Background: Damaged mitochondria is removed by autophagy.  This process includes engulf of mitochondria by autophagosomes and degradation of autophagosomes by lysosomes.  We recently reported that activation of SIRT1, a protein deacetylase, promotes autophagosome degradation and reduces damaged mitochondria in the heart of a mouse model of muscular dystrophy.  Here, we examined how SIRT1 participates in mitochondrial autophagy (mitophagy) in the cardiomyocyte.  

Methods and Results: Mitophagy was induced by CCCP (20 μM), a mitochondrial uncoupler, in H9c2 cardiomyocytes. Western blotting showed that levels of succinate dehydrogenase B and cyclophilin F, mitochondrial proteins, were reduced by CCCP. These reductions in mitochondrial proteins were significantly blocked by siRNA-mediated SIRT1 knockdown (KD). CCCP increased level of LC3-II, an autophagosome marker; however, LC3-II level was rather increased in SIRT1 KD cells, suggesting a role of SIRT1 in autophagosome degradation. Mitophagosomes defined as autophagosomes (LC3 dots) including fragmented mitochondria (Tomm20) in immunostaining were increased by CCCP. In contrast, SIRT1 KD promoted accumulation of mitophagosomes compared with control cells, suggesting disturbance of mitophagosome clearance. Finally, CCCP-induced autophagosome-lysosome fusion analyzed by colocalization of LC3 dot and LAMP1, a lysosome marker, was significantly suppressed by SIRT1 KD. 

Conclusion: These findings suggest that SIRT1 plays a role in mitophagy at autophagosome-lysosome fusion in cardiomyocytes.

Hyaluronan (HA) is one of the main components of the extracellular matrix. HA synthase (HAS) and hyaluronidase (HYAL) isoforms have been shown to influence malignant potential in cancer cells. Formation of a cohesive multicellular group has been reported to facilitate cancer progression, leading to distant metastasis without apoptosis. Previously, we demonstrated that the HAS inhibitor 4-methylumbelliferone (4-MU) inhibits cell proliferation and mobility. In this study, we used the canine mammary tumor cell line AZACB to investigate whether inhibiting HA production reduces cancer spheroid formation and induces apoptosis. In AZACB cells cultured under standard conditions, 4-MU decreased HA production, cell proliferation, and mobility, and increased Bim expression as an apoptosis marker. In addition, 4-MU inhibited the expression of HAS2 and the HA receptor RHAMM. The plastic ware was coated with poly (2-hydroxyethyl methacrylate) (poly-HEMA) to obtain a low adhesive scaffold. Cells cultured on the poly-HEMA-coated plastic ware exhibited spheroid formation without altering Bim expression. Addition of 4-MU decreased cell viability and increased Bim expression and the number of the annexin V/PI-positive (AP) cells. Moreover, exogenously applied HYAL decreased the number of spheroids and increased the number of AP cells. HA is likely necessary for spheroid formation and thus apoptosis evasion in cancer cells, suggesting that HA production could be a possible pharmacological target for tumors.

Cancer cells obtain anaerobic glycolysis and metabolic abnormalities due to reduced mitochondrial function. We have found that guaiazulene affects the metabolism of the nematode C. elegans and cultured mammalian cells. We investigated whether azulene derivatives synthesized from guaiazulene as a precursor have more effective interference with metabolism.

Adding azulene derivatives to cultured mammalian cells, cell growth was suppressed in immortalized cells and cancer cells, but not normal fibroblast. This result suggests that azulene derivatives affect cells with increased metabolic activity. Next, we investigated whether azulene derivative-treated cells did not proliferate due to cell cycle arrest or apoptosis. Whereas the treatment of azulene derivatives did not significantly change the cell cycle in flow cytometry, cleaved PARP was increased in HeLa cells in a time-dependent manner. These results indicate that the azulene derivative induces apoptosis instead of cell cycle arrest. Moreover, we observed the mitochondrial status using MitoProbe^TM JC-1. HeLa cells treated with azulene derivative were observed to undergo mitochondrial depolarization. Our findings indicate that azulene derivatives reduce mitochondrial function to induce apoptosis. We are analyzing where azulene derivatives act in the metabolic pathway.

The prenylated Rab acceptor 1 (PRA1) superfamily member PRAF3 plays crucial roles in membrane traffic as a GDI displacement factor via physical interaction with a variety of Rab proteins, as well as in the modulation of antioxidant glutathione through its interaction with EAAC1.  It is known that the toxicity of the host cell is induced by the overexpression of PRAF3, however, the factors capable of cancelling the cytotoxicity remained unknown.  Our findings demonstrate that Rab1a can protect from the toxicity of PRAF3-overexpressed cells.  Protective effects of Rab1a protein against the cytotoxicity could further suggest that PRAF3 and Rab1a are closely related to each other physiologically and genetically.

Neural stem cells (NSCs) play important roles in neurogenesis since they have self-renewal ability and pluripotentiality to differentiate into neurons and glial cells. Recently, it has been reported that differentiation of NSCs is regulated by endogenous metabolites. Therefore, we hypothesized that efflux transporters such as ATP-binding cassettes (ABCs) may regulate function of NSCs via excretion of metabolites. We first examined mRNA expression of ABCs in primary cultured NSCs derived from murine embryonic cortex: Quantitative PCR was performed at 3, 6, and 9 days after the primary culture. Abcb1b, Abcg2, Abcc1, Abcc4, and Abcc5 were expressed in the NSCs. Among them, expression of Abcc5 was the highest and increased in a culture period-dependent manner, whereas that of other ABCs was decreased. We also characterized the NSCs by evaluating mRNA expression of basic helix-loop-helix (bHLH) transcription factors which regulate neuronal differentiation. Especially, expression of Math1 and Mash1, activators of neuronal differentiation were increased, whereas that of Hes1 and Hes5, suppressors of neuronal differentiation were decreased during the culture period. Thus, some of the xenobiotic efflux transporters may be associated with differentiation of NSCs, and further studies are required to understand detailed regulatory mechanisms.

It is well known that some drugs cause pulmonary toxicities, such as interstitial pneumonia and it is important to minimize the risk of the drug-induced respiratory diseases for patient safety. However, it is difficult to obtain human lung cells and culture the cells for a long-term period to explore the mechanistic approach for the adverse effects. In this study, we tried to induce alveolar epithelial type II (AT2) cells from human induced pluripotent stem cells (iPSCs) using two-dimensional culture method. Differentiation was performed by mainly two steps; the first step was to generate lung progenitor cells and the second step was to induce AT2 cells from lung progenitor cells. The differentiated cells were collected, extracted RNA, and characterized by quantitative real-time PCR. We found that the differentiated cells from human iPSCs expressed AT2 cell markers, such as surfactant protein C, surfactant protein B, ATP binding cassette subfamily A member 3, and solute carrier family 34 member 2, suggesting the cells exhibit AT2-like properties. We are currently working on drug-induced pulmonary toxicities using AT2 cells.

Intestinal analysis has been usually performed using established cell lines or primary cells in 2D culture. However, these culture systems can not satisfy the complexity of 3D structure and diversity of composed cell types in the intestinal epithelial tissue.

