simulated, respectively) videos of the opposite party's face. Non-verbal communication through actions such as gaze, eye blinks, and facial expression was intrinsically only possible during real videos. After each trial, subjects individually subjectively discriminated whether the viewed video was real or simulated. Overall subjective discrimination accuracies were slightly but significantly above chance level. Statistical analysis of brain activity revealed a significant three way interaction between theta-band rhythm amplitude, video type, and subjective discrimination response in the right frontal cortex. Additionally, when subjects responded that videos were simulated, theta activity was significantly lower for real videos compared with simulated videos (p = 0.01). This result not only demonstrates the importance of right frontal theta activity during non-verbal communication, but also indicates the existence of unconscious, semi-automated neural processing during non-verbal communication that underlies one's ability to subjectively discriminate whether or not the opposite party is real.

Functional decline of the precuneus associated with mild cognitive impairment: Magnetoencephalographic observations.

Mild Cognitive Impairment (MCI) is a border or precursor state of dementia. To optimize implemented interventions for MCI, it is essential to clarify the underlying neural mechanisms. However, knowledge regarding the brain regions responsible for MCI is still limited. Here, we implemented the Montreal Cognitive Assessment (MoCA) test, a screening tool for MCI, in 20 healthy elderly participants (mean age, 67.5 years), and then recorded magnetoencephalograms (MEG) while they performed a visual sequential memory task. In the task, each participant memorized the four possible directions of seven sequentially presented arrow images. Recall accuracy for beginning items of the memory sequence was significantly positively related with MoCA score. Meanwhile, MEG revealed stronger alpha-band (8-13 Hz) rhythm desynchronization bilaterally in the precuneus (PCu) for higher MoCA (normal) participants. Most importantly, this PCu desynchronization response weakened in correspondence with lower MoCA score during the beginning of sequential memory encoding, a time period that should rely on working memory and be affected by declined cognitive function. Our results suggest that deactivation of the PCu is associated with early MCI, and corroborate pathophysiological findings based on post-mortem tissue which have implicated hypoperfusion of the PCu in early stages of Alzheimer disease. Our results indicate the possibility that cognitive decline can be detected early and non-invasively by monitoring PCu activity with electrophysiological methods.

The role of the ventromedial prefrontal cortex in automatic formation of impression and reflected impression.

Previous neuroimaging studies demonstrated that ventromedial prefrontal cortex (vmPFC) activity reflects how much an individual positively views each person (impression). Here, we investigated whether the degree to which individuals think others positively view them (reflected impression) is similarly tracked by activity in the vmPFC by using fMRI and speed-dating events. We also examined whether activity of the vmPFC in response to the faces of others would predict the impression formed through direct interactions with them. The task consisted of three sessions: pre-speed-dating fMRI, speed-dating events, and post-speed-dating fMRI (not reported here). During the pre-speed-dating fMRI, each participant passively viewed the faces of others whom they would meet in the subsequent speed-dating events. After the fMRI, they rated the impression and reflected impression of each face. During the speed-dating events, the participants had 3-min conversations with partners whose faces were presented during the fMRI task, and they were asked to choose the partners whom they preferred at the end of the events. The results revealed that the value of both the impression and reflected impression were automatically represented in the vmPFC. However, the impression fully mediated the link between the reflected impression and vmPFC activity. These results highlight a close link between reflected appraisal and impression formation and provide important insights into neural and psychological models of how the reflected impression is formed in the human brain.

Focused attention meditation training modifies neural activity and attention: longitudinal EEG data in non-meditators.

Focused attention meditation (FAM) is a basic meditation practice that cultivates attentional control and monitoring skills. Cross-sectional studies have highlighted high cognitive performance and discriminative neural activity in experienced meditators. However, a direct relationship between neural activity changes and improvement of attention caused by meditation training remains to be elucidated. To investigate this, we conducted a longitudinal study, which evaluated the results of electroencephalography (EEG) during three-stimulus oddball task, resting state and FAM before and after 8 weeks of FAM training in non-meditators. The FAM training group (n = 17) showed significantly higher P3 amplitude during the oddball task and shorter reaction time (RT) for target stimuli compared to that of the control group (n = 20). Furthermore, a significant negative correlation between F4-Oz theta band phase synchrony index (PSI) during FAM and P3 amplitude during the oddball task and a significant positive correlation between F4-Pz theta band PSI during FAM and P3 amplitude during the oddball task were observed. In contrast, these correlations were not observed in the control group. These findings provide direct evidence of the effectiveness of FAM training and contribute to our understanding of the mechanisms underpinning the effects of meditation on brain activity and cognitive performance.

Appearance and modulation of a reactive temporal-lobe 8-10-Hz tau-rhythm.

