Many patients treated with oxaliplatin experience sensory deficits in the distal extremities. This impairment may persist for several months after treatment is discontinued, resulting in a negative impact on quality of life. Statins, which are used to treat dyslipidemia, have a pleiotropic action, such as anti-inflammatory effect. Here we show that statins can be a therapeutic agent for oxaliplatin-induced peripheral neuropathy via the regulation of glutathione S-transferase (GST). The mechanical hypersensitivity induced by oxaliplatin was ameliorated on day 7 in mice repeated orally administered statins and lasted for 21 days. Furthermore, mechanical hypersensitivity was suppressed even when statins were administered after day 7 of oxaliplatin exposure. On the other hand, statins were not effective against cold hyperalgesia. Uptake of oxaliplatin in the DRG was not inhibited by statins. Analysis of gene association databases revealed that the expression of GST family members is regulated by statins. Co-administration of the GST inhibitor, ethacrynic acid, reversed the statin-induced suppression of oxaliplatin-induced mechanical allodynia. Statins might be potential therapeutic agents for the treatment of anticancer drug-induced chronic peripheral neuropathy that do not suppress the effects of oxaliplatin.

The flow of cerebrospinal fluid is considered to be a critical factor in the clearance of wastes in the brain. The dynamics of fluid has been investigated by several experimental methods such as fluorescence microscopy and magnetic resonance imaging. However, because probes used for these imagings are much larger compared to water itself, the dynamics of the fluid have been poorly understood. Here, we applied a multimodal multiphoton imaging system to the living brain tissue. Combining stimulated Raman scattering and two-photon fluorescent imaging, the system enables us to visualize spatiotemporal dynamics of deuterated water and fluorescent dyes simultaneously at a cellular level. We demonstrate that deuterated water diffuses faster than fluorescent dyes in the brain tissue. Detailed analysis reveals deuterated water rapidly exchanges inside and outside of cells, whereas fluorescent dyes only diffuse through extracellular spaces. Furthermore, we find that the dynamics of deuterated water is robust to changes under physiological and pathophysiological conditions; there is little change in the spatiotemporal dynamics of deuterated water during development and ischemia whereas fluorescent dyes are severely affected. Thus, our new approach reveals unique properties of the dynamics of the fluid in the living brain tissue.

Electrical activities of neuronal network with high-spatio-temporal resolution is useful for understanding brain functions and elucidating neurological disorders, and for drug screening and safety assessment of drugs. Complementary metal-oxide semiconductor micro-electrode array (CMOS-MEA) is excellent in detecting detailed electrical activity patterns of neural networks due to the large number of electrodes. In this study, we investigated the electrical activity characteristics of brain slices, brain organoids, and cultured neural networks using CMOS-MEA with 236,880 electrodes, which have the highest specifications in the world. In the measurement of brain slices, we succeeded in measuring the interregional propagation of the hippocampus and cerebral cortex area in detail, and detected changes of propagation patterns due to drug administration. In sensory neuron measurement, calculation of axon conduction velocity in single neuron and drug responses based on firing pattern of each neuron were detected. In human iPS cell-derived central nervous system networks and human cerebral organoids, network activity was detected on a cell-by-cell basis, and changes in propagation patterns due to drug administration were detected. It was found that CMOS-MEA with 236,880 electrodes and a large measurement area can measure the electrical activity characteristics of ex vivo and in vitro neural networks and single neuron in detail. It was suggested that big data with high temporal resolution is effective for elucidation of neural circuit function and drug evaluation based on new neural activity information.

Lactoferrin (LF) is reported to have various bioactivity such as bone growth stimulation and analgesic activity. Recently, LF has been reported to recover the fatigue in humans after the shift-work. In this study, we examined the effect of administration of LF on the functions of the circadian clock such as the free-running rhythm as well as the light pulse-induced phase shift of the behavioral rhythm using mouse model. Since an orally-administered LF is known to be largely degraded in stomach, we used the enteric microcapsule bovine lactoferrin (eMC-LF) which protects from stomach digestion, but can be absorbed in the small intestine. The wheel-running activity of mice at 6 min-bin was automatically recorded in PC and was analyzed by CLOCK Lab. The level of mRNA of several clock-related genes was quantified by in situ hybridization method. We found that oral single administration of eMC-LF at ZT24 promoted the re-entrainment of wheel-running rhythm to 8 hr advanced LD cycle in mice. Both acute and chronic administration of eMC-LF potentiated the light pulse-induced phase shift of the wheel-running rhythm. Furthermore, eMC-LF increased the light pulse-induced expression of Per1 mRNA in the restricted area of the suprachiasmatic nucleus (SCN). We also found that eMC-LF potentiated the Per1 mRNA upregulation by i.c.v. administration of gastrin-releasing peptide (GRP), a neuropeptide involved in the photic signaling in the SCN. These results suggest that LF promotes the light entrainment of the mouse circadian clock.

Amyloid-β proteins (A β), including Aβ42 and A β 43, are known pathogenesis factors of Alzheimer's disease (AD). Unwanted substances in the brain, including A β, are generally removed by microglia, astrocytes, or neurons via a phagocytosis receptor. We observed that neurons and astrocytes engulfed A β 42 and A β 43, which are more neurotoxic than A β 40. We previously showed that multiple-EGF like domains 10 (MEGF10) that is the mammalian homologue of Draper, a phagocytosis receptor of apoptotic cells in Drosophola, and is the type I transmembrane protein plays an important role in apoptotic cell elimination and is expressed in mammalian neurons and astrocytes. Therefore, we assessed whether MEGF10 is involved in A β42 and A β43 engulfment in MEGF10-expressing neurons and astrocytes. We found that MEGF10-expressing astrocytes and neurons engulfed A β42 and A β43 but not A β40. Furthermore, incubation of the neurons and astrocytes with A β42 and A β43 augmented MEGF10 phosphorylation; however, incubation with A β40 did not have this augmenting effect. Our findings suggest that MEGF10 plays a phagocytosis receptor function for A β42 and A β43 in neurons and astrocytes.

It is known that peripheral neuropathy as an unwanted side effect of the anticancer drug oxaliplatin, especially cold allodynia, which reacts hypersensitively to cold stimuli, greatly impairs the QOL of patients. Therefore, it is desired to develop preventive or therapeutic medicines for cold allodynia. Previous studies have shown that pituitary adenylate cyclase-activating polypeptide (PACAP) antagonists have potent effects in preventing cold allodynia in in vivo studies. In this study, we evaluated the adverse effects of oxaliplatin and their protective effects using an extracellular potential measurement system using rat E14 primary dorsal root ganglion cells (DRG). The effect of oxaliplatin and antagonist of PAC1, that is a PACAP receptor, on extracellular action potential was verified. The firing frequency of extracellular action potential increased in a concentration-dependent manner with oxaliplatin. On the other hand, the addition of PAC1 antagonists abolished the firing of the extracellular potential. Moreover, the firing of PAC1-treated DRG neuron decreased compared to before addition of oxaliplatin. The evaluation system for peripheral neuropathy caused by anticancer drugs by measuring the extracellular potential of DRG was able to show the usefulness as a system that can evaluate side effects in the acute phase.

Intracranial hemorrhage (ICH) is a type of hemorrhagic strokes that is caused due to bleeding in the brain parenchyma. Here we investigated the effects of Na₂S and Na₂S₃ on ICH in mice. Na₂S and Na₂S₃ were injected into two separate groups of mice (25 µmol/kg i.p.) 30 min before induction of ICH by collagenase injection into the striatum. Both agents attenuated the neutrophil infiltration (MPO), inhibited the upregulation of monocyte/macrophage chemokine (CCL-2) expression and maintained the axonal fibers transport function (APP). Moreover, pretreatment with Na₂S improved the motor function, prevented the decrease in the neuronal count (NeuN), maintained the integrity of axonal fiber structures (neurofilament-H) and lowered the upregulation of neutrophil chemokine (CXCL-2) expression after induction of ICH. On the other hand, pretreatment with Na₂S₃ significantly attenuated the activation of microglia/macrophages (Iba-1) in the perihematomal area. Both agents failed to prevent ICH-induced increase in the brain vascular permeability or to lower the pro-inflammatory cytokine (IL-6) expression significantly. In conclusion, these results suggest that Na₂S exhibits more potent neuroprotective effect than Na₂S₃ and promotes recovery of neurological functions after ICH.