Here, we report the generation of intestinal organoids using human iPS cells (iPSCs) by sequential treatment with various cytokines and compounds. We observed that almost cells were double positive for the definitive endoderm markers SOX17 and FOXA2 at day 3 of differentiation. The expression of CDX2, a marker of the mid/hindgut, was upregulated at day 7 of differentiation, and floating and semi-adherent spheroids were positive for CDX2. Within several days after floating spheroids were embedded in Matrigel and incubated in intestinal growth medium, round organoids were observed at day 21. Immunocytochemical analysis revealed that these organoids consisted of monolayer cells, which were positive for intestinal markers E-cadherin (ECAD) and KLF5. In addition, RT-qPCR analysis revealed that multiple epithelial cell markers, LGR5 (intestinal stem cells), VIL1 (enterocytes), MUC2 (goblet cells) and LYZ (paneth cells) were upregulated on day 21.

These data suggest that human iPSCs are successfully differentiated into intestinal organoids consisting of epithelial monolayers.

Muscle-resident stem cells called muscle satellite cells (MuSC) play an essential role in muscle regeneration. Mechanosensation is presumed to be critical for activation of MuSCs, but the molecular entity that determines the cell fate in MuSCs through converting the mechanical stimuli into biochemical signals remains to be elucidated. Here we identify PIEZO1, a mechanosensitive ion channel that is activated by membrane tension, as a critical determinant for activation of MuSCs. In silico analysis demonstrates that PIEZO1 is predominantly expressed in MuSCs but not in mature myofibers. By utilizing Piezo1-tdTomato mice where endogenous PIEZO1 is fused with a fluorescent protein tdTomato, our immunofluorescent analysis reveals that PIEZO1 is accumulated to the cleavage furrow during cell division of MuSCs. Moreover, a conditional deletion of Piezo1 leads to delayed myofibers regeneration after cardiotoxin-induced myofiber injury, at least in part due to the cell division delay in MuSCs. Thus, our results indicate that PIEZO1 is a bona fide mechanosensor whose ion channel activity is required for completion of cell division in MuSCs.

Triple-negative breast cancer (TNBC) is a highly aggressive cancer with fewer effective targeted therapy. Since growing evidence suggests that TNBC is originated from breast cancer stem cells (BCSCs), it is required to elucidate the molecular mechanism of BCSC proliferation for new drug development. We have previously reported that a lipid mediator lysophosphatidic acid (LPA) increased BCSCs via Ca²⁺ signaling pathway. In this study, we examined whether calcineurin/NFAT pathway is involved in the LPA-induced increase in BCSCs. We found that LPA stimulation increased the transcriptional activation of NFAT. The calcineurin inhibitor cyclosporine A inhibited both LPA-increased NFAT activation and increase in BCSCs. We next examined the downstream signaling pathway. To identify NFAT target gene which is involved in the LPA-induced increase in BCSCs, we performed RNA-sequencing using MDA-MB-231 cells. We identified that 428 transcripts were upregulated by LPA by two fold or more. Among them, we focused on proinflammatory cytokine IL-8 which promoter contains NFAT consensus sequence. We found that LPA increased IL-8 production in MDA-MB-231 cells. In addition, a selective IL-8 receptor antagonist inhibited the LPA-induced increase in BCSCs. These results suggested that LPA increases BCSC through the NFAT-mediated IL-8 production.

Drug-induced cardiotoxicity is critical in the non-clinical testing. Applications of iPS-derived cardiomyocytes (iPS-CMs) hold great promise as a human cell-based platform. To date, multielectrode array (MEA) system has been widely used as a standardized assay to detect proarrhythmia risk with iPS-CM. In addition, evaluation of inotropic effects in vivo is recognized as a safety pharmacology in drug development. Given its impact on drug development, it should be useful to detect the drug-induced effects on contractility in vitro. In the present study, we used the cell motion imaging system (CMI) and the measurement of cell-induced electrical impedance (IMP) for the contractility assessment. We confirmed the effects of isoprenaline and verapamil using these systems. Simultaneous recording of CMI and IMP showed clear correlation between CMI and IMP. Our results suggest that both CMI and IMP can monitor the contraction movement of iPS-CMs.

Evaluation of developmental toxicology has been an integral part of safety assessment issue for new compounds. One of the basic ideas behind developmental toxicity tests is that children differ significantly from adults in some aspects of their basic biology and responses to compound exposures.

Because current developmental neurotoxicity (DNT) guideline (OECD TG426) requires a lot of animals and costs, it is necessary to establish more predictable approach using human iPS cells (hiPSCs). Here, we tried to search suitable structural and functional endpoints by evaluating antifouling agent, such as tributyltin (TBT), as a positive compound using hiPSCs, which was expected to provide novel human cell-based applications for alternative in vitro testing approaches.

We focused on neural differentiation process (Structure) using hiPSCs. TBT reduced the expression of several genes, including OTX2, a marker of neurogenesis. We further focused on electrophysiological properties (Function) using hiPSC-derived neurons. TBT reduced the number of spikes and network burst neurons using microelectrode array (MEA) recordings. These data suggest that TBT inhibited neural differentiation from iPSCs and spontaneous firing of neurons. Our data indicate that integrated analyses using iPSCs and iPSC-derived neurons are useful for DNT assessment.

Human iPSC-derived neurons are expected to be applied to toxicity evaluations in nonclinical studies and drug screening. Microelectrode array (MEA) measurement system is suitable to evaluate the neuronal electrophysiological responses to drugs. We have previously reported the electrophysiological responses to convulsants using MEA in cultured hiPSC-derived neurons. In this study, we aimed to develop an analytical method enabling the evaluation of toxicity and the classification of MoA of convulsants using multivariate analysis and deep learning. hiPSC-derived cerebral cortical neurons were cultured on Micro-electrode array (MEA) plate, and the pharmacological responses over 10 drugs in spontaneous firings were obtained. We firstly constructed the raster plots of spontaneous firing and the divided image data. The 4096 feature vectors of the divvied image data in raster plots were extracted by pre-trained model. Next, CNN model was trained with feature vectors each drug name. Using this trained CNN model, we have succeeded in separating the responses between non-convulsive drugs and convulsants, and classifying the MoA of convulsants. This deep learning methods are useful for the prediction of seizure liability and the classification of MoA of new drugs.

Extracting teeth of patients treated with bisphosphonate (BP) occasionally induces the necrosis of jaw, but the cause of disease is still unclear. I have proved that the BPs taken into osteoblastic cells were gradually accumulated in lysosomes. In the present study, I investigated the mechanism of BP-induced cytotoxicity in osteoblast, focusing on mitochondria. MC3T3-E1 cells were used as osteoblastic cells. The uptake of BP into cells was observed by fluorescent BP. The intracellular reactive oxygenspecies (ROS) were evaluated by CM-H₂-DCFDA. Detection of autophagy and mitophagy was used DALGreen and mtphagy dye, respectively. The intracellular Ca²⁺ and mitochondrial Fe²⁺ were measured by Fluo 4-AM and Mito-FerroGreen, respectively. Zoledronate (ZD) impaired cells dose-dependently. BP taken into cells was accumulated into lysosomes. MC3T3-E1 cells were always occurred autophagy flux, but bafilomycin A1(BM), a lysosome inhibitor induced cell death, by inhibiting autophagy flux. ZD slightly suppressed the autophagy flux, however the combination of BM and ZD strongly enhanced cell death. ZD decreased intracellular Ca²⁺and mitochondrial Fe²⁺, and inhibited the response of intracellular ROS generation by oxidative stress, resulting in promotion of mitophagy. These results suggest that BP may form the chelate with Ca²⁺and Fe²⁺, and promote mitophagy of damaged mitochondria. Furthermore, the accumulation of BP into lysosomes indicates to induce cell death by inhibiting the autophagy flux.

