Pituitary adenylate cyclase-activating polypeptides (PACAPs) were discovered in the 1980s in the process of searching for unknown hypothalamic factors after the successive discovery of hypophysiotorpic hormones such as TRH, GHRH, and CRH. PACAP38, consisting of 38 amino acids, and PACAP27, consisting of 27 residues at its N-terminus, were isolated and identified from sheep hypothalamic extracts in 1989 using the stimulatory activity of cyclic AMP production in rat pituitary cells. Their structures show 68% homology to vasoactive intestinal peptide (VIP) and belong to the secretin-glucagon family. PACAP38 is widely distributed in the brain, with the highest concentration in the hypothalamus, where it promotes the secretion of almost all pituitary hormones. PACAP38 is widely distributed in the brain and is found in highest concentration in the hypothalamus, but it promotes the secretion of almost all pituitary hormones, and its full physiological function in the hypothalamus has yet to be elucidated. However, focusing on its function as a neurotrophic factor, we aim to apply it clinically as an agonist in the treatment of ischemic brain diseases such as stroke and neurodegenerative diseases. In the meantime, research is being conducted on the clinical application of antagonists for the treatment of intractable pain based on their function in pain transmission as neurotransmitters.

One cannot overstate that both vaccine and antiviral therapeutics are needed to control viral infections. The caveat in the initial response to COVID-19 was that the severity of COVID-19 varies much: some patients have no symptoms, but others die on respirators. While only large randomized studies can give insight into whether the "drugs" help patients, clinicians and researchers were fooled into thinking that those who recovered early were helped by the test drug. Indeed, panic and disorganization dominated the study of COVID-19 drugs in the early stages of our fight with COVID-19. Currently, there are only few to name in the list of possibly efficacious therapeutics. In the present presentation, I would discuss the development of orally available therapeutics for COVID-19 based on the knowledge and skills used in the development of therapeutics for HIV infection and AIDS and its perspective.

Sleep is a ubiquitous behavior in animals with a central nervous system. However, despite the fact that the executive neurocircuitry and neurochemistry for sleep/wake switching has been increasingly revealed in recent years, the fundamental mechanism for homeostatic regulation of sleep, as well as the neural substrate for ”sleepiness” (sleep need), remains unknown. To crack open this black box, we have initiated a large-scale forward genetic screen of sleep/wake phenotype in mice based on true polysomnographic (EEG/EMG) measurements. We have so far screened >8,000 heterozygous ENU-mutagenized founders and established a number of pedigrees exhibiting heritable and specific sleep/wake abnormalities. By combining linkage analysis and the next-generation whole exome sequencing, we have molecularly identified and verified the causal mutation in several of these pedigrees. Biochemical and neurophysiological analyses of these mutations are underway. Since these dominant mutations cause strong phenotypic traits, we expect that the mutated genes will provide new insights into the elusive pathway regulating sleep/wakefulness. Indeed, through a systematic cross-comparison of the Sleepy mutants and sleep-deprived mice, we have recently found that the cumulative phosphorylation state of a specific set of mostly synaptic proteins may be the molecular substrate of sleep need.

In recent years, technologies have been developed to degrade target proteins in cells, and drug discovery research has been actively conducted. Molecular glues such as lenalidomide, and chimeric compounds such as PROTACs (Proteolysis Targeting Chimeras) and SNIPERs (Specific and Nongenetic IAP-dependent Protein Erasers) are well known. These compounds degrade the target proteins in the cells, and thus exhibit different pharmacological properties from small molecule inhibitors. PROTACs/SNIPERs are chimeric compounds, in which a ligand that binds to a target protein is linked to a ligand that binds to the E3 ubiquitin ligase, forcing the target protein to be ubiquitylated and degraded by proteasome in the cells. The modular structure of PROTACs/SNIPERs allows us to rationally design the compounds to degrade protein of your interest by substituting target ligands, and therefore, it is expected to be a novel platform technology for drug discovery. In this talk, I will introduce our SNIPER compounds and the current status of proteolytic drug development.

The development of neuronal circuits in vivo depends on precise regulation of synapse formation, elimination, and remodeling. In vivo two-photon imaging confirmed the presence of two phases of synapse dynamics in the postnatal mouse cortex. In the first phase (until postnatal 20 days), synapse turnover is maintained to be high, while in the second phase (after three weeks postnatal), synapse dynamics are highly suppressed, leading to the maturation of the cortical neural network. The transition in synapse dynamics may underlie the pathophysiology of neurodevelopmental disorders and psychiatric diseases, but their precise mechanisms have not yet been clarified. Our laboratory has focused on (1) the diverse mechanisms in neural circuits and synapse formation, (2) the structure-function relationship in dynamic synaptic changes, and (3) the relationship between brain diseases and synaptic dysfunction. In particular, we have recently developed new tools for studying neural circuits, such as quantitative methods for analyzing the nano-structure of spine synapses and methods for measuring the molecular dynamics inside spines. Furthermore, we are applying these tools to the study of brain pathology. In this talk, I will introduce these researches and discuss the validity and prospects of understanding the pathogenesis of psychiatric disorders as synaptic dysfunction.

The Pharmaceuticals and Medical Devices Agency, PMDA, plays three key roles, namely relief services for persons injured by adverse reactions to drugs and regenerative medical products, product reviews, and safety measures.

Based on the concept of the regulatory science, PMDA has been conducting evidence-based review and evaluation, and has been providing citizens and healthcare professionals with rapid access to safer, more effective medical products.

To promote innovative products, PMDA took various measures, i.e. establishment of expedited review tracks and consultation services in response to the needs. Also, the efforts and challenges we face to combat COVID-19 would be introduced in this session.

Today, international collaboration and convergence is necessary and essential in the field of regulatory affairs. PMDA has been continuously contributing to the ICMRA, International Coalition of Medicines Regulatory Authorities, executive-level, strategic coordinating, advocacy and leadership entity of regulatory authorities. The COVID-19 related issues are hot topics of the ICMRA discussion.