We previously reported that protein tyrosine phosphatase delta (PTPd), one of type IIa receptor type protein tyrosine phosphatases, mediates Sema3A-induced dendritic growth of cortical pyramidal neurons. However, its endogenous substrates involved in cortical dendritic arborization have been yet identified. Phosphotyrosine-proteome analysis of PTPd knockout brains revealed the hyperphosphorylation of Signal Regulatory Protein alpha (SIRPa) at Tyr501 (Y501) residue. Immunohistochemistry with anti-phospho-Y501 SIRPa antibody showed that olfactory epithelium, cortical II to V layers, thalamic nuclei and axon-bundles including corpus callosum, fimbria, and pyramidal tract were hyperphosphorylated in PTPd knockout brains. Knockdown of SIRPa by siRNA transfection or the overexpression of cytoplasmic deletion mutant of SIRPa suppressed Sema3A-induced growth cone collapse response of mouse dorsal root ganglion neurons. Primary culture of mouse cortical neurons revealed that Sema3A-stimulation induced the dephosphorylation of SIRPa in the dendritic growth cones of wild-type but not in those of PTPd knockouts. Overexpression of non-phosphorylated SIRPa mutant Y501F in cultured cortical neurons attenuated Sema3A-induced dendritic growth. In utero electroporation of SIRPa-Y501F to mouse brains showed that the apical dendrites of cortical layer II/III pyramidal neurons were disoriented. Similar irregular projection of cortical apical dendrites was also observed in PTPd knockout brains. These results suggest that PTPd may regulate the phosphorylation of SIRPa in cortical dendritic growth.

Extracellular calcium ion ([Ca²⁺]o) change occurring during physiological and pathological conditions play important roles in regulation of brain functions, but has received limited attention due to the lack of easy to use device. Here, we show the development and application of highly selective calcium image sensor for the imaging of glutamate-induced [Ca²⁺]o change in hippocampal slices. Using this sensor, we found that glutamate decreased [Ca²⁺]o by more than 50 % with different spatiotemporal patterns. The glutamate-evoked decrease in [Ca²⁺]o was inhibited by a NMDA receptor antagonist D-AP5 but not AMPA receptor antagonist CNQX, and mimicked by NMDA. The spatial pattern of the glutamate-evoked [Ca²⁺]o decrease was associated with that of distribution of NMDA receptors in the hippocampus. Moreover, using an ATP-imaging fluorescent probe, we found that the stimulation with NMDA triggered increase in ATP release from astrocytes via either connexin hemichannels or chloride channels. The NMDA-evoked ATP release was mediated by decrease in [Ca²⁺]o because the astrocytic ATP release was mimicked by Ca²⁺-free medium. Taken together, using the newly developed Ca²⁺ image sensor, we demonstrated that [Ca²⁺]o is dramatically decreased during excitatory synaptic transmission by glutamate, and [Ca²⁺]o decrease act as a signal that transmits neuronal excitation to astrocytes via ATP release. The application of this Ca²⁺ sensor is expected to clarify the physiological and pathophysiological roles of [Ca²⁺]o , which have received limited attention so far.

Excessive or chronic social stress induces emotional and cognitive disturbances and precipitates mental illness. Altered neuronal morphology and functions in the medial prefrontal cortex (mPFC) underlie these behavioral abnormalities. However, its subcellular mechanisms remain elusive. Here we examined ultrastructural and multi-omics changes in the mPFC after social stress in mice. Social stress caused the loss of dendritic branches with morphological alterations of subcellular mitochondria and induced synaptic shrinkage selectively at the synapses with mitochondria. Multi-omics and functional analyses revealed that social stress deteriorated mitochondrial functions with altered mitochondrial proteome at synapses and dysregulated central metabolic pathways in the mPFC. Molecular biological and pharmacological manipulation targeting central metabolism and mitochondria attenuated the synaptic shrinkage and depression-related behaviors. These findings demonstrate that chronic social stress alters the central metabolism at mPFC synapses, leading to neuronal pathology and depression-related behaviors.

Stress caused by aversive and psychological stimuli induces sustained emotional and cognitive abnormalities and precipitates the onset and relapse of symptoms in psychiatric illnesses, such as depression. We have demonstrated that chronic social stress causes dendritic atrophy of pyramidal neurons in the medial prefrontal cortex (mPFC) only in mice susceptible to chronic social stress, whereas acute social stress leads to dendritic growth of these neurons concomitant with suppression of behavioral changes. However, it remains unknown how stress-induced dendritic remodeling of mPFC pyramidal neurons affects neural circuits associated with depressive behaviors. Here we utilized monosynaptic retrograde tracing with a G-deficient rabies viral vector to identify neural projections to the mPFC that can be anatomically altered by chronic social stress. A G-deficient rabies viral vector encoding a red fluorescent protein (RFP) was unilaterally infused into the mPFC to visualize neurons that directly input to the mPFC. Among 90 brain regions where RFP-positive cells were observed in mice subjected to chronic social stress, some particular regions showed a decrease or increase in RFP-positive cells. This finding suggests that chronic social stress anatomically alters neural projections to the mPFC, leading to sustained behavioral changes.

Diabetes mellitus and brain toxicity are closely linked. Oxidative stress, obesity, insulin resistance, and glucose toxicity can affect the brain. Orexin-A, also known as hypocretin-1, participates in many physiological processes through its activated receptor. Orexin-A has been associated with feeding behavior, obesity, and pathogenesis of Alzheimer's disease. We reported that high-dose thiamine in obese diabetic Otsuka Long–Evans Tokushima Fatty (OLETF) rats leads to reduced obesity and metabolic disorders. In addition, we found that plasma orexin-A levels in OLETF rats can be modulated by thiamine supplementation under conditions of oxidative stress. Herein, we focused on orexin-A in obese diabetic OLETF rats. At 58 weeks of age, the rats showed an increase in body weight and blood glucose levels. Plasma orexin-A was measured by ELISA and tended to be higher in obese diabetic OLETF rats than in non-obese diabetic control rats. We evaluated hypocretin receptor 1 (Hcrtr1, also orexin-A receptor) gene expression in the brain of diabetic OLETF rats by reverse transcription-polymerase chain reaction and found that diabetic OLETF rats exhibited higher orexin-A receptor gene expression in the brain than controls. The results presented here are expected to provide a better understanding of the role of orexin-A and its contribution to diabetic brain.

We previously established an animal model of Parkinson's disease (PD) induced by chronic low-dose rotenone as an environmental neurotoxic pesticide that reproducible central and enteric neurodegenerative features of PD, and also found that rotenone-induced enteric neurodegeneration is caused by dysfunction of enteric glia using primary cultured enteric cells. However, the mechanism of enteric neurodegeneration and inflammation are still obscure. In this study, we examined changes in enteric cellular environment in the enteric epithelium and myenteric plexus of the rotenone-induced PD model mice. Chronic subcutaneous administration with low-dose rotenone (2.5 mg/kg/day) for 4 weeks using an osmotic mini pump reduced the number of dopamine neurons in the substantia nigra and the intestinal myenteric neurons and glial cells of mice. Furthermore, it produced disruption of mucosal epithelial barrier and marked translocation of HMGB1 to the cytosol beside nuclear membrane towards the apical lumen side. These results suggest that the rotenone-induced dysfunctions of epithelial barrier and HMGB1 transportation are involved in the inflammatory reactions and dysfunction of enteric glia and consequent enteric neurodegeneration.