Clioquinol, extensively used as an amebicide to treat indigestion and diarrhea in the mid-1900s, was withdrawn from the market due to an increase in the incidence of subacute myelo-optic neuropathy (SMON). Yet, the pathogenesis of SMON has not been fully elucidated. Since clioquinol is known as a chelator and ionophore of copper and zinc ions, we focused on proteins involved in homeostasis of these metal ions. A global analysis on human neuroblastoma cells demonstrated that among 4 isoforms of metallothionein (MT), a family of metal-binding proteins, 7 subclasses of MT-1 and MT-2A were remarkably up-regulated by clioquinol. Clioquinol-induced up-regulation of SLC30A1 (zinc exporter ZNT1) was further verified by quantitative PCR. Up-regulation of these proteins suggested that clioquinol activated metal regulatory transcription factor 1 (MTF1)-dependent transcription. We alsoexamined antioxidant 1 (ATOX1), a copper chaperone which has a redox-sensitive metal binding motif and is known to promote neuronal survival. Monitoring the redox state of ATOX1 showed clioquinol-induced thiol oxidization, possibly resulting in the inactivation of ATOX1. Collectively, dyshomeostasis of copper and zinc may be involved in the neurotoxicity of clioquinol.

Cigarette smoke is divided in tar phase containing nicotine and gas phase. The gas phase of cigarette smoke is prepared by passing cigarette smoke through Cambridge filter. The tar phase is extracted from Cambridge filter by 2-propanol. We have previously elucidated that the gas phase induce cell death by intracellular Ca²⁺- and protein kinase C (PKC)-dependent manner, and identified acrolein and methyl vinyl ketone as the major cytotoxic compounds in the gas phase (Mai et al., 2012; Noya et al., 2013; Higashi et al., 2014). In this study, we examined molecular mechanism(s) for cigarette smoke tar phase-induced cell death in lung cancer cells. Lung adenocarcinoma, small cell carcinoma, and non-small cell carcinoma cell lines are all sensitive to cigarette smoke tar phase. Tar phase-induced cell death is intracellular Ca²⁺- and PKC-independent, whereas intracellular Ca²⁺ chelator and PKC inhibitor effectively suppressed gas phase-induced cell death. These results indicate that the molecular mechanisms for cell death induction by cigarette tar phase is different from that of cigarette smoke gas phase.

To detect adverse effects of toxic substances on neurons, we quantitated neuron number, dendrite length and synaptic status of cultured neurons. An actin-binding protein, drebrin accumulated in the postsynaptic sites of glutamatergic synapses and a tubulin-binding protein, MAP2 were used as markers to detect synaptic changes and to visualize neuronal cell body and dendrites, respectively. We have applied this method for high-throughput analysis and showed that glutamate treatment for 10 min significantly reduced drebrin cluster density of 21-days-in-vitro (DIV) neurons in a dose-dependent manner. In this study, we examined the effects of other toxic substances. Treatment of 0.5-50 µM latrunculin A, which sequesters monomeric actin, for 5 min significantly reduced drebrin cluster density of 21-DIV neurons in a dose-dependent manner. We also confirmed that exposure of 1 Gy X-irradiation to 1-DIV neurons reduces neuron number, dendrite length and drebrin cluster density in the neurons at 21-DIV. In addition, our analysis could efficiently detect staurosporine-induced neuronal cell death in mature neurons. 24 hours exposure of 0.3 and 1.0 µM staurosporine to 21-DIV neurons significantly reduced neuron number. These results suggest that our high-content imaging analysis is useful for analyzing the effects of various toxic substances.

The developing brain is extremely sensitive to many chemicals. Various screening methods have been used to assess the developmental neurotoxicity (DNT) of chemicals. However, assessment of toxicity during progenitor cell differentiation into neurons, astrocytes, and oligodendrocytes often requires immunohistochemistry, which is a reliable but labor-intensive and time-consuming assay. Here, we report the development of a triple-transgenic zebrafish line that expresses distinct fluorescent proteins in neurons (Cerulean), astrocytes (mCherry), and oligodendrocytes (mCitrine), which can be used to detect DNT during neuronal differentiation. Using in vivo fluorescence microscopy, we could detect DNT by 6 of the 10 neurotoxicants tested after exposure to zebrafish from 12 h to 5 days' post-fertilization. Moreover, the chemicals could be clustered into three main DNT groups based on the fluorescence pattern: (i) inhibition of neuron and oligodendrocyte differentiation and stimulation of astrocyte differentiation; (ii) inhibition of neuron and oligodendrocyte differentiation; and (iii) inhibition of neuron and astrocyte differentiation, which suggests that reporter expression reflects the toxicodynamics of the chemicals. Thus, the triple-transgenic zebrafish line developed here may be a useful tool to assess DNT during neuronal differentiation.

In vitro Microelectrode array (MEA) assay systems using human iPSC-derived neurons and rodent primary cultured cells are expected to be useful for drug discovery and pre-clinical studies for toxicity and efficacy. However, as experimental problems, there are problems that the sample is likely to aggregate, it takes time until functional maturation, and dispersion culture has random structure and does not reflect the tissue structure. This is particularly remarkable for iPSC-derived neurons. In addition, since the actual state of nerve function is not yet elucidated, the fact that evaluation parameters are not established also contributes to difficulty.

As one of the methods to solve these problems, we are working on the construction of an evaluation method in which neurons are cultured on an oriented nanofiber device (NFD). When human iPSC-derived central neurons were cultured on NFD, it was found that aggregation was suppressed and synchronous burst firing, which is an indicator of maturation, was detected early. Since this NFD forms a neural network along the fiber, it can give direction to activity propagation in the network. When excitatory drugs acting on synapses were administered, the propagation speed in the network changed. The change in propagation speed reflects synaptic function, suggesting that it is useful as a drug efficacy evaluation parameter. In addition, rat DRG neurons, which are peripheral nerves, were cultured on NFD and measured by CMOS-MEA. As a result, we succeeded in measuring the axonal conduction of one cell along the fiber in multiple points. A change in conduction speed due to drug administration was detected, suggesting that it is also effective in evaluating peripheral neurotoxicity such as axon disorder.

In this study, we examined to develop evaluation system to judge degree of risk of the process of dependence for the purpose of the prevention of the dependence using questionnaire methods because we would like to reveal the tendency of various addiction and the psychological characteristic in undergraduate students.

For 167 university students (male:64, female:103), we carried out this study with the approval of the Saitama Prefectural University Ethical Review Board (No.28095). For this study, we examined association and the correlation with the addiction tendency using two kinds of following originals (dependence recognition test, dependence risk degree test) and existing evaluation four kinds (GHQ-30, STAI, SACL, RSES).