The latest international trends of the regulatory affairs, and current situation and challenges of the development of the products would be introduced in this session.

Oncolytic viruses (OVs) are promising candidates for innovative cancer treatment technologies. We developed an original platform technology of "Conditionally replicating adenovirus that regulated with multiple factors" (m-CRA) for efficiently developing the next-generation OVs. Survivin-responsive m-CRAs (Surv.m-CRAs), which we developed using the technology, demonstrated the innovative therapeutic effects, including the potent effectiveness against most of malignant tumors in the strictly cancer-specific (safe) manner and the increased effectiveness against cancer stem cells, which are resistant to conventional therapies. After manufacturing GMP products and preforming GLP nonclinical studies in accordance with ICH guidelines, we completed the First-In-Human phase I clinical trial of Surv.m-CRA-1 (no transgene) for malignant tumors in bone or soft tissue region and cofirmed the high safety and the remarkable effectiveness. Currently, we are performing two Phase II clinical trials of Surv.m-CRA-1 for malignant bone tumors toward approval and for pancreatic cancer. Moreover, we are performing nonclinical development of Surv.m-CRA-2 (armed with a cytokine gene), which more strongly induced systemic antitumor immunity, and developing further novel technologies. In this lecture, I would like to present mainly these projects and also briefly growth factor gene therapy for intractable diseases and viral vector technology for pluripotent stem cell-based regenerative medicine.

The importance of P2Rs has increased in various fields in recent years, but this time I will focus on research on pain. Chronic pain is a debilitating condition that often occurs following peripheral tissue inflammation and nerve injury. Especially neuropathic pain is a significant clinical problem because there are few clinically effective drugs. I will review the findings for the role of ATP signaling through P2X3Rs and P2X2/3Rs in primary afferent neurons and through P2X4Rs, P2X7Rs, and P2Y12R in spinal microglia in chronic pain. Many discoveries have strongly accelerated the search for new drugs that target P2Rs, and several compounds have been developed so far in the world. Gefapixant is the only P2X3R antagonist that has finished clinical trials as a refractory chronic cough, and it is under clinical trial for endometriosis-related pain. NP-1815-PX and NC-2600 are recently identified as novel P2X4R antagonists in Japan. NC-2600 has been finished the Phase I study without serious side effects. I think we'll soon get new drugs against pain.

Drug addiction can be viewed as a stable form of drug-induced neural plasticity, whereby long-lasting changes in gene expression mediate some of the stable behavioral abnormalities that define an addicted state. Our laboratory has focused on transcriptional pathways in addiction, deduced from large RNA-sequencing datasets of RNAs that show altered expression in brain reward regions of mice as a consequence of drug self-administration, withdrawal, and relapse. Activation or induction of certain transcription factors represent homeostatic adaptations that oppose drug action and mediate aspects of drug tolerance and dependence. In contrast, induction of other transcription factors exerts the opposite effect and contributes to sensitized responses to drug exposure. We are also characterizing a range of chromatin mechanisms that act in concert with these transcription factors to control gene expression. These studies are identifying many of the molecular targets of drug self-administration in brain reward regions and the biochemical pathways most prominently affected. Parallel work has focused on homologous regions in the brains of addicted humans examined postmortem. These advances can now be mined to develop improved diagnostic tests and treatments for addictive disorders.

Funded by the National Institute on Drug Abuse

Over the past four decades, no class of drugs has had more impact on cardiovascular health than the HMC-CoA reductase inhibitors or statins. Developed as potent lipid-lowering agents, statins were shown to reduce mortality and morbidity of patients who are at risk for cardiovascular disease. However, retrospective analyses of some of these clinical trials have uncovered some aspects of their clinical benefits that may be additional to their lipid-lowering effects. In cell culture and animal studies, these effects alter the expression of endothelial nitric oxide synthase, the stability of atherosclerotic plaques, the production of pro-inflammatory cytokines and reactive oxygen species, the reactivity of platelets, and the development of cardiac hypertrophy and fibrosis. Such cholesterol-independent or "pleiotropic" effects of statins generated intense interest as to their potential mechanism and created debate over their relative contribution to cardiovascular risk reduction. One potential mechanism for statin pleiotropy is through inhibition of isoprenoid synthesis and protein prenylation. In particular, the prenylation of Rho GTPases such as Rho, Rac, and Cdc42 is critical to their cellular localization and function. Thus, inhibition of Rho and its downstream effector, Rho kinase, by statins may constitute an important pleiotropic mechanism that could be exploited therapeutically for non-lipid conditions beyond cardiovascular disease.

Transporters are membrane proteins that contribute to the distribution of compounds in the body. Drugs that target them can achieve various metabolic regulations. In the cells involved in pathogenesis, such as cancer cells, changes in the metabolic system due to metabolic reprogramming often play an essential role in maintaining their function and survival. In particular, nutrient transporters that contribute to the regulation of cellular metabolism can be targets for drugs that intervene in intracellular metabolism and prevent the development of pathological states. We discovered the amino acid transporter LAT1 (SLC7A5), upregulated in cancer cells. We revealed that it contributes to maintaining cancer cell-specific functions through the amino acid signaling centered on mTORC1. Inhibitors generated based on the structure-activity relationship analysis showed antitumor effects and regulated the function of cells other than cancer cells that express LAT1 in the course of pathogenesis, such as vascular endothelial cells and immune cells. In addition, the structure of LAT1 was recently elucidated by cryo-EM, showing the mechanism of substrate recognition and action of inhibitors. In this lecture, the role of transporters in metabolic reprogramming revealed by LAT1 inhibitors and the significance of LAT1 as a drug discovery target will be discussed.