Febrile seizures (FSs) are the most common convulsive seizure in early childhood. About 30% of them are "complex", with repetitive seizures, and related to adult temporal lobe epilepsy (TLE). In this study, we investigated the role of membrane-associated prostaglandin E synthase-1 (mPGES-1), an inducible terminal enzyme for PGE₂ synthesis, in hippocampal inflammation induced by repetitive FSs (RFSs) in mouse pups as a model of complex FSs.

Wild-type (WT) and mPGES-1 knockout (ES1KO) mice at P9-11 were given intraperitoneal injections of lipopolysaccharide (100 µg/kg) and exposed to heat lamp to induce hyperthermia and FSs. The induction of FSs was repeated twice at 4 h-interval (RFSs).

In WT mice, mPGES-1 mRNA was significantly up-regulated in hippocampus after RFSs. The production of hippocampal PGE₂ observed after RFSs in WT mice was completely absent in ES1KO mice. The seizure score and increase in rectal temperature during the hyperthermia induction in ES1KO mice were slightly but significantly lower than those in WT mice. The inductions of IL-1β, TNF-α and GFAP observed significantly in WT mice were less in ES1KO mice even in which the seizure scores were almost the same level.

These results suggest that mPGES-1 contributes to inflammatory hyperthermia, convulsive events, glial activation and production of inflammatory cytokines through PGE₂ production in hippocampus. Thus, mPGES-1 may contribute to the complex FSs-induced adulthood TLE and may be a potential therapeutic target for the development of epilepsy after RFSs.

My research focuses on the relationship between peripheral inflammation and microglia. Lipopolysaccharide (LPS) is used to induce peripheral acute inflammation. It has been reported that LPS application induces microvesicle (MV) release from microglia in vitro, and these MVs contain inflammatory cytokines such as IL-1b. These data indicate that microglia detect peripheral inflammatory signals and react by releasing MVs. However, MVs released from microglia had not previously been observed in vivo. To observe microglial MVs, we made cranial windows into the heads of Iba1-GFP mice and performed in vivo live imaging using 2 photon-microscopy. GFP+ MVs were observed 24h after LPS injection (1 mg/kg, ip), and the peak of the increase in GFP+ MVs was at 48h after LPS injection. Because Iba1 is expressed in not only microglia but also some macrophages, we treated mice with clodronate (33 mg/kg, ip) to deplete peripheral macrophages. Clodronate treatment did not affect the increase of LPS-induced GFP+ MVs. This indicates that the GFP+ MVs were released from Iba1+ cells like microglia, but not macrophages. Interestingly, the GFP+ MVs were also observed in contact dermatitis model mice with chronic skin inflammation. These results provide a framework to study the role of microglial MVs in peripheral inflammatory mouse models.

Recently we have shown that delta opioid receptor agonists (DORs) shorten sleep time during the light period. However, the impact of DOR agonists on sleep quality has not been determined. In the present study, we investigated the effects of the DOR agonist KNT-127 on delta wave power (1-4 Hz), which is an indicator of non-rapid eye movement (REM) sleep depth. The vigilance states (e.g., wakefulness, REM and non-REM sleep) of the ddY-mice (6-10 weeks) were classified based on the hippocampal local field potential (LFP) and neck muscle electromyogram. KNT-127 (10-30 mg/kg, i.p.) significantly decreased the mean REM and non-REM sleep periods, and prolonged the mean wakefulness period during 5 hr after its injection. KNT-127 significantly increased delta wave power during non-REM sleep compared to saline, and this effect was also observed at 3 mg/kg without the arousal. KNT-127 (3 mg/kg), when administered in the urethane-anesthetized mice, increased delta wave power, indicating that the action is not a rebound due to sleep suppression. Together, KNT-127 promotes deeper non-REM sleep independently of its arousal effects.

Propolis (Brazilian green propolis) is a chemically complex resinous substance that is expected to be beneficial to therapy for Alzheimer's disease (AD). To reveal its beneficial effect on impaired cognition, we first performed three memory-related behavior tasks in mice aged 4 and 12 months: Y-maze task, novel object recognition task, and passive avoidance task. Oral dosages of 300-1000 mg/ kg once daily for 8 weeks, did significantly prevent the cognitive decline in the APP-KI mice aged 4 months, but not 12 months. Consistent with the observations from behavioral tasks, impaired hippocampal long-term potentiation (LTP) was markedly ameliorated in the acute brain slices prepared from the mice that underwent the repeated propolis administration. In addition, increased phosphorylation of CaMKII and AMPAR subunit (GluA1) was simultaneously observed in the CA1 of the mice. Similar to CaMKII activation, the propolis administration also increased CaMKIV and CREB phosphorylation and BDNF production in the CA1 of the mice. Finally, we confirmed that the presence of 30 μg/ mL propolis significantly elevated intracellular Ca²⁺ concentration in Neuro2A cells. These findings suggest that propolis is capable of rescuing the cognitive dysfunction via both upregulated activities of CaMKII and CaMKIV in the CA1 of the APP-KI mice.

Cardiac fibrosis is a leading cause of heart failure, particularly heart failure with preserved ejection fraction (HFpEF). Currently, there is no effective treatment for HFpEF. We previously reported that doxorubicin-induced cardiac fibrosis was suppressed in mice deficient in Nox1, a non-phagocytic isoform of superoxide-producing NADPH oxidase. In this study, the role of NOX1 in the development of cardiac fibrosis was investigated in cultured cells using a rat cardiomyoblast cell line H9c2 and cardiac fibroblasts isolated from adult male mice. Increased proliferation was demonstrated when cardiac fibroblasts were exposed to homogenates from wild-type H9c2. On the other hand, increased proliferation was significantly attenuated in cardiac fibroblasts exposed to homogenates from Nox1-disrupted H9c2. In Nox1-disrupted H9c2 cells, the expression of osteoglycin (Ogn), and podocan (Podn), which are small leucine-rich proteoglycans and known to regulate cardiac remodeling, were up-regulated. When homogenates from Nox1-disrupted H9c2 with disruption of Ogn or Podn exposed to cardiac fibroblasts, the proliferation of fibroblasts was significantly restored compared to those exposed to Nox1-disrupted H9c2. These findings suggest that NOX1 promotes cardiac fibrosis via down-regulation of Ogn or Podn in cardiomyocytes.

Background: Atrial fibrillation (AF) is the most common arrhythmia. AF is highly correlated with multiple risk factors including heart failure, age, obesity, and type 2 diabetes. Among risk factors, the incidence in obesity is increasing worldwide. Recently, it was reported that SGLT2 inhibitors reduced the incidence of atrial fibrillation. However, it is unclear how the treatment with SGLT2 inhibitors has effects on vulnerability to AF. In this study, we examined the effects on the inducibility and duration of AF by treatment with SGLT2 inhibitors in diet-induced obese mice.

Methods: Mice were fed a normal chow diet (NCD) or high-fat diet (HFD). Following diet-loading, we randomly divided the animals into groups: NCD+vehicle, HFD+vehicle, and HFD+ SGLT2 treatments. Induction of AF was performed by transesophageal atrial burst pacing. Furthermore, we evaluated cardiac function, blood pressure, atrial fibrosis, and glucose tolerance at the end of the treatments.

Results: The results showed that HFD-fed mice increased the inducibility of AF compared to NCD mice. In addition, treatment with the SGLT2 inhibitor in HFD-fed mice dose-dependently reduced the inducibility and duration of AF. There were no significant differences in cardiac function, blood pressure, and fibrosis among all groups. Impairment of glucose tolerance in HFD-induced obesity was improved by treatment with the SGLT2 inhibitor.

Conclusion: Treatment with the SGLT2 inhibitor reduced the inducibility of AF and shortened the duration of AF without affecting atrial structural remodeling, suggesting that the SGLT2 inhibitor effectively prevents AF in obesity.