As a result, the correlation with some psychological characteristics considered to participate in the process of dependence was accepted by around 9-10 questions paper. In addition, from the result according to the group of the addiction tendency, the possibility that an associated psychological characteristic varied according to a difference for the addiction was shown. Thus, the possibility as the evaluation standard that we detected an addiction tendency than the above, and could clarify degree of risk of the process of dependence was shown.

[Background] Magnesium oxide is an osmotic laxative widely used for treatment of constipation in children and the elderly. However, it has been administered indiscriminately, and side effects have been reported. [Methods] Using medical records, the present study investigated children and the elderly who had been prescribed magnesium oxide, in order to clarify the associated risk factors and side effects. Children aged 0-14 years and elderly patients aged 65 years or older who received magnesium oxide were studied. Univariate analysis was carried out, and the risk factors of side effects were investigated. Fisher's exact test was performed to calculate the odds ratios. [Results] Children who developed side effects were significantly younger than those who did not. It was also clarified that lean children were 6.5 times more likely to develop side effects than normal to obese children (P<0.05). On the other hand, elderly individuals who developed side effects had significantly higher Cr and BUN levels than those who did not. Fisher's exact test also revealed that patients with low body weight, poor renal function, and a history of hyperuricemia had higher risks of developing hypermagnesemia than those who did not (P<0.05). [Conclusion] These results suggest that younger children have a higher risk of developing side effects when taking magnesium oxide preparations, whereas elderly individuals with a lower body weight and poor renal function are also more at risk.

In present study, we aimed to investigate the feasibility of machine-learning-based classification using clinical features of patients for risk predication of anesthesia-related anaphylaxis.

After data pre-processing, the performance of four classification methods, which were integrated with four feature selection methods, were evaluated using two-layer cross-validation. Linear Discriminate Analysis in conjunction with Recursive Feature Elimination presented the best performance, with accuracy of 0.867 and Matthews correlation coefficient of 0.558 with 25 features used in the classification.

This study presents initial proof of the capability of a machine-learning-based strategy for forecasting low-prevalence anesthesia-related anaphylaxis. In future, we plan to utilize an extended database including preoperative information and vital-sign streams to define personalized risk status for anaphylaxis.

A microdose (MD) of antibody drugs for clinical study is still not defined by any guidance.

In this survey, we studied retrospectively on the starting dose in the first in human (FIH) clinical trials of antibody drugs. We survey 52approved antibody drugs that were listed in the National Institute of Health Sciences' website as of October, 2018. We used the database survey of Pharmaceuticals and Medical Devices Agancy (PMDA) web homepage and reviewed the information from package insert, interview form, and review report. We defined the lowest dose of a human-equivalent dose of no-observed-adverse-effect levels (NOAEL) calculated from nonclinical studies as NHD and the smallest start dose treated for clinical trial as FHD. NHD in 10 items of the antineoplastic drugs was unknown as a toxicity appeared at the minimum dose set in toxicity studies. FHD in 3 items was less than 100 micrograms. FHD is normally selected and assumed the lower than the dose expected not to exert any pharmacological actions. The MD is also expected to be lower than the effect level. When NHD is unknown, 100 micrograms seems to be low enough for MD dose of antibody.

Type 2 diabetes mellitus (T2DM) is a risk factor for depression. Since brain insulin resistance plays a potential role in depression, the future risk of depression in patients with T2DM may be altered depending on the class of oral hypoglycemic agent (OHA) used for T2DM therapy. The aim of the present study was to determine if specific classes of OHAs are associated with a risk for co-morbid depression in T2DM. Japanese adult patients with T2DM (n = 40,214) were divided into a case group (with depression; n = 1,979) and control group (without depression; n = 38,235). After adjustment for age [adjusted odds ratio (AOR) for 10 years: 1.03; 95% confidence interval (CI): 0.99 – 1.07; P = 0.1211], sex [AOR for female: 1.39; 95% CI: 1.26 – 1.53; P < 0.0001], hemoglobin A1c [AOR for 1.0%: 1.18; 95% CI: 1.11 – 1.26; P < 0.0001], duration of T2DM [AOR for 1 year: 1.00; 95% CI: 0.99 – 1.01; P = 0.4089], and history of seven medical conditions, the odds ratios for the development of depression was significantly lower for dipeptidyl peptidase-4 (DPP-4) inhibitors [AOR: 0.31; 95% CI: 0.24 – 0.42; P < 0.0001]. However, there was no significant association for the other classes of OHAs. Therefore, this study finds that there is less risk of depression associated with the use of DPP-4 inhibitors for the treatment of T2DM.

Therapeutic drugs have been classified based on pharmacodynamics and disease indications. However, it has gradually been revealed that profiling of side effects can be used to classify therapeutic drugs and to find novel disease indications of drugs. In this study, we generated multidimensional vectors for each therapeutic drug based on the cosine similarity of indications or side effects in the US FDA Adverse Event Reporting System (FAERS). Using the spatial density, the multidimensional vectors were clustered based on the indications and side effects in FAERS. By comparing these clusters, we were able to identify several sets of therapeutic drugs that were common in the two clusterings, including a few sets comprising of therapeutic drugs with different pharmacodynamics and different disease indications. These findings suggest that clustering therapeutic drugs based on similarities of indications and side effects reported in public databases can be useful to find new functions of therapeutic drugs.

In recent years, medical care by multidisciplinary team has become increasingly important in order to achieve higher quality medical care. Educational institutions those train for medical professionals are also introducing interprofessional education (IPE) to help students understand the need for team practice. In 2018, we started IPE in collaboration with St. Marianna University School of Medicine and Showa Pharmaceutical University, and we conducted a questionnaire survey on students and faculty members to clarify student responses and future challenges.

For the 4th year students from both universities, the IPE program held a small group discussion using simulated cases, followed by a presentation. At the end of the IPE, questionnaire surveys were conducted on students and faculty members in charge of IPE on the day, and responses were compiled.

This survey gave a positive response regarding the implementation and content of IPE and the addition of undergraduate students other than medicine and pharmacy. On the other hand, 33% answered "difficult" regarding the difficulty level of the task. It is not easy to conduct IPE, but 89% of respondents answered that they understood the importance of collaborate in multidisciplinary team. It is necessary to continue IPE and improve the program, though creating an educational program is challenging.

1. Background

Japan Radioisotope Association is an organization promoting the beneficial use of radioisotopes to contribute to the development of science and technology in Japan. Facing a decreasing trend of the use of radioisotope (RI) reagents, we conducted customer surveys. The result shows there is a potential demand in the fields of molecular imaging and cancer research. It is also found that the services providing information and supplying short-lived RI are required to encourage the use in these fields. We carried out further investigation to know what information and which nuclides the researchers in these fields need.

2. Information service

We conducted a questioner survey to know what type of information is needed. Based on the result of the survey, we created the user's guides about 1) usage of RI in the field of molecular imaging, 2) fundamental method of life-science experiment using RI and 3) safety handling of RI reagents in cooperation with the experts.

3. Supply of short-lived RI

We carried out another survey about the needs for the short-lived RI. The result shows there is an increasing interest in short-lived RI such as Cu-67, Zr-89, I-124, At-211, Ra-223, and Ac-225. To date, we have established the supply routes of these short-lived RI products except for At-211.