Cancer stem cells (CSCs) are the cells that are the origin and play a central role in maintenance of cancer tissue. Clinically, the most important feature of CSCs is that they are resistant to various treatments and consequently lead to recurrence and metastasis. However, the therapeutic resistant mechanism of CSCs is diverse and depend on the cell of origin and their microenvironment. CSCs are thought to be established by two mechanisms; somatic stem cells change to CSCs by a driver gene mutation (stem cell origin type), and progenitor cells change to CSCs under the background of chronic inflammation (progenitor cell origin type). We have clarified that the differentiation control mechanism, the reactive oxygen species suppression and cell death suppression mechanism are involved in therapeutic resistance of CSCs. In this talk, I would like to explain these mechanisms elucidated by basic research data and propose new treatment strategies based on drug repositioning.

Recapitulating the three-dimensional organ structure in vitro is a major challenge for developmental biology and regenerative medicine. The kidney develops by the reciprocal interactions between the nephron progenitor and ureteric bud, and the origins of these two types of precursors are spatially distinct. Based on these developmental findings, we previously established the induction protocols of the nephron progenitors from pluripotent stem cells (PSCs). Induced nephron progenitors robustly formed nephrons: glomeruli and renal tubules. By generating iPS cell-derived nephron organoids from a patient with the congenital nephrotic syndrome, we reproduced the glomerular abnormalities that represent the initial phase of this disease. We also established the protocols to induce the ureteric bud from mouse and human PSCs. Mouse organoids reassembled from the differentially induced ureteric bud and nephron progenitors developed the inherent architectures of the embryonic kidney. Humans ureteric bud organoids were applied to autosomal dominant polycystic kidney disease to successfully reproduce cyst formation in vitro. Thus, kidney organoids will serve as useful basis to analyze human kidney development and disease.

The N-methyl-D-aspartate receptor (NMDAR) ketamine has produce rapid-acting and sustained antidepressant actions in treatment resistant patients with depression. Furthermore, ketamine can reduce suicidal ideation in severe patients with depression. The Johnson and Johnson in the USA developed esketamine as new antidepressant for treatment-resistant depression. In 2019, USA and Europe approved J&J esketamine nasal spray. In contrast, we reported that arketamine, another enantiomer of ketamine, could produce greater potency and longer-lasting antidepressant-like actions in rodent models of depression than esketamine. The Ph2 study of arketamine in depressed patients is underway by Perception Neuroscience (New York, USA). Otsuka Pharmaceutical company will start Ph1 study in Japan. Here, the author will discuss recent findings of novel antidepressant arketamine.

Differentiation-inducing factor (DIF) produced by Dictyostelium discoideum inhibits the growth, motility (migration and infiltration) and adhesion to endothelial cells of many types of cancer cells, which leads to the inhibition of cancer growth and metastasis. Its mechanism of action has long remained unclear, however recent studies have revealed that AMP-activated protein kinase (AMPK)-mediated inhibition of mTORC1 activity is the early signal of DIF. When mTORC1 is inhibited, S6 kinase is suppressed, resulting in the suppression of cell proliferation and cell motility through the suppression of STAT3 and Snail. And moreover, analysis of DIF-binding proteins has suggested that DIF binds to and inhibits type 2 malate dehydrogenase (MDH2), which may lead to the activation of AMPK. Although DIF synthase has not been found in organisms other than Dictyostelium, the target molecule MDH2 is conserved in almost all organisms. This is probably why the effects of DIF extend to mammals. Many drugs have been made from natural toxins, such as plant alkaloids, however drugs made from signal molecules of other organisms, such as DIF, are rare. In this talk, I would like to consider the possibility of clinical application of DIF.

The application of IT technology to medicine accelerates the improvement of medical quality, the development of diagnostic modality, and the application of precision medicine. The nicotine dependence treatment app & CO checker were approved as the first program medical device in Japan. A new category of medical care, digital therapy, is about to begin. Apps used for digital treatment are commonly referred to as digital medicine. Many of these apps are related to cognitive behavioral therapy and are also called behavior modification apps. US venture companies are leading the development of digital medicine. Japanese pharmaceutical companies and venture companies are following them. Since the approval of new digital drugs will be subject to examination by the Pharmaceuticals and Medical Devices Agency (PMDA), a new program medical device examination department has been established in PMDA to strengthen the examination system. In the future, it is expected that the development of digital drugs will progress and many products will be used in clinical practice. However, there are many issues to be resolved, such as the mechanism of action and the interaction between drugs and digital medicine. The pharmacology research on the analysis of the effects of digital drugs will progress in future.

Plasticity of central synaptic transmission has been considered as the mechanism of learning and memory. Its disruption causes neuropsychiatric and neurocognitive disorders. The molecular mechanism of synaptic plasticity has been the center of research since its discovery. I have been particularly interested in the postsynaptic signaling process regulating synaptic surface receptor and found that activity-dependent trafficking of various receptor and intracellular components during synaptic plasticity. This is triggered by the raise in intracellular Ca²⁺ concentration, which in turn triggers various signaling cascades. Among them, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) has been considered pivotal signaling molecule in the synaptic plasticity. However, its characteristic dodecameric structure and abundance at the synapse have been mystery. I found that CaMKII can indeed undergo liquid-liquid phase separation in a manner dependent on Ca²⁺/calmodulin- with its substrate proteins and cross-link them together. This modulates the distribution of synaptic surface receptors and define their nanodomains. In this way, Ca²⁺-dependent liquid-liquid phase separation of CaMKII and other synaptic proteins is a mechanism to modulate synaptic transmission persistently during synaptic plasticity.