Gain-of-function mutations in type 2 ryanodine receptors (RyR2) are known to cause severe arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT). Conventional antiarrhythmic drugs are sometimes insufficient to suppress these arrhythmias. To seek more effective drugs, we aimed to provide a basis for quantitative evaluation of the two important effects of antiarrhythmic drugs, i.e., prevention and treatment/stopping of arrhythmia, using multiple RyR2 mutant mouse lines (R420W, I4093V and K4750Q) with varying degrees of enhanced Ca²⁺ release activity. Short term ECG was recorded from the limb leads under isoflurane anesthesia, and arrhythmia was induced by administration of adrenaline alone or adrenaline/caffeine mixture. For monitoring basal arrhythmia during everyday activities, long range ECG was recorded by telemetry system. The R420W mice having moderately activated channels showed little basal arrhythmia but exhibited severe ventricular arrhythmias by adrenaline/caffeine induction. On the contrary, the RyR2-I4093V and K4750Q strains having highly activated channels showed frequent basal arrhythmia without adrenergic induction. Na channel blockers, Ca channel blockers and beta blockers suppressed the induced arrhythmia and basal arrhythmia to varying degrees. Interestingly, the preventive effect and the stopping effect seemed to differ depending on their mechanism of action. The usage of multiple lines of mice with different degrees of activity are useful for the evaluation of therapies for CPVT.

Although abnormalities of function in femoral artery, including aberrant vascular reactivity to various vasoactive mediators, are common in many chronic disorders including hypertension and diabetes, their inducible and/or progressive factors remain unclear. On the other hand, methylglyoxal (MGO), a highly reactive dicarbonyl compound, has been implicated in the pathogenesis of several chronic disorders. Although our previous studies demonstrated that uridine 5'-diphosphate (UDP)-induced contraction in the femoral artery is increased in hypertensive rat model, the direct relationship between MGO and UDP-mediated contraction is currently unknown in rat femoral artery. We therefore investigated the acute effect of MGO (4.2 × 10⁻⁴ M for 60 min) on UDP-induced contraction in the rat femoral artery. MGO amplified the UDP-induced contraction in the Wistar rat femoral artery. This augmented response was not abolished in all conditions, including nitric oxide synthase inhibition by L-NNA (10⁻⁴ M), cyclooxygenase inhibition by indomethacin (10⁻⁵ M), or endothelial denudation. Moreover, in the endothelium-denuded arteries, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (10⁻⁵ M) reduced the UDP-induced contraction in both control and MGO-treated groups, while MGO enhanced the p38 MAPK activation regardless of the UDP presence. Moreover, in the endothelium-denuded arteries, the Syk tyrosine kinase inhibitor piceatannol (10⁻⁵ M) reduced the UDP-induced contraction in both control and MGO-treated groups. These results suggest that MGO enhances UDP-induced contraction in rat femoral arteries and that this enhancement may be partly due to increases in the activities of Syk tyrosine kinase and p38 MAPK in femoral arterial smooth muscle.

This study investigated whether soluble guanylate cyclase (sGC) activators (activating the heme-oxidized and heme-free sGC) have potential as therapeutic drugs for coronary artery spasm. Isolated canine and porcine coronary arteries were suspended in organ chambers for isometric tension recording. The contractile responses of canine coronary arteries to potassium chloride (3×10^-2 M), endothelin-1 (3×10^-11 to 3×10^-8 M), prostaglandin F_2α (10^-8 to 10^-5 M), and 5-hydroxytryptamine (10^-9 to 10^-6 M) were suppressed by previous exposure to the sGC activator BAY 60-2770 (10^-10, 10^-9 and 10^-8 M). In porcine coronary arteries, the addition of BAY 60-2770 (10^-10, 10^-9 and 10^-8 M) concentration-dependently prolonged the cycle length of 3,4-diaminopyridine (10^-2 M)-induced phasic contractions and reduced the peak tension. As for vessel-size-dependent difference in vasoreactivity, BAY 60-2770 (10^-12 to 10^-7 M) caused a greater relaxation of porcine coronary arteries precontracted with endothelin-1 (3×10^-8 M) in small arteries (#9 in AHA classification) than in large arteries (#6 in AHA classification). These findings suggest that sGC activators are beneficial for the treatment of vasospastic angina. In addition, anti-spasmolytic efficacy of sGC activators may be expected to be observed even in microvascular angina.

Introduction: Vernakalant was approved as an anti-atrial fibrillatory drug by EMA and Health Canada but not by FDA due to its cardiovascular adverse events including bradycardia and hypotension. We characterized its electropharmacological profile in vivo with the isoflurane-anesthetized beagle dogs and in vitro with the rat aorta.

Methods: Vernakalant (0.3 and 3 mg/kg/10 min) was intravenously administered to the dogs in the absence (n=5) and presence (n=4) of α-adrenoceptor blocker phentolamine. Next, the in vitro vascular effect of vernakalant on the rat aorta was assessed by its cumulative application in concentrations of 0.001-100 μmol/L (n=13 preparations). 

Results: Vernakalant suppressed the sinus automaticity, ventricular contractility and atrioventricular nodal as well as intraventricular conduction, whereas it increased the total peripheral vascular resistance, preload to the left ventricle and mean blood pressure. It delayed the ventricular repolarization in a reverse frequency-dependent manner; the extent of prolongation of early and late repolarization was similar. It also prolonged the atrial and ventricular effective refractory period similarly. Pretreatment of phentolamine hardly affected those results. Meanwhile, vernakalant did not induce the contraction of aorta in vitro.

Conclusion: Vernakalant exerted α-adrenodceptor-independent vasoconstrictor action only in vivo. It also showed electrophysiological effects on the atria and ventricles to a similar extent, which resembles those of dl-sotalol and bepridil.

【Background】

Mammalian cardiomyocytes (CMs) retain proliferative capacity shortly after birth, though these cells are differentiated with contractile activity. By using Runx1 as a dedifferentiation marker, recent studies have demonstrated that CMs are dedifferentiated prior to cell proliferation; however, biological significance of Runx1 remains to be fully elucidated. The aim of this research is to clarify the regulatory mechanisms of Runx1 expression and its biological functions in CM proliferation.

【Method/Result】

CMs were prepared from neonatal rats. Cell proliferative activity was estimated by immune-fluorescent microscopic analysis with anti-Ki-67 antibody. CM exhibited proliferative activity in response to fetal bovine serum (FBS). Previously, since we demonstrated that STAT3 plays an important role in CM proliferation, the effects of STAT3 on CM proliferation was analyzed by using siRNA. STAT3 knockdown reduced the frequency of Ki-67⁺ CM, accompanied by the decrease in Runx1 expression. Importantly, Runx1 knockdown also suppressed CM proliferation in response to FBS.

【Conclusion】

Runx1 expression is regulated by STAT3, and promotes CM proliferation, indicating the functional importance of Runx1 in cardiac proliferation.

Myocardial ischemia/reperfusion (MI/R) injury leads to aggravated cardiac remodeling and heart failure. Previously, we reported that 2,5-dimethylelcelecoxib (DMC), a derivative of celecoxib without cyclooxygenase-2 inhibition, prevents cardiac remodeling in a non-ischemic cardiac fibrosis model. In this study, we examined whether DMC inhibited myocardial remodeling associated with MI/R injury. The left anterior descending coronary artery was ligatured for 0.5 hours and subsequently subjected to reperfusion for MI/R injury in male C57 BL/6 mice. Vehicle or DMC was administered orally (DMC: 150 mg/kg) immediately after awakening and followed by feeding (DMC: 1000 ppm). Echocardiographic evaluation showed significant improvement of left ventricular ejection fraction in the DMC-treated group compared to the Vehicle-treated group at 1-4 weeks after MI/R injury. In MI/R-injured hearts, protein expression of alpha-smooth muscle actin (myofibroblast marker) was significantly reduced by DMC treatment, as were mRNA expressions of fibronectin, connective tissue growth factor, and matrix metalloproteinase-9, 3 days after injury. Masson trichrome staining indicated that DMC significantly reduced cardiac fibrosis area 4 weeks after MI/R injury. This study revealed that DMC decreased myofibroblast appearance, and suppressed fibrosis and cardiac dysfunction associated with MI/R injury. DMC might be useful for preventing the development of heart failure associated with reperfusion therapy for acute myocardial infarction.