4. Future development

As a future development, we are planning the service to introduce the news and reviews on molecular images and cancer therapy researches. Also, we will continue the survey on the customer's needs for the information and short-lived RI to meet the demand timely.

Purpose

This study aims to obtain suggestions on factors affecting the total score of evaluation tools based on the results of pretests for the development of a medication adherence assessment tools focusing on the autonomy of the elderly.

Methods

A survey using an anonymous self-administered questionnaire was conducted on 30 nurses working at acute hospitals and home-visit nursing stations. Recalling a case of elderly patients aged 65–74 having difficulties in self-management of medications, and another case in which self-management was successful, the subjects were asked to answer the same questions. The survey comprised the personal attributes of the recalled patient and 91 questions regarding autonomous adherence. A t-test or one-way analysis of variance for total scores and background factors was performed.

Results

Based on the responses of 19 nurses (recovery rate, 63.3%), we obtained data for a total of 38 cases (19 in the difficult group and 19 in the appropriate group). The total medication adherence score significantly reduced with the cognitive function of the elderly. However, it was not associated with gender, age, number and types of medications and doses taken per day, medication management method, and the use of psychiatric and external medications.

Conclusion

Cognitive function was shown to influence medication adherence. Hence, its utility as a scale to measure management ability based on patient autonomy was shown. Further evaluation of reliability and validity as a medication adherence assessment tool through an expanded survey is needed.

Histamine N-methyltransferase (HNMT), which inactivates histamine to 1-methylhistamine, plays an important role in the regulation of histamine concentration and brain functions. Although previous studies indicated the possible involvement of neurons and astrocytes in brain histamine inactivation, the contribution of each cell type to the histamine inactivation was not fully elucidated. In the present study, we phenotyped astrocytes-specific Hnmt knockout mice (cKO) to reveal the importance of astrocytic histamine inactivation for brain functions. First, we generated cKO mice by crossing Hnmt flox mice and Gfap-Cre mice which expressed Cre recombinase specifically in astrocytes. Increase in brain histamine concentration of cKO mice was modest compared to that of conventional Hnmt knockout mice, indicating the limited contribution of astrocytes to histamine metabolism. Behavioral test battery showed the lower locomotor activity of cKO mice in novel environment and home cages, although anxiety-like behaviors and depression-like behavior were not changed by Hnmt deletion in astrocytes. These results demonstrated that astrocytic Hnmt maintained normal locomotor activity despite of its minor role in histamine clearance.

Depression is the major mental disorder characterized by the decrease of motivation, interest and activity and over one million patients are suffering from this disease. It was also reported that the number of patients showing resistance toward anti-depressant, i.g. SSRI and SNRI, got increase nowadays. We have already known that molecular mechanism underlying depression is heterogeneous so that it is hard to estimate the efficacy of anti-depressant depends without molecular rationale. Postmortem human brain analysis indicated that the number of AMPA receptors (AMPARs), major molecule controlling synaptic functions, varied among depression patients compared to healthy subjects and these results were not consistent. To clarify the dynamics of AMPARs in depression patients, we developed the PET (positron emission tomography) imaging drug to measure the density of AMPARs in depression patients. This result showed that depression patient decreased AMPARs expression broadly throughout the brain. This fact motivated us to develop novel AMPARs potentiator in order to cure the depression. To find the compound showing high affinity to AMPARs and high BBB penetratability, we modified the compound A, previously known to bind specifically to and activate AMPARs, and finally succeeded in synthesizing the seed compound B, showing higher BBB penetratability compared with compound A. This compound B could exert the anti-depressant effect quickly and sustained for a week after withdrawal from repetitive one-week administration. Furthermore, this anti-depressant effect was significantly stronger that another AMPARs potentiators already under development in clinical trials.

Quinolinic acid phosphoribosyltransferase (QPRT) metabolizes quinolinic acid (QA) to nicotinamide adenine nucleotide (NAD⁺) via kynurenine pathway. QA is a excitotoxic substance that activate N-methyl-D-aspertate (NMDA) receptors and NAD⁺ is essential for cell survival. In this study, we evaluated QPRT knock out (KO) mice to explore the physiological role of QPRT in central nervous system. QPRT KO mice demonstrated motor deficits (decrease of locomotor activity, decrease of duration time to maintain balance on the rotarod, wide stance in footprint pattern test) and cognitive deficits (decrease of spontaneous alternation behavior in Y-maze test, and prolongation of latency to enter the target hole in the Barnes-maze test). But emotional change was not observed except for decrease in number of buried marbles in marble burying test. Dopaminergic dysfunction was observed in prefrontal cortex, nucleus accumbens and striatum of QPRT KO mice. Dopamine D₁ receptor agonist (SKF81297)-induced hyperactivity is not observed in QPRT KO mice. Dopamine D₂ receptor antagonist (raclopride)-induced catalepsy is more sensitive in QPRT KO mice. The activation of dopaminergic function by methylphenidate attenuated the impairment of short-term memory and hypoactivity of QPRT KO mice. QPRT KO mice showed increased level of QA in serum but normal level of NAD+ in brain. QA-mediated NMDA receptor signaling (phosphorylation of CaMK2 and activation of calpain) and oxidative stress were enhanced in prefrontal cortex, nucleus accumbens and striatum of QPRT KO mice. These results suggested that deficiency of QPRT lead motor and cognitive deficits associated with dopaminergic dysfunction via QA-induced calpain activation and oxidative stress.

Oxytocin (OXT) is a peptide hormone synthesized in the hypothalamic paraventricular nucleus. OXT has been reported to be involved in regulation of learning and memory performance. However, there is no report that shows the effect of OXT on the amyloid-beta (Aβ)-induced impairment of synaptic plasticity. Here, we examined whether OXT have effects on the Ab-induced impairment of synaptic plasticity in mice.

Methods: Male ddY mice were used. To investigate the effect of OXT on synaptic plasticity, we prepared acute hippocampal slice for extracellular recording, and assessed long-term potentiation (LTP) with Aβ_25-35 perfusion in the absence and presence of OXT.

Results: In the present study, we found that OXT recovered the LTP impaired by perfusion of Aβ_25-35 in the mouse hippocampus. These effects were blocked by the pretreatment with a selective OXT receptor antagonist L-368,899. Further, the pretreatment with an ERK inhibitor U0126 and a selective Ca²⁺-permeable AMPA receptor antagonist NASPM were completely antagonized the effects of OXT, respectively.

Conclusion: These results suggested that OXT recovered Aβ–induced impairment of hippocampal synaptic plasticity through the OXT receptors in the mice. We proposed that ERK phosphorylation and Ca²⁺-permeable AMPA receptors are involved in these effects of OXT.

The involvement of reactive oxygen species (ROS) in psychiatric disorders has been reported. However, the source of ROS has not been identified yet. NADPH oxidase is a superoxide-generating enzyme composed of multiple subunits including a membrane-spanning catalytic subunit, NOX. We investigated the role of NOX1/NADPH oxidase in the anxiety-like behavior using mice deficient in Nox1(NOX1-KO).