Spontaneous 8- to 10-Hz "tau-rhythm" in magnetoencephalographic (MEG) recordings has been reported to originate in the auditory cortex and be suppressed by sound. For unknown reasons however, tau-rhythm is often difficult to detect. In this study, we sought to characterize its emergence and auditory reactivity. Using a 306-channel MEG on 26 right-handed participants, we delivered six-second-long, natural, monaural sounds with pleasant, unpleasant, or neutral emotional valence. In eight participants, a clear, sound-related bilateral suppression of 8-10 Hz tau-rhythm occurred in the temporal areas, close to the source of the 100-ms auditory response. Moreover, these eight "tau subjects" exhibited significantly larger temporal-lobe theta-band (4-8 Hz) power over the entire experimental period compared to the remaining 18 "non-tau subjects". As it is known that larger theta power is one of signs of drowsiness, this result is consistent with a previously proposed idea that tau-rhythm emerges during drowsiness. Tau-rhythm was furthermore significantly affected by emotional valence in the right hemisphere, where it was respectively suppressed by unpleasant and neutral sounds 8% and 6% more than by pleasant sounds, significantly. Altogether, our results reveal characteristics of tau-rhythm appearance and modulation which have hitherto been difficult to detect non-invasively.

WT1 peptide-based immunotherapy for advanced thymic epithelial malignancies.

Thymic epithelial tumors are rare malignancies, and no optimal therapeutic regimen has been defined for patients with advanced disease. Patients with advanced thymic epithelial tumors, which were resistant or intolerable to prior therapies, were eligible for this study. Patients received 9 mer-WT1-derived peptide emulsified with Montanide ISA51 adjuvant via intradermal administration once a week as a monotherapy. After the 3-month-protocol treatment, the treatment was continued mostly at intervals of 2-4 weeks until disease progression or intolerable adverse events occurred. Of the 15 patients enrolled, 11 had thymic carcinoma (TC) and 4 had invasive thymoma (IT). Median period from diagnosis to the start of treatment was 13.3 and 65.5 months for TC and IT, respectively. No patients achieved a complete or partial response. Of the 8 evaluable TC patients, 6 (75.0%) had stable disease (SD) and 2 had progressive disease (PD). Of the 4 evaluable IT patients, 3 (75.0%) had SD and 1 (25.0%) had PD. Median period of monotherapy treatment was 133 and 683 days in TC and IT patients, respectively. No severe adverse events occurred during the 3-month-protocol treatment. As adverse events in long responders, thymoma-related autoimmune complications, pure red cell aplasia and myasthenia gravis occurred in two IT patients. Cerebellar hemorrhage developed in a TC patient complicated with Von Willebrand disease. Induction of WT1-specific immune responses was observed in the majority of the patients. WT1 peptide vaccine immunotherapy may have antitumor potential against thymic malignancies.

Autistic traits modulate the activity of the ventromedial prefrontal cortex in response to female faces.

Previous findings have revealed abnormal visual attention or processing of faces among individuals with autism spectrum condition (ASC). However, little attention has been paid to the relationship between autistic traits and neural mechanisms associated with representing facial values. Using fMRI, we investigated the patterns of brain activity in the vmPFC and VS in response to faces of elderly males, elderly females, young males, and young females. During fMRI, subjects with a relatively high autism quotient (high group) and those with a relatively low autism quotient (low group) were presented with a face and asked to rate its pleasantness. After fMRI, the subjects were presented with pairs of faces and asked to select the face that they preferred. Our results indicate a dissociable modulatory effect of autistic traits on the vmPFC and VS: The vmPFC activity in the low group was more sensitive to age differences in female faces compared to that in the high group, whereas VS activity did not show differences between groups. These results suggest that, in the BVS, autistic traits selectively modulate the vmPFC activity associated with facial value representation.

Association of WT1 IgG antibody against WT1 peptide with prolonged survival in glioblastoma multiforme patients vaccinated with WT1 peptide.

We previously evaluated Wilms' tumor gene 1 (WT1) peptide vaccination in a large number of patients with leukemia or solid tumors and have reported that HLA-A*24:02 restricted, 9-mer WT1-235 peptide (CYTWNQMNL) vaccine induces cellular immune responses and elicits WT1-235-specific cytotoxic T lymphocytes (CTLs). However, whether this vaccine induces humoral immune responses to produce WT1 antibody remains unknown. Thus, we measured IgG antibody levels against the WT1-235 peptide (WT1-235 IgG antibody) in patients with glioblastoma multiforme (GBM) receiving the WT1 peptide vaccine. The WT1-235 IgG antibody, which was undetectable before vaccination, became detectable in 30 (50.8%) of a total of 59 patients during 3 months of WT1 peptide vaccination. The dominant WT1-235 IgG antibody subclass was Th1-type, IgG1 and IgG3 . WT1-235 IgG antibody production was significantly and positively correlated with both progression-free survival (PFS) and overall survival (OS). Importantly, the combination of WT1-235 IgG antibody production and positive delayed type-hypersensitivity (DTH) to the WT1-235 peptide was a better prognostic marker for long-term OS than either parameter alone. These results suggested that WT1-235 peptide vaccination induces not only WT1-235-specific CTLs as previously described but also WT1-235-specific humoral immune responses associated with antitumor cellular immune response. Our results indicate that the WT1 IgG antibody against the WT1 peptide may be a useful predictive marker, with better predictive performance in combination with DTH to WT1 peptide, and provide a new insight into the antitumor immune response induction in WT1 peptide vaccine-treated patients.