［Background］Though the importance of myeloid cells in the cardiac remodeling after myocardial infarction (MI) is widely accepted, it remains to be fully elucidated how myeloid cells regulate post-infarct inflammation, at least partially, because subpopulation-specific cell knock-out methods are not available.

［Methods and Results］We generated transgenic mice expressing diphtheria toxin receptor (DTR)/GFP fusion protein under the control of CD11b promoter in a Cre recombinase-expressing cell-specific manner (CD11b-DTR TG mice). Double TG mice (DTG mice) were generated by crossing CD11b-DTR TG mice with LysM-Cre mice that express Cre recombinase preferentially in monocytes/macrophages. The MI model was created in DTG mice by ligation of the left anterior descending branch. Flow cytometry analysis revealed that monocytes were labeled with GFP in the peripheral blood 4 days after MI. Consistently, immunofluorescent microscopic analysis showed that GFP⁺ cells infiltrated into the infarcted heart. Importantly, the administration of diphtheria toxin resulted in the depletion of GFP⁺ cells in peripheral blood and post-infarct myocardium.

［Conclusion］CD11b-DTR TG mice are useful for labeling and/or depleting subpopulation of myeloid cells in MI model.

Ferroptosis is defined as an iron-dependent regulated necrosis that is caused by massive lipid peroxidation-mediated cell membrane damage. The present study examined whether ferroptosis is involved in endothelial dysfunction by nicotine- and tar-free cigarette smoke extract (CSE) prepared from heated tobacco products (HTPs). The CSE of HTPs (Ploom X, IQOS 3, and IQOS ILUMA) and a combustion cigarette (1R6F) was prepared according to Health Canada Intense smoking method (55 mL puff volume, 2 sec puff duration, and 1 puff every 30 sec) using an analytical vaping machine LM5E (Borgwaldt KC GmbH). The cytotoxicity of CSE of HTPs and 1R6F to human umbilical vein endothelial EA.hy926 cells was evaluated by measuring mitochondrial metabolic activity and lactate dehydrogenase (LDH) leakage. CSE from cigarettes except for Ploom X, and erastin, a ferroptosis inducer by inhibiting cystine-glutamate exchange transporter (system X_C^-), triggered a decrease in mitochondrial metabolic activity and an increase in LDH leakage. The cytotoxic effects of CSE of IQOS 3 and 1R6F were reduced by an iron chelator deferoxamine mesylate (DFO), but not by a ferroptosis inhibitor UAMC-3203, which scavenges lipid reactive oxygen species (ROS). On the other hand, erastin cytotoxicity was inhibited by both DFO and UAMC-3203. These results suggest that erastin-induced, iron-dependent ferroptosis leads to cell damage characterized by a decrease in mitochondrial metabolic activity and an increase in LDH leakage, in EA.hy926 cells. CSE of IQOS 3 and 1R6F causes iron-dependent mitochondrial and cell membrane damage, both of which are independent of lipid peroxidation by ROS.

We previously revealed that glucocorticoids such as cortisol and cortisone may also regulate sodium and fluid balance in healthy subjects. We also reported that dexamethasone administration to mice initially increased urinary sodium excretion and urine volume and reduced skin sodium and water content in the early phase of administration and that chronic dexamethasone injection only decreased skin sodium and water content. However, it remains to be clarified which organ, kidney or skin, is responsible for the initial sodium and water loss at initial dexamethasone injection. In the present study, we examined the effects of dexamethasone on skin sodium and water content in bilateral nephrectomized mice. In the sham-operated group, dexamethasone (1 mg/kg/day, s.c.) significantly increased urinary sodium excretion and urine volume and decreased skin sodium and water content 24 hours after the injection. Dexamethasone did not affect plasma sodium concentration and osmolarity. In bilateral nephrectomy groups, dexamethasone did not alter skin sodium and water content. These findings suggest that glucocorticoid originally increases urinary sodium excretion and urine volume, which decreases skin sodium and water loss to compensate for renal sodium and water loss. In order to elucidate the mechanisms of sodium and fluid homeostasis, it may be necessary to examine skin and glucocorticoids in addition to the known hormones and kidney systems.

Aquaporins (AQPs) are the water channel that facilitate water transport through plasma membrane. Among 13 AQP isoforms, AQP5 is selectively expressed in apical membrane of exocrine glands, airway and alveolar epithelial cells, and plays important role to maintain water secretion and clearance in airway tracts. Recently, it is revealed that AQPs have new functions, as well as water transport. In our previous study, AQP5 enhances TNF-α-induced chemokine expression in vitro experiments. It has also known that the expression of AQP5 is markedly reduced in airway inflammation mouse model. Therefore, changes in AQP5 expression may be contribute to the pathogenesis of inflammatory respiratory diseases. In this study, we have established a transgenic (Tg) mouse in which AQP5 is highly expressed specifically in the airway epithelial cells, to confirm the significance of the AQP5-madiated regulation in cytokine expression. Intratracheal treatment of LPS increased the expression of inflammatory cytokines, such as KC, TNF-α and IL-6 in WT mice. In AQP5-Tg mice, the increase in the expression of these cytokines by LPS was considerably less than that in WT. These results indicated that AQP5 can enhance airway inflammation not only in vitro, but also in vivo conditions.

Subgroups of severe asthma patients with marked increases in sputum eosinophils and neutrophils are insensitive to corticosteroids. Exogenous administration of IL-10 negatively regulates both migration of eosinophilic and neutrophilic into tissues. This study evaluated whether intratracheal IL-10 administration suppresses asthmatic responses in a severe model of mice. Ovalbumin (OVA)-sensitized mice were intratracheally challenged with OVA. Dexamethasone (DEX, 1 mg/kg, intraperitoneal) or IL-10 (25 ng/mouse, intratracheal) was administered during the challenges. The number of leukocytes, expressions of adhesion molecules and IL-10 receptor, and development of airway hyperresponsiveness (AHR) were evaluated after the challenges. Although DEX hardly suppressed the development of AHR, the infiltration of eosinophils and neutrophils, and the development of AHR were significantly inhibited by intratracheal IL-10 administration. Moreover, IL-10 administration markedly decreased the numbers of ICAM-1⁺ and VCAM-1⁺ pulmonary vascular endothelial cells, which express IL-10 receptor 1. IL-10 could suppress eosinophil and neutrophil infiltration by inhibiting the proliferation of ICAM-1⁺ and VCAM-1⁺ pulmonary vascular endothelial cells, resulting in inhibition of AHR in severe asthmatic mice.

Virus infection may affect the epigenetic regulations in host cells, including post-translational histone modifications. Ubiquitination of histone H2B, has been reported to be involved in transcription activation. However, it remains unknown the role of histone ubiquitination in the pathology of virus infection (e.g. influenza virus, SARS-CoV2). CNOT4 that is a component of the CCR4-NOT complex has a ubiquitin transferase activity at the RING domain (L16). Here we show that CNOT4 is responsible for histone H2B ubiquitination in the host cells, which was linked to H3K4 methylation. Upon influenza virus or SARS-CoV2 virus infection CNOT4 interacted to virus protein, resulting in the loss of H2B ubiquitination and H3K4 methylation, which suppress interferon-related gene expression. The cells with a ubiquitination activity site of L16 of CNOT4, have increased virus replication. These results suggest that the CNOT4 is involved in the virus replication through histone H2B ubiquitination.