The communication between the brain, immune and vascular systems is a key contributor to the onset and progression of neurological diseases. We discovered the coagulation factor fibrinogen as a blood-derived driver for neuroinflammation and inhibitor of repair in a wide range of neurologic diseases, such as multiple sclerosis, Alzheimer's disease and brain trauma. We showed that fibrinogen is necessary and sufficient for neurodegeneration and a new culprit for microglia-mediated oxidative stress-dependent spine elimination and cognitive impairment. By developing Tox-Seq, we reported the oxidative stress innate immune cell atlas in neuroinflammation. We developed cutting-edge imaging tools to study brain network synchronization and the neurovascular interface. We discovered a first-in-class fibrin-targeting immunotherapy to selectively target inflammatory functions of fibrin without interference with clotting with potent therapeutic effects in autoimmune- and amyloid-driven neurotoxicity. High throughput drug screens identified small molecule compounds to block fibrin-induced activation of microglia with therapeutic effects in neuroinflammatory disease. These findings could be a common thread for the understanding of the etiology, progression, and new treatments for neurologic diseases with neuroimmune and cerebrovascular dysfunction. As fibrin is a global activator of toxic innate immune responses in the brain and periphery, these studies could provide the basis for the development of a new class of therapeutics for autoimmune and inflammatory diseases¹.

1. Akassoglou, K. The immunology of blood: Connecting the dots at the neurovascular interface. Nat Immunol 2020, 21:710-712

Refractory neurological diseases, such as Parkinson's disease and fibromyalgia, may progress and become more severe due to multiple factors triggered by alterations in different neural types. Research on disease mechanisms using human iPS cells has allowed us to infer integrated neural cell fragility by inducing differentiation from one iPS cell to different neural subtypes and observing the intracellular changes caused by the disease. On the other hand, based on information obtained from studies on human iPS cells, tailor-made disease model mice can be produced, and an in vivo analysis of pathophysiological phenotypes can be achieved through the use of "reverse-translational neuroscience research". Hopefully, the development of pharmacological therapeutic approaches using these cell-specific genetically modified disease model animals will lead to tailor-made drug therapies that will be able to control disease onset and intractability. With these research efforts, I intend to perform "reverse-translational neuroscience research" through the integrated analysis of disease iPS cell differentiation-induced cells and would like to contribute to the development of the pharmacology field.

We developed radiopharmaceuticals for positron emission tomography (PET tracers) such as [¹⁸F]THK-5351 for imaging tau tangles, which is one of key neuropathological hallmarks in Alzheimer's disease (AD). Clinical PET studies revealed that [¹⁸F]THK-5351 possessed off-target binding. From validation studies such as imaging-autopsy correlations, we identified monoamine oxidase-B (MAO-B) as a dominant off-target binding substrate of [¹⁸F]THK-5351. MAO-B was predominantly expressed in astrocytes and elevated in various neurological conditions. Therefore, MAO-B would be an attractive target for imaging reactive astrogliosis. Though compound optimization from [¹⁸F]THK-5351, recently we developed a selective and reversible MAO-B PET tracer, [¹⁸F]SMBT-1. These translational and reverse-translational research from the development to validations of PET tracers lead to the generation of a new biomarker of reactive astrogliosis.

Neural circuits are mainly formed by genetic programs in early development but reorganized after birth under the influence of external factors. We have focused on social and environmental stress as an external factor and analyzed stress-induced changes in neural circuits and their functional significance. Long-term (chronic) stress attenuates dopaminergic function in the medial prefrontal cortex, leading to depressive-like behavior. Along with this behavioral change, long-term stress causes dendritic atrophy of pyramidal neurons in the medial prefrontal cortex. On the other hand, we found that short-term stress (acute) stress suppresses depressive-like behavior via dopamine response and dopamine D1 receptor signaling in the medial prefrontal cortex and induces dendritic hypertrophy of pyramidal neurons in a D1 receptor-dependent manner. Concomitantly, short-term stress increases the neural activity of the extended amygdala via dopamine D1 receptors in the medial prefrontal cortex. We have also revealed that rapid antidepressant effects of ketamine are mediated by neural circuits similar to those activated by short-term stress. In this presentation, I would like to introduce our recent findings and future directions on neural circuit plasticity in stress and depression.

The number of human patients with chronic kidney disease (CKD) is increasing with the aging of society. Similarly, the incidence of CKD in animals, especially dogs and cats, is also increasing with the aging of individuals.

In order to achieve "One Health" for humans and animals, it is important to study useful targets for the diagnosis and treatment of CKD in these mammals. However, the species specific-differences of renal pathogenesis complicate it; dogs tend to show renal glomerular lesions as humans, but most cats manifest tubulointerstitial lesions. Therefore, there is a need to develop therapeutic and diagnostic methods based on the elucidation of species-specific renal pathogenesis, and we are conducting comprehensive research targeting human as well as model animals and companion animals.

In particular, we continue to focus on the unique role of "urine" in various kidney diseases. Our previous studies have shown that urine from model or companion animals with CKD contains candidate disease markers such as cytokines and non-coding RNAs, which are involved in their renal pathogenesis. In addition, as for a unique recent topic, we found that urine is a biological fluid that alters the local immune status of urogenital tissues. Briefly, tertiary lymphoid structures in the renal pelvis, called "urinary tract-associated lymphoid structures (UTALSs)", were formed in humans and mice with chronic nephritis, regardless of infections. In the development of UTALSs, aging and altered immune status as wells as urine played a role in UTALS formation. In a nephritis mouse model, urine leaked from the lumen of the renal pelvis into its parenchyma. Based on obtained findings, we consider that altered urine-urothelium barrier-based UTALS formation may represent a novel mechanism underlying the pathogenesis of chronic nephritis, regardless of urinary tract infection.

Further studies focusing on urinary biomarker candidates and unique roles of urine in renal pathogenesis would be crucial for clinical development and understanding the mechanism of kidney disease progression, to realize One Health in the field of human and animal nephrology.

Recently, the use of neonicotinoid pesticides (NNs) has been increasing in place of organophosphorus pesticides, because NNs have very low toxicity to humans, and their toxicity is considered negligible in daily life, as long as it is below a certain standard. In Japan, however, food residue limits for fruits, vegetables, and tea leaves are set several to several dozen times higher than in other countries.

In this study, to clarify the exposure to NNs in Japan, we collected urine samples from newborns and infants and measured the amount of NNs in the urine to assess the exposure.