Induction of eEF2-specific antitumor CTL responses in vivo by vaccination with eEF2-derived 9mer-peptides.

Eukaryotic elongation factor 2 (eEF2) is an essential factor for protein synthesis. Previous studies have shown that the eEF2 gene was overexpressed and plays an oncogenic role in various types of cancers and that eEF2 gene product elicited both humoral immune responses to produce eEF2-specific IgG autoantibody in cancer-bearing individuals and cellular immune responses to induce eEF2 peptide-specific cytotoxic T lymphocytes (CTLs) in vitro. The purpose of the present study was to induce eEF2-specific, antitumor CTL responses in vivo by vaccination with MHC class I-binding eEF2-derived peptide. First, two mouse MHC class I-restricted eEF2­derived, 9-mer peptides, EF17 (17-25 aa, ANIRNMSVI) and EF180 (180-188 aa, RIVENVNVI) were identified as eEF2-specific CTL peptides, and mice were vaccinated intradermally eight times with either EF17 or EF180 peptide emulsified with Montanide ISA51 adjuvant. Cytotoxicity assay showed that eEF2-specific CTLs were induced in both EF17­and EF180­vaccinated mice, and histological study showed no detectable damage in the organs of these mice. Next, to examine in vivo antitumor effects of eEF2 peptide vaccination in a therapeutic model, mice were vaccinated four times with one each of the two eEF2 peptides at weekly intervals after implantation of eEF2-expressing leukemia cells. The vaccination with eEF2 peptides induced eEF2-specific CTLs and suppressed tumor growth, and disease-free survival was significantly longer in EF180-vaccinated mice compared to control mice. The survival was associated with the robustness of eEF2-specific CTL induction. These results indicate that vaccination with MHC class I-binding eEF2 peptide induced eEF2-targeting, antitumor CTL responses in vivo without damage to normal organs, which provided us a rationale for eEF2 peptide-based cancer immunotherapy.

Neural activity associated with enhanced facial attractiveness by cosmetics use.

Previous psychological studies have shown that make-up enhances facial attractiveness. Although neuroimaging evidence indicates that the orbitofrontal cortex (OFC) shows greater activity for faces of attractive people than for those of unattractive people, there is no direct evidence that the OFC also shows greater activity for the face of an individual wearing make-up than for the same face without make-up. Using functional magnetic resonance imaging (fMRI), we investigated neural activity while subjects viewed 144 photographs of the same faces with and without make-up (48 with make-up, 48 without make-up, and 48 scrambled photographs) and assigned these faces an attractiveness rating. The behavioral data showed that the faces with make-up were rated as more attractive than those without make-up. The imaging data revealed that the left OFC and the right hippocampus showed greater activity for faces with make-up than for those without make-up. Furthermore, the activities of the right anterior cingulate cortex, left hippocampus, and left OFC increased with increasing facial attractiveness resulting from cosmetics use. These results provide direct evidence of the neural underpinnings of cosmetically enhanced facial attractiveness.

The translation elongation factor eEF2 is a novel tumor­associated antigen overexpressed in various types of cancers.

Recent studies have shown that cancer immunotherapy could be a promising therapeutic approach for the treatment of cancer. In the present study, to identify novel tumor-associated antigens (TAAs), the proteins expressed in a panel of cancer cells were serologically screened by immunoblot analysis and the eukaryotic elongation factor 2 (eEF2) was identified as an antigen that was recognized by IgG autoantibody in sera from a group of patients with head and neck squamous cell carcinoma (HNSCC) or colon cancer. Enzyme-linked immunosorbent assay showed that serum eEF2 IgG Ab levels were significantly higher in colorectal and gastric cancer patients compared to healthy individuals. Immunohistochemistry experiments showed that the eEF2 protein was overexpressed in the majority of lung, esophageal, pancreatic, breast and prostate cancers, HNSCC, glioblastoma multiforme and non-Hodgkin's lymphoma (NHL). Knockdown of eEF2 by short hairpin RNA (shRNA) significantly inhibited the growth in four eEF2-expressing cell lines, PC14 lung cancer, PCI6 pancreatic cancer, HT1080 fibrosarcoma and A172 glioblastoma cells, but not in eEF2-undetectable MCF7 cells. Furthermore, eEF2-derived 9-mer peptides, EF786 (eEF2 786-794 aa) and EF292 (eEF2 292-300 aa), elicited cytotoxic T lymphocyte (CTL) responses in peripheral blood mononuclear cells (PBMCs) from an HLA-A*24:02- and an HLA-A*02:01-positive healthy donor, respectively, in an HLA-A-restricted manner. These results indicated that the eEF2 gene is overexpressed in the majority of several types of cancers and plays an oncogenic role in cancer cell growth. Moreover, the eEF2 gene product is immunogenic and a promising target molecule of cancer immunotherapy for several types of cancers.