The chemokine CCL28 is mainly expressed in mucosal tissues such as colon and salivary gland, and migrates IgA-secreting cells via its receptor CCR10. Because IgA protects mucosal tissues from pathogenic microorganisms, CCL28 is thought to play an important role in mucosal immunity. However, CCL28 has been shown to be also expressed in non-mucosal tissues. Although some previous studies reported that CCL28 was increased in liver of patients with chronic liver disease, the detailed involvement in the pathology remains unclear. In this study, we examined the influence of CCL28 deficiency on carbon tetrachloride (CCl₄)-induced chronic hepatitis in mice. Chronic treatment with CCl₄ increased CCL28 expression levels in the liver. CCL28-deficient mice showed increased serum ALT levels and fibrotic areas of the liver. CCl₄ treatment also increased IgA-secreting plasma cells (PC), which expressed CCR10, in the liver of wild-type mice, but not CCL28-deficient mice. The hepatic IgA-secreting PC, but not IgA-negative PC, expressed IL-10, FasL, and PD-L1. Furthermore, CCL28-deficient mice showed decreased apoptosis and activity of hepatic stellate cells which are a key player in the progression of liver fibrosis. These findings suggest that CCL28 would suppress the pathology of chronic hepatitis through the migration of IgA-secreting PC.

Aging is associated with a progressive decline in cognitive function. While obesity accelerates aging process of the brain,physical activity preserves and improves cognitive performance in the elderly. The expression of a chemokine CX3CL1 has been reported to be increased in both conditions. CX3CL1 is known to chemoattract T cells and monocytes. Moreover, it promotes survival of neurons in the brain and b-cells in the pancreas. In adipose tissues, it is expressed in adipose cells and attenuates effects of obesity-induced chronic inflammation. However, the role of the lifestyle-induced CX3CL1 expression in cognitive aging is unknown. Here, we administered CX3CL1 into the peritoneal cavity of aged mice (15-16 months old) to investigate its impact on the aging process of cognition. In the hippocampus, CX3CL1 increased the number of Type-2 neural stem cells and promoted brain-derived neurotrophic factor (BDNF) expression. This treatment, furthermore, improved novel object recognition memory impaired with advancing age. Intraperitoneal transplantation of peritoneal cells from CX3CL1-treated aged mice improved novel object recognition memory in recipient aged mice. Vagotomy inhibited the CX3CL1-induced increase in BDNF expression. Thus, our results demonstrate that a novel connection among peritoneal cells, the vagal nerve and the hippocampus can reverse the age-associated decline in recognition memory.

Introduction: Interleukin (IL)-34, a macrophage (Mø) mediator, is expressed by tubular epithelial cells. However, the influence of IL-34 on tubulointerstitial fibrosis remains to be fully elucidated. We investigated the effect of IL-34 on renal fibrosis caused by unilateral ureteral obstruction (UUO). Material and Methods: 10-week-old male C57BL/6 (B6) mice (n=16) were induced UUO. Groups of animals were given either anti-mouse IL-34 antibody (UUO+anti-IL-34 Ab, 400 ng/kg, n=8) or vehicle (UUO+V, equal volume of saline, n=8) daily by intraperitoneal injection. Four age-matched male B6 mice received sham operation as control. All mice were sacrificed on day 10. Results: Compared to the control, the UUO+V mice exhibited remarkable intrarenal expressions of IL-34 and its two receptors (cFMS and PTP-ζ), which were significantly suppressed by anti-IL-34 Ab treatment. Compared to the UUO+V mice, tubular injury and sirius red positive area were significantly attenuated in the UUO+anti-IL-34 Ab mice. Treatment with anti-IL-34 Ab significantly suppressed the number of F4/80⁺ Mø and α-SMA⁺ myofibroblast in damaged kidneys of UUO. The renal cortical transcript levels of TGF-β, COL-1, TNF-α, IL-6, MCP-1/CCL2, and MIP-1/CCL3 were significantly lower in the UUO+anti-IL-34 Ab mice. Conclusion: Elevated IL-34 expression was related to renal fibrosis. Inhibition of IL-34 with neutralizing Ab suppressed expressions of inflammatory cytokines and fibrogenetic genes via reducing the Mø infiltration, which might lead to attenuate the development of renal fibrosis.

It is widely known that cannabinoid type 2 (CB2) receptor deficiency enhances neuroinflammation and pain development in the animal model of nerve injury-evoked neuropathic pain. We previously proposed the upregulated leptin signaling at the peripheral nerve as one of the underlying molecular mechanism, as nerve-injured CB2 receptor knockouts (CB2-KO) displayed robust upregulation of leptin receptors in both injured and non-injured nerve tissue. Those leptin receptors seemed to be expressed on the macrophages which is recruited to the nerve by the tissue injury, indicating the infiltration of leptin receptor-expressing macrophages. Thus, Due to these past results we also hypothesized that lack of CB2 receptor might also enhance the high fat diet (HFD)-induced peripheral neuroinflammation. However, surprisingly, CB2-KOs showed the significant resistance to the HFD-induced neuroinflammation. Namely, 5-week feeding of HFD induced substantial hypersensitivity in WT mice, while tactile sensitivity of HFD-fed CB2-KO remained intact. In the same animals, we further found the significant upregulation of infiltrated macrophages and chemokine receptor CXCR4 expression in HFD-fed WT animals, but not in either HFD-fed CB2 knockout mice or standard fat diet (SFD)-fed WT and CB2-KO controls. Based on these results, we will propose that CB2 receptors might have the bipolar regulatory role to chemokine receptor-mediated inflammatory response, which in the end enhance or inhibit the development of neuroinflammation depending on its cause.

Atopic dermatitis (AD) is a chronic skin disease with severe inflammation and pruritus. Traditional Kampo medicine Orengedokuto improves AD symptom in human patients and atopy-like symptoms in AD mouse model. Berberine is a major component of Orengedokuto. This study investigated the effects and molecular mechanisms of berberine on AD-like symptoms in mice. In NC/Nga mice with atopy-like dermatitis (dermatitis mice), intermittent oral administrations of berberine inhibited skin symptom, itching, cutaneous infiltration of eosinophils and mast cells, and the cutaneous expression of eotaxin, macrophage migration inhibitory factor (MIF) and IL-4. Berberine also inhibited both IL-4/MIF-induced eotaxin in fibroblasts and allergen-induced MIF and IL-4 in mast cells. In mast cells, the GeneChip^® microarray analysis showed that antigen increased the expression of EIF3F and MALT1, inhibited by berberine. The regulation of these factors by siRNAs for them showed antigen-induced the expression of MIF and IL-4. These results suggest that berberine inhibits AD-like symptoms through at least downregulation of EIF3F and MALT1 in mast cells.

Oral tolerance (OT) was immune regulatory system for foods. Food allergy (FA) patients are induced allergy by inhibition of acquiring OT or overriding OT. Recently, it has been hypothesized that immune tolerance is induced for foods taken orally and that allergy is caused by exposure via skin. In this study, we tried to override OT through exposure of food antigen to skin. We established three murine FA models to analyze the relationship between OT and percutaneous sensitization. IP model: Mice were injected ovalbumin (OVA) intraperitoneally (IP) followed by induction of food allergy. EC model: Mice were pasted a filter paper containing OVA on shaved back skin for epicutaneous sensitization (EC). ID model: Mice were injected OVA intradermally (ID) for sensitization via skin. OT was induced by oral OVA treatment before the sensitization in each model. FA was estimated by drop of body temperature, diarrhea, and OVA-specific IgE level in plasma. In all FA models, we confirmed FA symptoms involving elevation of OVA-specific IgE. OT induction inhibited the increasing in IgE level and suppressed FA in IP model. In EC model with OT induction, we detected increasing in IgE level. In ID model with OT induction, we confirmed FA symptoms and increasing in IgE level. The data indicated that exposure of OVA via skin could override OT.

Background: The clinical efficacy of Japanese cedar (JC) pollen SLIT tablets for allergic symptoms during Japanese cypress (JCY) pollen dispersal has been controversial in actual clinical settings. JC and JCY both belong to the Cupressaceae family and the major allergens of the two species have strong amino acid homology. The efficacy of JC pollen SLIT tablets against seasonal allergic rhinitis during the JC pollen and JCY pollen dispersal periods was investigated.