On the other hand, NNs have been reported to have effects on experimental animals even at lower exposure levels than NOAEL. For example, Hirano et al. reported that NNs induced anxiety behavior in mice at a dose level of 5 mg/kg, which is lower than the NOAEL of 9.7 mg/kg for clothianidin. Therefore, we sought to develop sensitive biomarkers to conduct appropriate toxicity assessment. Monoamine neurotransmitters (MAs) such as dopamine (DA) and serotonin (5-HT) regulate brain functions such as behavior, memory, and learning, and their abnormalities are said to be involved in the development of neurological diseases such as depression and Parkinson's disease. In order to clarify the distribution and concentration of MA in the brain, we developed a comprehensive, highly sensitive, and accurate analytical method using mass spectrometry, and attempted to clarify the effects of NN administration on MA in the brain.

As a result of quantification of MAs in various brain regions, no change in DA concentration in the striatum was observed after IMI administration, but decreases in 3-MT and DA in the olfactory bulb, and decreases in 5-HT and histamine in the striatum were observed.

Considering the fact that NNs disrupt neurotransmitters in the brain and that infants are exposed to multiple NNs, the exposure level of infants and newborns is not negligible, and it is necessary to elucidate the toxicity mechanism in more detail.

In our laboratory, we have applied lipidomics technology to elucidate the mechanisms of disease progression, and to find novel bioactive lipid, to search diagnostic biomarker for various types of diseases. We usually search for target molecules and obtained proof of concept using highly reproducible animal experimental models, and based on the results, we apply them to human clinical practice. For example, the urinary biomarkers (tetranor-PGDM) for food allergies that we discovered using animal models have been proved to be disease specific and symptom predictable in human clinical studies. In this symposium, I would like to introduce the biomarkers and physiologically active lipids of food allergies, atopic dermatitis, and allergic rhinitis that we have discovered, and their applications to humans.

Membrane transport proteins, such as transporters, pumps and channels, cannot fully exert their transport activity simply by being translated as polypeptide chains and inserted into membranes, but demonstrate the physiological transport activity only when the proteins are properly folded and localize to membrane domains where the proteins should function. Heterodimeric Amino acid Transporters (HATs) family consists of a light chain, which has a transport function, and a heavy chain, which is essential for the localization to the plasma membrane. HATs play important roles in various physiological processes. The rBAT (heavy chain)-b^0,+AT (light chain) complex, a member of the HAT family, transports cystine and basic and neutral amino acids. Mutations in either b^0,+AT or rBAT lead to cystinuria (renal cystine stones). Since the identification of the rBAT and b^0,+AT complex in the late '90s, hundreds of pathological mutations have been found. The studies of mutations improved the understanding of the biosynthesis and transport mechanisms of the complex. However, the pathological mechanisms of mutations outside the predicted substrate binding sites remain largely unknown. By combining biochemical analysis and cryo-EM structures, we solved the structure and function of rBAT-b^0,+AT and examined their biogenesis. As a result, we found that b^0,+AT recognizes the substrates at different amino acid residues depending on the type of substrate. Furthermore, we identified a Ca²⁺binding site in rBAT, and showed that Ca²⁺-mediated super-dimerization (dimer x dimer) is a key for the formation of higher-order structures of the complex and its localization to the plasma membrane. Accordingly, we elucidated the pathogenesis of HAT by mutations related to biosynthesis, which is different from mutations in the substrate-binding site. This study provides an understanding of the HAT biogenesis and serves as a guide to develop a new therapeutic approach. At molecular basis, the study lights up a novel role of Ca²⁺ on membrane protein biogenesis.

Voltage-gated K⁺ channels are tetramer, and four α (main) subunits form a single K⁺ channel. Each α subunit possesses a single voltage sensor domain consisting of four transmembrane segments (S1-S4) and contributes voltage-dependent gating. The fourth transmembrane segment (S4) is a central part of voltage-sensing. Membrane depolarization induces upward movement of the S4 segments, which leads to the channel opening. Most of the voltage-gated K⁺ channels have auxiliary subunits. These subunits could modulate gating properties and even change the affinity of agonist/antagonist. Therefore, it is essential to understand the structures of the macromolecular complex and how the auxiliary subunits modify the voltage-dependent gating. KCNQ1 channel is one of the most well-studied ion channels for gating modulation because its auxiliary subunit KCNE drastically changes the gating properties. For example, KCNE3 makes the KCNQ1 channel constitutively open. Recent cryo-EM structures clearly show KCNE3 interacts with the voltage sensor domain via the S1 segment of KCNQ1. By introducing mutations on putative interaction sites, we identified that the tight interaction between the S1 segment and KCNE3 is required to stabilize the channel's open state. This KCNQ1-KCNE3 complex could be an excellent model to study how auxiliary subunits modulate the gating behavior of voltage-gated ion channels.

Changes in cytosolic Ca²⁺ are involved in various biological responses such as neurotransmission, muscle contraction, and hormone secretion. Therefore, Ca²⁺ channels and their downstream molecules make supramolecular complexes to activate specific signal pathways. We have shown that caveolin (cav)-1, an essential component of caveolae, and junctophilin (JP)-2, a protein that bridges the plasma membrane and sarcoplasmic reticulum (SR), accumulate Cav1.2 voltage dependent Ca²⁺ channels, large conductance Ca²⁺-activated K⁺ (BK) channels and ryanodine receptors within caveolae-SR junctions. This molecular complex supports an effective conversion of Ca²⁺ signals to membrane hyperpolarization by the activation of BK channels and thus, control vascular tone. In addition, we have recently revealed that the activation of a complex of Cav1.2, Ca²⁺/calmodulin-dependent kinase kinase (CaMKK)-2 and CaMK1a localized in caveolae causes transcription of pro-inflammatory genes, which promotes macrophage recruitments to the adventitia and vascular remodeling. In summary, the caveola-based supramolecular complexes convert Ca²⁺ signals to the changes in membrane potential and gene transcription, which is involved in the regulation of vascular tone and the adaptation to increased circumferential stretch, respectively.