When anxiety-like behaviors were evaluated in elevated plus-maze and open field tests, no difference in anxiety levels was observed between wild-type mice (WT) and NOX1-KO. Increased anxiety-like behavior was demonstrated in WT subjected to two hour-restraint stress, but it was markedly ameliorated in NOX1-KO. Delivery of miRNA against NOX1 to the hypothalamus suppressed the anxiety-like behavior in WT. An increase in oxidative stress induced by restraint stress was blunted in the hypothalamus of NOX1-KO. Concomitantly, elevated levels of plasma ACTH as well as corticotropin-releasing hormone (CRH) and c-fos mRNA in the hypothalamus were significantly attenuated in NOX1-KO subjected to restraint stress. In hypothalamic slice cultures, the increase in CRH mRNA induced by a protein kinase A activator, forskolin, was suppressed in NOX1-KO. Moreover, the levels of phosphorylated CREB in the hypothalamus caused by stress were ameliorated in NOX1-KO.

Taken together, NOX1/NADPH oxidase appear to play a key role in stress-induced anxiety,possibly by regulating activation of the PKA-CREB pathway in the hypothalamus.

Current animal behavioral studies have much room for improvement. Data-driven drug discovery research and AI research using big data have become hot topics in human clinical research. Radiotelemetry provides an alternative means of obtaining physiological measurements from awake and freely moving laboratory animals, without introducing stress artifacts. For researchers, especially those in the fields of pharmacology and toxicology, the technique may provide a valuable tool for predicting the effectiveness and safety of new compounds in humans. The current embedded type sensor has a built-in battery and is therefore large, and cannot be individually identified, so that simultaneous measurements cannot be made in multiple animals at the same time. We developed a compact telemetry system using a new electromagnetic power supply system. The new system can continuously measure long-term biometric data such as locomotor activity and body temperature in a plurality of individually identified mice. The excellent feature of this device is that biometric data can be measured by five individuals over a long period without changing the sensor embedded in the body. The newly-developed technology is an important tool for the stress-free collection of these physiologic data in small rodents, including mice.

Exposure to pesticides can induce neurobehavioral effects in humans, as well as in other mammals, including rodents. However, the effects of the toxicity of pesticides on the central nervous system (CNS) remain largely unclear. We previously developed Arc-promoter-driven luciferase transgenic (Tg) mouse strains for non-invasive monitoring of the neuronal-activity-dependent gene expression in mouse brain under physiological and pathological conditions. In this study, we examined the effects of glufosinate-ammonium (GLA), one of herbicides used in a variety of crops, on neuronal activity using Arc-Luc Tg mice and detected a decrease in bioluminescence signal at juvenile stage after chronic treatment with GLA. Next, we performed transcriptome analysis of primary cultured neurons and identified differentially expressed genes related to axonal guidance signaling between GLA-treated and saline-control neurons. Linked to these results, we further found disturbance of synapse formation after low dose exposure to GLA. Our results provide valuable evidence to understand the mechanistic basis for the effect of GLA on the CNS.

Very recently, the immunotherapies against cancer, autoimmune diseases, and infection have been feasible and promising. Thus, we have examined the possibility whether or not human gamma delta T cells can be applied for the novel immunotherapies. We previously established the cells stably maintaining NFkB-driven human secreted embryonic alkaline phosphatase (SEAP) expression. The cells can be used to determine the transcription activity of NFkB with high-standard dynamic range and accuracy. Because IL-18 is a kind of cytokines that enhances cytotoxicity and activity of human gamma delta T cells through NFkB activation, we have focused on the activity and signaling of IL-18. In this study, we modified the previous reporter cell that can determine the transcription activity of NFkB to express two subunits consisted of human IL-18 receptor. The modified cells secreted SEAP in response to treatment with human recombinant IL-18 in a concentration-dependent manner. We also observed the concentration-dependently enhancement of NFkB activity in the cells treated with mouse recombinant IL-18 although the affinity was lower compared to human recombinant IL-18. We also previously established the cells stably expressing and secreting human recombinant IL-18 and then validated whether or not the conditioned medium from the cells activate NFkB transcription activity using this assay. We demonstrated drug screening using number of extracts derived from marine bacteria and synthetic compounds.

Ca²⁺-sensing receptor (CaSR) belongs to family C of G protein-coupled receptors and is activated by the endogenous agonists such as Ca²⁺. Stimulation of CaSR expressed in vascular endothelial cells through the increase in extracellular Ca²⁺ concentration ([Ca²⁺]_o) is reported to induce vasorelaxation via the production of nitric oxide (NO). The purpose of the present study is to characterize the CaSR-mediated NO production in human vascular endothelial cells. In human endothelial EA.hy926 cells, the increase in [Ca²⁺]_o from 0.2 to 2 mM induced a concentration-dependent increase in intracellular Ca²⁺ concentration, which was significantly inhibited by NPS 2143 (a CaSR antagonist) and YM-254890 (a G_q/11 protein inhibitor). Stimulation with 2 mM Ca²⁺ for 4 h elicited an increase in the phosphorylation level of eNOS at Ser¹¹⁷⁷, which was significantly depressed by NPS 2143, YM-254890, and removal of Ca²⁺ from the medium. Ca²⁺ (2 mM) induced an increase in NO production, which was inhibited by NPS 2143, YM-254890, removal of Ca²⁺ from the medium, and L-NAME (a competitive eNOS inhibitor). These results provide evidence that activation of CaSR with extracellular Ca²⁺ facilitates NO release from human vascular endothelial cells via a G_q/11 protein-eNOS-dependent pathway.

Large-conductance Ca2+-activated K+ (BKCa) channels are expressed in vascular smooth muscle cells and regulate the membrane excitability. Activation of BKCa channels following membrane depolarization and/or cytosolic [Ca2+] increase causes membrane hyperpolarization and subsequent cytosolic [Ca2+] decrease. Therefore, BKCa channels are recognized as a key factor for the negative feedback regulation of vascular tone. Pimaric acid is a common resin acid naturally contained in pine rosin. We previous reported that pimaric acid activated BKCa channels and slightly blocked voltage-dependent Ca2+ channels. However, the effects of pimaric acid on contractile response of vascular smooth muscles are still unclear. In the present study, we examined the effects of pimaric acid on contraction of pulmonary artery. Pulmonary arteries were isolated from male Sprague-Dawley rats and contracted with high K+ solution. The high K+-induced contraction was reduced by pimaric acid in a concentration-dependent manner (1-100 mM). Quantitative real-time PCR data revealed that the a and b1 subunits of BKCa channel were highly expressed in human pulmonary arterial smooth muscle cells. These results indicate that pimaric acid enhances the activity of BKCa channels and results in the relaxation of pulmonary arterial smooth muscles.

Melatonin is synthesized in and secreted from the pineal gland as a neurohormone. Secreted melatonin regulates the circadian rhythm. It has been reported that melatonin acts on ion channels directly, or indirectly via the melatonin receptors. The voltage-dependent potassium channel family is expressed in the most types of tissues and contributed to the physiological functions including the regulation of resting membrane potential and the formation of action potential. In this study, the effects of melatonin on voltage-dependent potassium (Kv4.2) channels were analyzed by whole-cell patch clamp techniques. In HEK293 cells stably expressed with Kv4.2 channels, outward currents with fast activation and inactivation were observed by membrane depolarization to +100 mV from the resting potential of -80 mV for 500 ms. The outward currents were clearly reduced by the application of 1 mM melatonin and partly recovered by wash-out. Quantitative real-time PCR data revealed that Kv4.2 channels were highly expressed in pineal glands from the rats. These results suggest that melatonin regulates the activity of Kv4.2 channels in pineal glands, potentially contributing to the regulation of circadian rhythm.