Methods: A post-hoc analysis was conducted in a phase II/III study (JapicCTI no. 142579). Patients with JC pollinosis (aged 5-64 years) were included (placebo n=159, 5000 JAU n=158). Patients in the active 5000 JAU treatment group was treated with JC SLIT tablet daily by self-administration for the duration of the trial. Clinical efficacy was evaluated by the total nasal symptom and medication score (TNSMS) during the peak symptom periods of each pollen season over 3 years.

Results: The daily average TNSMS in the 5000 JAU group was consistently lower than in the placebo group during both the JC and JCY pollen dispersal periods in all three seasons. The larger reduction in TNSMS in the 5000 JAU group compared with the placebo group was observed in a treatment duration-dependent manner.

Conclusions: JC SLIT tablet treatment showed sustained clinical efficacy on allergic symptoms during 3 consecutive JCY pollen seasons. Further studies are required to examine the immunological responses and extent of antibody cross-reactivity towards the homologous major allergens from different Cupressaceae family species including JC and JCY.

Oxaliplatin is a platinum-based chemotherapeutic agent treating colorectal and gastric cancer. The most common dose-limiting adverse event of oxaliplatin treatment is chronic neuropathic pain, while the detailed mechanisms remain unknown. The present study investigated the molecular mechanisms of microglial regulation of the oxaliplatin-induced neuropathic pain. Intrathecal (I.t.) treatment with inducible nitric oxide synthetase (iNOS) inhibitor 1400W attenuated the oxaliplatin-induced cold and mechanical hypersensitivity. In addition, pharmacological and chemogenetical inhibition of macrophage/microglia also attenuated the oxaliplatin-induced cold and mechanical hyperalgesia. I.t. treatment with STAT3 inhibitor stattic attenuated the oxaliplatin-indued cold and mechanical hypersensitivity. Oxaliplatin induced the increased phosphorylation of neural STAT3 in the dorsal root ganglion (DRG), which was attenuated by i.t. pretreatment with 1400W. Since STAT3 phosphorylation was regulated by PTEN, effects of oxaliplatin on the PTEN activity in the DRG were examined. PTEN expression was decreased by oxaliplatin treatment. Our present study suggested that oxaliplatin induces neural STAT3 activation through the iNOS induction by macrophage/microglial activation in the DRG, which resulted in the oxaliplatin-induced neuropathic pain.

Background: Although glioblastoma (GBM) stem like cells (GSCs), which exhibit chemo-radio resistance and recurrence, are key prognostic factors in GBM patients, molecular mechanisms of GSCs development are largely unknown. Recent studies revealed that extrinsic ribosome incorporation into somatic cells played key roles in cell reprogramming process towards stem cell properties. In this study, we sought to elucidate the mechanisms underlying GSCs development by focusing extrinsic ribosome incorporation into GBM cells.

Results: The ribosome incorporation into GBM cells significantly increased ribosome induced cancer cell spheroid (RICCS) formation, and showed the stem like cell characters. In RICCS, phosphorylation and protein expression of RPS6, an intrinsic ribosomal protein, and STAT3 phosphorylation were involved in regulating cell spheroids formation. Interestingly, glioma-derived extrinsic ribosome also promoted GBM-RICCS formation through the intrinsic RPS6 phosphorylation. Moreover, in glioma patients, RPS6 phosphorylation was observed in higher grade glioma tissues, and predominantly up-regulated in GSCs niches, such as perinecrosis niche and perivascular niche.

Conclusion: Our results suggest the potential biological & clinical significance of extrinsic ribosomal proteins in GSCs development.

Cells in our body are often exposed to hypoxic environment. In such environment, cells regulate respiration and metabolism for adaptation (hypoxic response). Hypoxic response not only plays a key role in maintaining homeostasis, but also in different diseases, such as cancer. Hypoxia-Inducible Factor (HIF) is a central transcription factor in hypoxic response. HIF is promptly up-regulated in hypoxia, and forms adapted cellular state. HIF has multiple target genes, and they coordinately regulate hypoxic response. Recently, HIF specific inhibitor was developed, and it is currently under clinical trial. Alternatively, we have demonstrated that HIF is downregulated, and CREB and NF-kB become activated during chronic phase of hypoxia. However, it remains unclear how the expression pattern of hypoxia-responsive genes changes depending on the timing.

HIF becomes activated by a heterodimer formation of a and b subunits. There are three a subunit isotypes, and they commonly bind to b subunit, ARNT. We have established ARNT knockout (KO) colon cancer HCT116 cells. These cells showed a clear inhibition of typical HIF target gene LDHA. In contrast, MMP1, which is induced under chronic hypoxia, was equally up-regulated in both wild type and KO cells, but its expression level is significantly reduced in KO cells. Altogether, these results indicate that induction of hypoxic genes is dependent on transcription factors which are activated during chronic phase, however, basal expression of them is mediated by a HIF-dependent machinery.

Despite notable advances in chemotherapy protocols and targeted therapies, ensuing drug resistance limits the efficacy of cancer treatments, calling for the identifying druggable targets that can overcome chemo-resistance. Here we show that the mitochondria-shaping protein OPA1 takes part in the resistance against the tyrosine kinase inhibitor gefitinib in lung adenocarcinoma cells. In gefitinib-resistant lung cancer cells, OPA1 levels were increased, mitochondrial cristae structures were narrower and mitochondrial respiration increased. Genetic and pharmacological OPA1 inhibition in the resistant lung cancer cells sensitized them to gefitinib-induced cytochrome c release and apoptosis. In vivo, orthotopic tumors formed by the injection of gefitinib-resistant lung cancer cells were insensitive to gefitinib treatment, but a combination of gefitinib and OPA1 inhibitor reduced tumor size and increased apoptosis. Our data identify the mitochondrial protein OPA1 as a downstream factor that sustains gefitinib resistance and can be targeted to overcome chemo-resistance.

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy via gene amplification and protein overexpression. Previously, we developed an anti-human EGFR (hEGFR) monoclonal antibody, EMab-134, which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity by flow cytometry, western blotting and immunohistochemistry. In this study, we produced a defucosylated mouse–dog chimeric anti-EGFR monoclonal antibody, E134Bf. Kinetic analysis of the interactions of E134Bf with the canine osteosarcoma cell line (D-17) and canine fibroblastic cell line (A-72) cells was conducted by flow cytometry. The K_D for the interaction of E134Bf with the D-17 and A-72 cells was 5.5 × 10⁻¹⁰ M and 6.0 × 10⁻¹⁰ M, respectively, indicating that E134Bf exhibits high affinity for D-17 and A-72 cells. Furthermore, E134Bf highly exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 and A-72 cells. In vivo administration of E134Bf significantly suppressed the development of D-17 and A-72 compared with the control dog IgG in mouse xenografts. These results indicate that E134Bf exerts antitumor effects against dEGFR-expressing canine cancers and could be valuable as part of an antibody treatment regimen for dogs.

Hyaluronic acid (HA) nanogel is derived by the partial modification of hyaluronicacid with cholesterol, causing the formation of self-assembled nanometer-scalehydrogel in water. Thus, HA nanogels might contribute to alter drug delivery ofpoorly water-soluble drugs. In this study, we tried to investigate the effect ofHA nanogels by using cyclosporine (CyA) in rats. HA nanogels (Asahi Kasei Co, 5mL/kg) containing CyA (2.5-15 mg/kg) were subcutaneously administered in male SDrats (Charles River) and whole blood samples were collected at the designatedtime-points up to 28 days. The concentrations of CyA were measured usingLC-MS/MS 6045 (Shimadzu). In rats administered CyA without HA nanogel (controlgroup), the concentration of CyA was below the detection limit (3 ng/mL) on 7days after administration. Some formulas of HA nanogel showed thesustained-release properties of CyA. Interestingly, one formula could bedetectable up to at least 10 days after administration. HA nanogels would beuseful as a tool to alter drug delivery of poorly water-soluble drugs such asCyA.