In skeletal muscle excitation-contraction coupling, depolarization of transverse tubule membrane causes conformational change in dihydropyridine receptor (DHPR), which in turn opens type 1 ryanodine receptor (RyR1) to release Ca²⁺ from sarcoplasmic reticulum. This 'depolarization-induced Ca²⁺ release' (DICR) occurs through a supramolecular complex composed of several proteins including RyR1, Cav1.1, β1a, Stac3, and junctophilin. However, it remains so far unclear about molecular mechanism of DICR, especially conformational change in RyR1. Recently, Perni et al. reported successful reconstitution of DICR by co-expressing these essential components in tsA201 cells (PNAS, 114: 13822-13827, 2017). In this study, we developed a high-throughput platform of reconstituted DICR in HEK293 cells. Baculovirus infection of essential components into HEK293 cells expressing RyR1 greatly increased their transduction efficiency, and fluorescent ER Ca²⁺ indicator (R-CEPIA1er) quantitatively measured Ca²⁺ release without contaminant of Ca²⁺ influx. We demonstrated [K⁺]_o-dependent Ca²⁺ release by chemical depolarization in the baculovirus-infected cells, indicating a successful reconstitution of DICR. Our high-throughput platform will accelerate elucidation of molecular mechanism of DICR.

Sepsis is one of the leading cause of death worldwide. Recently, several studies suggested that free-hemoglobin and heme derived from hemolysis are important factors which may be associated with severity of septic patients including COVID-19. In other words, hemolysis-derived products enhance the inflammatory responses as damage-associated molecular patterns (DAMPs) in both intravascular and extravascular space. In addition, hemoglobin has vasoconstrictive activity by depleting nitric oxide, whereas heme or Fe²⁺ produce reactive oxygen species (ROS) through Fenton reaction leading to tissue injury. At present, we have no therapeutic options against sepsis-related hemolysis in clinical settings, however, there are might be two therapeutic strategies in this regard. One is supplemental therapy of depleted scavenging proteins such as haptoglobin and hemopexin, the other is activation of the internal scavenging system including macrophage-CD163 pathway. These novel targets against sepsis are also critical for the next pandemic. We will show you our recent data aiming at anti-hemolytic therapy.

Histidine-rich glycoprotein (HRG) is a plasma glycoprotein and exists in blood at a concentration of approximately 100 µg/ml. Although many previous reports already revealed that HRG has a wide range of activities against coagulation-fibrinolysis, immune and blood vascular system, we newly identified that HRG ameliorates a septic condition due to its inhibitory activity against immunothrombus formation starting from neutrophil-endothelial cell interaction. Additionally, we demonstrated that HRG regulates the translocation of HMGB1, damage-associated molecular pattern (DAMP) protein, from nuclei to cytosol and neutralizes the toxicity of heme, a kind of DAMP, and LPS, a kind of pathogen-associated molecular pattern (PAMP). Based on the above, HRG was thought to contribute the amelioration of a septic condition, caused by its regulatory activities against not only blood cell functions but DAMPs/PAMPs actions. Because DAMPs and PAMPs were associated with the exacerbation of not sepsis alone but a variety of inflammatory diseases, HRG, which regulates those reactions, has the potential of therapeutic drug for many kinds of diseases.

In recent years, it has been elucidated that a series of inflammatory factors called DAMPs (damage-associated molecular patterns) are involved in the onset and exacerbation of lifestyle-related diseases and age-related diseases. It is known that DAMPs molecules are released extracellularly in response to cell death or stress, and stimulate some pattern recognition receptors in immune cells to induce the inflammatory responses. However, at present, few reports have been made on the existence form of DAMPs molecules and the factors capable of interacting with DAMPs to cause functional changes. In this symposium, we discuss the extracellular existence form of DAMPs, the regulation of their activities by complex formation and their pathophysiological roles.

It was well known in the end of 1980s that NADPH oxidase generates superoxide as respiratory burst in phagocytes (later named as NOX2), and that its genetic mutations could be causative for chronic granulomatous disease. Since the finding of NOX1 in 1999, a catalytic subunit highly expressed in colon epithelium, seven isoforms of NOX/DUOX family (NOX1 to NOX5 and DUOX1/2) were identified. Novel cytosolic components or maturation factors have also been identified, and isoform-specific activation mechanisms have been clarified. Using genetically modified mice, reactive oxygen species derived from NOX/NADPH oxidase have been found to be involved in various physiological functions and pathogenesis in multiple tissues in the body. An age ago, reagents that were used as inhibitors of NADPH oxidase were non-specific ones like diphenyleneiodonium, a broad-spectrum flavoprotein inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride, an inhibitor of serine proteases, or an antioxidant apocynin. In the past two decades, selective NOX inhibitors have been developed. Among them, Setanaxib (GKT137831), a NOX1/4 inhibitor, is under Phase II clinical trial in patients with primary biliary cholangitis (liver fibrosis), Type 1 Diabetes and Kidney Disease (kidney fibrosis), and idiopathic pulmonary fibrosis, suggesting its potential as a new antifibrotic agent. Thus, NOX/NADPH oxidases are expected as novel targets of pharmacotherapeutics.