Adverse Outcome Pathway (AOP) is developed for the prediction of the adverse effects. Epithelial-mesenchymal transition (EMT) plays an important role in the acquisition of cancer stem cell (CSC) feature and drug resistance, which are main hallmarks of cancer malignancy. Although previous findings have shown that Wnt/beta-catenin signaling pathway is activated in the cancer progression, the precise mechanism of Wnt/beta-catenin signaling in EMT and CSCs are not fully understood. To reveal the network pathways in EMT, gene expression in mesenchymal stem cells (MSCs) and diffuse-type gastric cancer (GC) as well as intestinal-type GC have been analyzed and compared. The network pathways in MSCs and GC were analyzed with Ingenuity Pathway Analysis (IPA). The gene expression profiling demonstrated that gene expression of cadherin 1 (CDH1), Wnt family member 9A (WNT9A) and catenin beta 1 (CTNNB1) were up-regulated in diffuse-type GC compared to MSCs. The gene expression of growth factor receptor bound protein 7 (GRB7) and erb-b2 receptor tyrosine kinase 2 (ERBB2) were up-regulated in intestinal-type GC compared to diffuse-type GC. Wnt/beta-catenin signaling, as well as ERBB signaling networks, involved in EMT, CSCs and drug resistance, have been investigated and profiled in bioinformatics. In conclusion, the Wnt/beta-catenin signaling pathway was included in EMT-related molecular network pathways in MSCs and GC, which may contribute into the elucidation of mechanism in the drug resistance of CSC population. AOP related to Wnt/beta-catenin signaling pathway is discussed.

p13 is mitochondrial protein widely expressed in central and peripheral tissues. Recently, we generated mice lacking p13 (p13-/- mice), and found that p13-/- genotype was smaller than the expected Mendelian ratio at 3 weeks of age (approx. 40% of the expected ratio). Here, we investigated the possible mechanisms underlying the loss of p13-/- mice. At postnatal day 0 (P0), Mendelian segregation of pup genotypes from heterozygous breeding was observed (n = 294, P = 0.25, χ² analysis), suggesting a significant loss of p13-/- pups specifically during the postnatal period. Kaplan-Meier survival analysis demonstrated that more than half of p13-/- mice died during the first 2 postnatal days. At P0, we observed the presence of milk in p13-/- pups stomach, however, their blood glucose levels were significantly lower than that of wild-type littermates. Taken together, the present results suggest that p13 contributes to early postnatal survival and maintenance of the normal blood glucose levels.

Excessive intake of sugar (sucrose) has been considered to be related to obesity, diabetes mellitus and lipid metabolism disorder, but there are not many reports on blood glucose level and insulin secretion affected by the way of sucrose charge. In this study, we investigated the effects of different types of sweeteners and their charge methods. Male ddY mice were divided into three groups: water-group, sucrose-group, and glucose-group. Furthermore, they were divided into three groups: intraperitoneal administration (IP)-group, oral administration (PO)-group, and free intake-group. The free intake-group was divided into those with and without food, and each drink and food were freely taken for 1 hour. Blood glucose level and insulin concentration were measured at 0, 30, and 60 minutes after the start of the experiment. Blood glucose levels and insulin levels of IP-group, PO-group, and free-intake-group (without food) increased more in the glucose-group than in the sucrose-group. The blood glucose level in the free intake-group (with food) did not differ depending on the type of sweetener, but insulin was secreted in large amounts, and the insulin secretion in the glucose-group was particularly high. Therefore, taking sweet drinks with food promoted insulin secretion as compared to taking sweet drinks alone.

Small extracellular vesicles (sEV) are lipid-bilayer-capsuled particles with a 50-150 nm diameter. They contain various molecules, such as proteins, lipids, nucleic acids, and metabolites. sEV affect cellular function via signal transduction through binding to cell surface receptors or delivering contents by phagocytosis, pinocytosis or membrane fusion. Therefore, sEV are recognized mediating cell-to-cell communication. Recent studies suggest that sEV play a key role during various disease states. However, little is known about changes in sEVs characters by aging. We then aimed to compare the size distribution of plasma sEV from young and aged Wistar rats. We isolated sEV from plasma of male Wistar rats (6- and 15-week-old; 6w-sEV and 15w-sEV) by polyethylene glycol precipitation and ultracentrifuge method. sEV particle distribution was measured by a tunable resistive pulse sensing method. Mean diameter in 15w-sEV was higher than 6w-sEV. Particles with less than 150 nm of diameter in 15w-sEV were lower than 6w-sEV. The present study for the first time revealed that the size of plasma sEV in Wistar rat increases by aging. Further studies are needed in order to clarify physiological significance of the increase in particle diameter by aging.

Pulmonary arterial hypertension (PAH) is pathophysiologically characterized by vasoconstriction and vascular remodeling of the pulmonary artery. Pulmonary vascular remodeling is mainly mediated by the enhanced cell proliferation of pulmonary arterial smooth muscle cells (PASMCs). In this study, we examined the pharmacological effects of fingolimod on the development of PAH. The proliferation rate of PASMCs from idiopathic PAH (IPAH) patients was much higher than that of PASMCs from normal subjects. In normal-PASMCs, fingolimod at low concentrations did not affect the cell proliferation, whereas higher concentrations partly reduced the cell proliferation. On the other hand, the application of fingolimod clearly inhibited the proliferation of IPAH-PASMCs and the inhibitory effect was in a concentration-dependent manner. In monocrotaline-induced pulmonary hypertensive rats, intraperitoneal administration of fingolimod ameliorated both pulmonary vascular remodeling and right ventricular hypertrophy. In addition, fingolimod improved the mortality rate. Our results suggest that fingolimod blocks the development of PAH through inhibiting the excessive proliferation of PASMCs. Fingolimod may be a novel option for the treatment of PAH.

Hepatic stellate cells are liver-specific pericytes that play central roles in the development of liver fibrosis. During liver injury, hepatic stellate cells transdifferentiate from the quiescent phenotype into the myofibroblast-like phenotype, resulting in high proliferation and extracellular matrix production. Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are expressed in many types of tissues and involved in the regulation of membrane potential, intracellular Ca²⁺ concentration, and cell proliferation. However, the involvement of BK_Ca channels on liver fibrosis remains unclear. In this study, we investigated the pathophysiological roles of BK_Ca channels in a human hepatic stellate cell line, LX-2. The mRNA expression analysis revealed that LX-2 cells highly expressed the α subunit of BK_Ca channels. In LX-2 cells, extracellular Ca²⁺ restoration in the presence of thapsigargin induced store-operated Ca²⁺ (SOC) entry, which potentially mediated by Orai/STIM channels. The SOC entry was significantly reduced by a specific inhibitor of BK_Ca channels, paxilline. In addition, the proliferation of LX-2 cells was clearly attenuated by paxilline. These results suggest that BK_Ca channels are functionally expressed in LX-2 cells and contribute to cell proliferation by regulating intracellular Ca²⁺ signaling.