In skeletal muscle, depolarization of the plasma membrane triggers Ca²⁺ release from the sarcoplasmic reticulum (SR),referred to as depolarization-induced Ca²⁺ release (DICR). DICR occurs via the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including β1a, Stac3 adaptor protein and junctophilins. It has recently become clear that mutations in these components cause various skeletal muscle diseases. However, no specific treatment has been developed yet. In this study, we established a high-throughput platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced using baculovirus vectors, and Ca²⁺ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca²⁺ indicator. High [K⁺] depolarization triggered rapid Ca²⁺ release, indicating successful reconstitution of DICR. We tested several known drugs modulating DICR. Whereas RyR1 inhibitors, dantrolene and Cpd1, suppressed DICR, twitch potentiators, e.g., perchlorate, accelerated DICR. These results well reproduced the findings with the muscle fibers and the cultured myotubes. The reconstituted DICR platform will be highly useful for drug discovery for skeletal muscle diseases.

We have been searching for mechanism to induce smooth muscle contraction that is not associated with phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. We report that arachidonic acid (AraA) stimulates ATPase activity of unphosphorylated smooth muscle myosin with maximal stimulation (Rmax) of 6.84 + 0.51 relative to stimulation by the vehicle and with a harf-maximal effective concentration (EC50) of 50.3 + 4.2 μM. In presence of actin Rmax was 1.72 + 0.08 and EC50 was 26.3 + 2.3 μM. Our experiments with eicosanoids consisting of the AraA cascade suggested that they neither stimulated nor inhibited the activity. Under conditions that did not allow RLC to be phosphorylated. AraA stimulated contraction of smooth muscle tissue and culture cells with an Rmax of 1.45 + 0.07 and EC50 of 27.0 + 4.4 μM. In addition to the ATPase activities of the myosin, AraA stimulated those of heavy meromyosin, subfragument 1 (S1), S1 from which the RLC was removed, and a recombinant heavy chain consisting of the myosin head. The stimulatory effects of AraA on these preparations were about two fold. The site of AraA action was indicated to be the step-releasing iorganic phosphate (Pi) from the reaction intermediate of the myosin-ADP-Pi complex. The enhancement of Pi relese by AraA was supported by computer stimulation indicating that AraA docked in the actin- binding cleft oh the myosin motor domain. The stimulatory effect of AraA was detectable with both unphosphorylated myosin and the myosin which RLC was fully phosphorylated. The AraA effect on both myosin forms was suggested to cause excess contraction and such as vasospasm.

One of the most dynamic processes during skeletal myogenesis is cell-cell fusion, which promotes skeletal myoblast to form a large multi-nucleated myofiber equipped with EC-coupling machinery. Recently discovered transmembrane protein Myomixer (Mymx) executes cell-cell fusion, although the physiological significance of Mymx-mediated fusion on myogenic cell differentiation remains largely unknown. The current study focuses on the intracellular Ca²⁺ signaling mechanism to understand the physiological importance of Mymx mediated cell fusion in regulating voltage-induced Ca²⁺release in the developing skeletal muscle cells. C2C12 cells were used as an in vitro myogenesis model of skeletal muscle cells. We investigated intracellular Ca²⁺ release upon electric field stimulation applied to differentiated Mymx-KO cells and Mymx-rescued cells. We found that the efficiency of Ca²⁺ response was dependent on the Mymx gene expression. Importantly, expression levels of MyoD and myogenin were almost unaltered by the gene rescue of Mymx, suggesting that the Mymx-dependent Ca²⁺ response is regulated independently of these transcription factors. In conclusion, we found a novel regulatory linkage between Mymx expression and the voltage-induced Ca²⁺ release essential for EC coupling.

Lipopolysaccharide (LPS), a component of the Gram-negative bacterial cell wall, activates Toll-like receptors (TLRs). Porphyromonas gingivalis (Pg) appears to play a role in the development of periodontal disease. In vitro studies have suggested that unlike LPS derived from Escherichia coli (Ec-LPS), which stimulates TLR4, LPS derived from Pg (Pg-LPS) may inhibit the TLR4. Mice exposed to a novel environment show hyperlocomotion that is inhibited by systemic administration of Ec-LPS. However, whether Pg-LPS influences novelty-induced locomotion is unknown. Therefore, we carried out an open field test to analyze the effects of Pg-LPS. For comparison, effects of Ec-LPS were also studied. Male ddY mouse (25-30 g) were used. The movement of each mouse in the open field was recorded for 30 min using a commercially available behavioural analysis system and the distance travelled (cm) was determined. Each compound was given intraperitoneally 4h before the open field test. Ec-LPS 500 and 840 µg/kg, but not 100 µg/kg, inhibited novelty-induced increases in distance travelled. Inhibition of hyperlocomotion by 840 µg/kg Ec-LPS was counteracted by co-administration of the TLR4 antagonist TAK-242 (3.0 mg/kg). Pg-LPS (100, 500 or 840 µg/kg) failed to alter novelty-induced locomotion. The present results provide in vivo evidence that Ec- and Pg-LPS induce different effects. Thus, Ec- but not Pg-LPS inhibits novelty-induced locomotor activity in mice by activating TLR4.

Differentiation-inducing factor-1 (DIF-1) isolated from the cellular slime mold Dictyostelium discoideum has been shown to promote glucose uptake in mouse 3T3-L1 fibroblasts and differentiated 3T3-L1 adipocytes ⁽¹⁾. DIF-1 promotes glucose uptake in the cells, at least in part, via an AMP kinase-dependent pathway (a PI-3 kinase/Akt-independent pathway), which is different from the insulin-induced glucose uptake pathway. In this study, we investigated the actions of DIF-1 in skeletal muscle, the largest glucose-metabolizing tissue, using C2C12 myotube cells.

　During 15 h of incubation, DIF-1 at 1–20 μM promoted glucose consumption (uptake) in a dose-dependent manner in C2C12 myotube cells, while DIF-1 at 10–20 μM was slightly toxic to the cells. DIF-1 (2 μM)-induced glucose consumption was hardly inhibited with wortmannin (0.1 μM), a PI3K inhibitor, but was partially inhibited with compound C (30 μM), an AMP kinase inhibitor. These results suggest that DIF-1 promotes glucose uptake in skeletal muscle cells via the same mechanisms as those in adipocytes and also that DIF-1 may have therapeutic potential in the treatment of obesity and/or diabetes.

(1) Omata, W. et al. FEBS J. 274, 3392-3404. (2007).

Background. The role-play for pharmacological education (RPPE) provides a practical training focused on the basis of drug therapy. Learning achievement in RPPE, however, has not been researched precisely. In this study, we assessed the learning achievement in RPPE by using a self-assessment rubric.

Methods. Participants: Fourth grade medical students at Tohoku Medical and Pharmaceutical University who took the online RPPE (oRPPE) in 2022. Measurements: Self-assessment rubric was presented to participants on the first day of the course. Four following categories were scored on a five-point scale: I) Understanding: a. health problems of the case in charge; b. drug therapy of the case in charge; c. role-play performed by the others, II) Preparation, III) Performance and IV) Discussion and dialogue. Students were directed to assess themselves during preparation and performance of oRPPE. Data collection and analysis: Scores were collected after course completion. All data are presented as the mean ± standard error of the mean (S.E.M.).

Results. The self-assessment showed high scores in the following 3 categories: I) Understanding (a. 4.10 ± 0.06; b. 4.10 ± 0.06; c. 3.94 ± 0.07), II) Preparation (4.32 ± 0.06) and III) Performance (4.21 ± 0.07). These results indicate that RPPE could provide basic practice for drug therapy. It also implies that specification of expected outcomes through rubric beforehand might be useful to guide self- and peer-learning during preparation and performance. On the other hand, the score in IV) Discussion and dialogue (3.75 ± 0.09) might reflect difficulties to discuss the whole process of clinical practice in each case, which requires advanced professional competencies.

Conclusion. The self-assessment rubric is a useful tool for assessing achievement of learning in RPPE.