The NOX family NADPH oxidases deliberately generate reactive oxygen species (ROS) such as superoxide. The human genome contains seven members of the family: NOX1 to NOX5 and DUOX1/DUOX2. These enzymes are membrane-integrated flavocytochromes that transfer electrons from NADPH to molecular oxygen for ROS production. The founder of the family is the phagocyte oxidase, presently known as NOX2, which is dormant in resting cells but produces superoxide, a precursor of microbicidal oxidants, during phagocytosis of invading microbes. The significance of NOX2 is evident from the fact that recurrent and life-threatening infections occur in patients with chronic granulomatous disease (CGD) because of a hereditary defect of the Nox2-encoding gene. The thyroid oxidase DUOX2 is required for thyroid hormone synthesis and its genetic inactivation causes congenital hypothyroidism. Recent studies have revealed that NOX-produced ROS also have various regulatory functions: NOX2 defect causes not only severe infections but also noninfectious granuloma formation, indicating its role in immune system regulation; NOX1 and DUOX2 are highly expressed in gastrointestinal epithelial cells, and their gene variations are associated with inflammatory bowel disease (IBD); and NOX4 is cytoprotective against heart damage.

Reactive oxygen species (ROS) have been implicated in the development of psychiatric disorders. We presently report NOX1/NADPH oxidase regulates the emotional behavior demonstrated in chronic pain model as well as maternal immune activation (MIA) model. There was no difference in the mechanical allodynia developed by spared nerve injury between Nox1 deficient mice (NOX1-KO) and wild type mice (WT). Increased anxiety- and depressive-like behaviors in WT by chronic pain were markedly ameliorated in mice deficient in NOX1-KO. Similarly, the impaired recognition by chronic pain demonstrated in WT was significantly suppressed in NOX1-KO. These affective and cognitive impairment were ameliorated in mice selectively suppressed the expression of hippocampal NOX1 mRNA. In MIA model of gestational polyinosinic-polycytidylic acid, increased serum levels of IL-6 were observed in both genotypes, however, impairment of social preference and defects in motor coordination were observed in WT but not in NOX1-KO. MIA up-regulated NOX1 mRNA in the cerebral cortex and cerebellum of the fetus but not in the offspring. The dropout of Purkinje cells in lobule VII of MIA-affected offspring was significantly ameliorated in NOX1-KO. Taken together, NOX1 may therefore play a key role in the development of behaviors related to the psychiatric disorders.

Hearing loss (HL) is one of the most common sensory impairments. Acquired HLs, such as age-related (ARHL), noise-induced, and drug-induced HL, are classified based on the underlying mechanism. The most common form of sensorineural HL (SNHL) is ARHL, affecting 25~40% of individuals over 65 years of age. Noise is one of the most common occupational hazards. In 2019, WHO warned that half of young people risk damaging their hearing through excessive use of audio devices. However, treatment options for SNHL rely on medical instruments, with no reliable pharmacological interventions.

Nox family is one of the main sources of reactive oxygen species (ROS). Recently, we have reported transgenic mice expressing NOX4 exhibit hearing vulnerability after noise exposure, demonstrating ROS contribute to SNHL. Nox3, activated by a Rho-family GTPase Rac, was discovered as an inner ear specific Nox; however, Nox3-expressing cells were ambiguous. We generated Nox3-Cre;tdTomato mice, in which tdTomato fluorescence is regulated by the Nox3 promoter-driven Cre. Nox3-expressing cells in cochleae included outer hair cells (OHCs) and supporting cells (SCs), and they increased with aging, noise, and cisplatin. Moreover, increased Nox3 expression in OHCs and SCs played essential roles in ROS-related SNHL through OHC apoptosis. Thus, ROS are promising targets for therapeutics development for acquired SNHLs. I will discuss our hearing research focused on ROS and Rho-family GTPases.

Discovery of receptor activator of NF-κB ligand (RANKL) gave a great impact on identification of the mechanisms regulating osteoclast differentiation and function, establishment of research field bridging bone and the immune system (osteoimmunology), and development of a fully human anti-RANKL neutralizing antibody (denosumab). Soluble RANKL and anti-RANKL antibody have been used in vitro and in vivo as valuable research tools. We have reported a novel and rapid bone loss model by administration of glutathione-S transferase (GST)-RANKL to mice, and continuous inhibition of RANKL for several weeks by a single injection of a denosumab-like anti-mouse RANKL neutralizing monoclonal antibody (OYC1) to mice. It is known that RANKL is a multifunction protein that plays a variety of roles such as regulation of bone metabolism. For example, exploration of its pivotal role in immunology is a recent topic of increasing interest. In this presentation I will talk about the discovery of RANKL and the current and future impact of its research.

In the late 1980s, it was found that receptor activator of nuclear factor-κB ligand (RANKL) is expressed by osteoblasts has a crucial role in osteoblastogenesis, which had been a dogma for long. Later studies, however, have revealed that osteocytes functions as the major source of RANKL in many biological processes, including unloading and unloading onto the bone. Osteocytes are known as a mechanosensor in the bone tissue and to respond to mechanical stimuli by expressing various molecules, such as RANKL, Sclerostin (Scl) and insulin–like growth factor (IGF)-1. These cells employ cell surface molecules and intracellular signaling molecules as mechanosensors and mechanotransducers. Mechanosensors detects the flow of tissue fluid and the strain of bone matrix. These sensors then mediate intracellular signals using mechanotransducers, resulting in the regulation of the expression of effector molecules. Although many molecules have found as such sensors and transducers, there remains room for investigation. In this session, I would like to overview of the recent findings on mechanobiology of the bone, focusing on osteocyte RANKL.

Periodontitis, one of the most common infectious diseases in humans, is a unique "osteoimmune" disease in which antibacterial immune response causes alveolar bone destruction. Recently, it was revealed that the osteoclastic bone damage that occurs during periodontitis is dependent on the receptor activator of NF-kB ligand (RANKL) produced by osteoblastic cells and periodontal ligament cells (Tsukasaki et al., Nature Commun 2018, Nature Rev Immunol 2019). To understand the pathogenesis of and develop future therapeutic strategies for periodontal bone loss, it is vitally important to clarify precise molecular mechanisms underlying osteoclastic bone erosion. Recently, we unveiled the importance of the tight regulation of RANKL activity by the local OPG production in bone and immune systems by generating OPG-floxed mice (Tsukasaki et al., Cell Rep 2020), and deciphered the stepwise cell fate decision pathways during osteoclastogenesis at single-cell resolution (Tsukasaki et al., Nature Metabolism 2020). In this talk, I will summarize the recent progress in the fields of periodontology and RANKL biology.