The hippocampus is functionally segregated along the dorso-ventral axis in rodents. The dorsal hippocampus is involved in spatial memory whereas the ventral is in emotional responses. Consistently, each region receives afferents from distinct brain regions. Furthermore, within the dentate gyrus (DG), the proximal and distal portions of granule cell (GC) dendrites receive medial and lateral perforant pathway axons, respectively, which are originating from different regions of the entorhinal cortex. Differences in dendritic morphology and spine density presumably reflect differences in activities of their partner axon termini. Here, we investigate the morphological features of the GC in the dorsal and the ventral DG, and their responses to social defeat stress as an example of emotional inputs. Visualization of single neurons by microinjection of Lucifer Yellow revealed that the branching patterns of GC dendrites are distinct between dorsal and ventral DGs; the peak of the number of intersections in Sholl analyses localized more proximal in the dorsal than in the ventral DG. Spine density of ventral DG was higher than dorsal DG. Social defeat stress was found to suppress the dendritic branching and spine densities of GCs both in dorsal and ventral DGs to similar extent. Mushroom and thin spines decreased significantly whereas stubby spines did not. The data suggest that the dendritic branching pattern and spine density of GCs are distinct between the dorsal and ventral hippocampus, but undergo uniform morphological remodeling in response to an emotional stress.

The prevalence of mental disorders in diabetes mellitus is reported to be higher than that in general population. However, its mechanism is unclear. Since glucose is metabolized to L-lactate in the astrocytes of the brain and L-lactate suppresses AMP-activated protein kinase (AMPK), we investigated whether L-lactate and AMPK in the brain are involved in fear memory in streptozotocin (STZ)-induced diabetic mice. In the conditioned fear test, L-lactate injection increased freezing. In addition, injection of the AMPK inhibitor compound C also increased freezing. Freezing induced by both L-lactate and compound C was inhibited by the AMPK activator AICAR. We next examined the levels of L-lactate and AMPK in the amygdala and the hippocampus, which are known to play important roles in fear memory. L-lactate was increased in the amygdala and the hippocampus in STZ-induced diabetic mice. In contrast, phosphorylated AMPK, which is an active form of AMPK, was reduced in the amygdala and the hippocampus in STZ-induced diabetic mice. In addition, the increase of freezing in STZ-induced diabetic mice was suppressed by AICAR. These results suggest that L-lactate production is increased in the amygdala and the hippocampus in diabetes, which enhances fear memory through inhibition of AMPK.

Redox imbalances by overproduction of reactive oxygen species (ROS) are known to play an essential role in the pathological events of cerebral and cardiac ischemic injury, hypertension, inflammation and cancer. Widely used calcium channel antagonists such as nifedipine, nicardipine, amlodipine are not effective for ischemia-reperfusion (I/R) injuries at clinical used low dose on animal models.

BBB permeable Ca²⁺ /calmodulin antagonist CV-159, 1,4- Dihydro -2,6-dimethyl- 4-(3-nitrophenyl) -3,5-pyridinedicarboxylic acid methyl 6-(5-phenyl-3-pyrazolyloxy)hexyl ester characterized by markedly inhibitory effects for infarct size and edema on cerebral I/R injury. In cyclic voltammetry and ESR studies, hydroxyl radical scavenging ability of CV-159 was detected 100 times stronger than that of nicardipine, and it also suppressed mitochondrial superoxide and iNOS generation. Radical spin trapper G-CYPMPO (CAS No.1350616-52-2) and CV-159 markedly relaxed the high concentration of K⁺-induced contractions in isolated endothelium-denuded rat aortic strips, suggesting the existence of a novel role of oxygen radical in smooth muscle signal transduction.

Aims: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) causes chronic pain and/or storage symptoms. This study aimed to evaluate whether bladder sensation is deteriorated in a carrageenan induced CP/CPPS model by a direct measurement of the bladder mechanosensitive single-unit afferent nerve activity.

Methods: Male adult Sprague-Dawley rats were used. Fifty μL of 3% λ-carrageenan was injected into both lobes of the ventral prostate and for the control rats, fifty μL of saline was used. Seven days after injection, histology was examined along with cystometry and mechanosensitive single-unit afferent nerve activity. Statistical significance was determined using an unpaired Student's t-test with a two-sided significance level of 0.05.

Results: In the carrageenan group, weight increase and inflammatory cell infiltrations in the prostate were confirmed, basal and threshold-pressures of the bladder were remarkably increased, when compared to the sham group. Regarding Aδ- or C-fibers, the mechanosensitive afferent nerve activities revealed no differences in either group.

Conclusions: The carrageenan-induced CP/CPPS rat model showed edema and inflammation in the prostate, whereas little change was detected in bladder sensation. These findings, which were evaluated using a direct measurement of the mechanosensitive single-unit afferent nerve activity, suggest that the bladder sensation is unlikely deteriorated in this model.

[Background] Glutamate-cysteine ligase modifier subunit (GCLM) null mice display a 70-80% reduction in total glutathione (GSH) level (Killoy et al. Exp Neurol 2018 Apr;302:129-135). The GCLM null mice causes motor neuron degeneration in mice like SOD1 mutant mice. We here investigated whether oral GSH administration can rescue neurons form neuroinflammation in Alzheimer's disease model mice.

[Methods] After 3 weeks administration of glutathione (100 or 500 mg/kg/day, p.o.) in 12 month-old wild and APP-NL-GF knock-in mice, oxidative stress, neuroinflammation and cognition were investigated. We also assessed the GSH levels in mouse brain.

[Results] APP-NL-GF knock-in mouse brain display a 50% reduction in total GSH as compare to wild mouse brain. The lipid oxidation assessed by 4-hydroxy-2-nonenal (4-HNE) was also markedly increased in APP-NL-GF knock-in mouse brain. The GSH administration dose-dependently diseased the oxidative stress and suppressed microglial activation in the hippocampus. Likewise, the GSH administration improved cognitive impairment observed in APP-NL-GF knock-in mice.

[Conclusion] Taken together, the oral GSH administration rescues neurons from oxidative stress and neuroinflammation in neurodegenerative disorders and should be try in Alzheimer disease patients.

We report that tumor necrosis factor-alpha (TNF-α) prodction via histamine H₄ receptors contributes to cisplatin-induced fatigue. Previous studies reported the activation of histamine H₃ receptors also have the potential to reduce the inflammatory peptides. In this study, we investigated the effects of the H₃ receptor agonist on the development of chemotherapy-induced fatigue in mice. Cisplatin (7.5 mg/kg, i.p.) induced anorexia and decrease of voluntary wheel running within 24 hours of its administration and they continued for 3 days, and daily administration of a selective H₃ receptor agonist (immethtridine, 10 mg/kg, s.c.) significantly inhibited the development of anorexia and decrease of voluntary wheel running. Cisplatin significantly increased TNF-α mRNA expression in the hypothalamus and spleen, and the period of expression increase paralleled the onset period of anorexia and decrease of voluntary wheel running. Pretreatment with immethridine inhibited splenic TNF-α mRNA expression. These results suggest that peripheral TNF-α mRNA expression via H₃ receptors may contribute to the development of cisplatin-induced fatigue.