In bone tissue, RANKL acts as a positive regulator of bone turnover through both RANKL forward signaling, which induces osteoclast maturation, and RANKL reverse signaling, which promotes early osteoblast differentiation. Inhibition of osteoclast maturation by blocking RANKL forward signaling inevitably reduces the supply of osteoclast-derived coupling factors, leading to the suppression of bone formation. If we can design an agent that blocks RANKL forward signaling while simultaneously activating RANKL reverse signaling, the influence of reduced supply of osteoclast-derived coupling factors can be mitigated, which may be useful in the treatment of bone destructive diseases. Our previous study has shown that the cross-linking of RANKL molecules on the cell surface triggers the activation reverse signaling. Therefore, we screened various structures of IgG Fc-fusion protein constructs with multivalent binding sites for RANKL by arranging anti-RANKL single-chain antibody variable regions (scFv). Consequently, it was found that a structure in which the domain order inside the scFv was VH-VL order was advantageous for its ability to activate RANKL reverse signaling. Finally, the optimal structure was selected, and its pharmacological activity was evaluated using the ovariectomized mouse model. Results confirmed that the activation of RANKL reverse signaling could mitigate the decrease in bone formation associated with the suppression of osteoclast maturation.

The production of angiotensin II (Ang II) in the brain plays important roles as neurotransmitter and neuropeptide. Central Ang II is involved in regulating various physiological processes, such as blood pressure and water homeostasis, via Ang II type 1 (AT1) receptors. We have demonstrated that Ang II induces frequent urination via AT1 receptors in the brain even at doses that does not seem to affect the blood pressure in animal experiment. Intracerebroventricular administration of Ang II was also found to reduce the bladder capacity without affecting the maximum voiding pressure, post voiding residual urine volume or voiding efficiency. Additionally, the activation of AT1 receptor downstream signal pathway (phospholipase C/protein kinase C/NADPH oxidase/superoxide anion) and suppression of GABAergic nervous system in the brain are involved in the mechanism underlying the central Ang II-inducted frequent urination. AT1 receptor antagonists have been widely used to treat hypertension. We demonstrated that peripherally administered AT1 receptor antagonist telmisartan, which can penetrate blood brain barrier, exerted an inhibitory effect on central Ang II-inducted frequent urination. At this symposium, we would present and discuss the possible drug therapy targeting AT1 receptors in the brain against frequent urination on the results obtained from our recent research work.

Atrial fibrillation reduces the quality of life and increases mortality from cardiovascular disease. The prevention of AF is of major health importance because the incidence, prevalence, and lifetime risk of AF are increasing globally. However, the number of studies directed at the primary or secondary prevention of AF is limited. Recent evidence suggests the association of inflammation with the incidence and progression of AF. NLRP3 inflammasome is central to the innate immune system. Although several studies suggest the association of NLRP3 with AF, the precise mechanism linking activated NLRP3 to AF remains to be elucidated. Gut-derived metabolites, such as lipopolysaccharides and trimethylamine N-oxide (TMAO), have been shown to play a role in AF susceptibility. Lipopolysaccharides are derived from gut microbiota and may enter the circulation through the gut mucosa. TMAO is produced in the liver by oxidation of trimethylamine, which is derived from dietary choline and L-carnitine. Although these gut-derived metabolites have been shown to increase AF susceptibility, it is unclear as to how lipopolysaccharides and TMAO increase the risk of AF and whether lowering circulating levels of lipopolysaccharides and TMAO lead to the prevention of AF. This presentation will discuss the association of inflammation with AF and the potential of drug discovery to prevent AF by focusing on the involvement of gut-derived metabolites in inflammation.

The roles of the nitric oxide synthases system (NOSs) in inter-organ communication of cardiovascular diseases remain elusive. In this symposium, we introduce our three studies in which we examined this issue in our mice lacking all three NOS isoforms (triple n/i/eNOSs^-/- mice) (PNAS 2005).

There is no experimentally useful model that develops myocardial infarction (MI). We previously reported that our triple NOSs^-/- mice spontaneously develop MI. However, it takes a long time (1 year) to develop MI (Circulation 2008). We then revealed that 2/3-nephrectomized triple NOSs^-/- mice suddenly die due to early onset of MI with high incidence, succeeding in an experimentally useful model of developing MI. These results suggest the protective role of NOSs in reno-cardiac communication (JMCC 2014).

We studied the role of bone marrow (BM) NOSs in vascular lesion formation. Constrictive vascular remodeling and neointimal formation at 2 weeks after carotid artery ligation were markedly accelerated in wild-type (WT) mice transplanted with triple NOSs^-/- BM as compared with those with WT BM. These results suggest the protective role of NOSs in BM-vascular communication (Nitric Oxide 2011).

We investigated the role of NOSs in pulmonary hypertension (PH). The extents of PH at 3 weeks after hypoxic exposure were markedly exacerbated in WT mice transplanted with triple NOSs^-/- BM as compared with those with WT BM, suggesting the protective role of NOSs in BM-lung communication (AJRCCM 2018).

These lines of evidence indicate the protective role of NOSs in inter-organ communication.

The gastrointestinal tract plays an important role in maintaining of our various physiological functions. Although the gastrointestinal tract is inside of our body, it is the "outside world". In fact, large number of bacteria coexist and are involved in maintaining of mucosal function. Unbalanced diet, several drugs, decreased blood flow, stress may cause the damage of this balance. Once the balance is destroyed, dysfunction of mucosa and increased permeability are induced. Such excessively increased permeability of intestinal mucosa is defined as "Leaky gut syndrome (LGS)", and various large-molecular weighted substances are transferred into the blood circulation. The LGS is considered to be a trigger for various systemic diseases such as non-alcoholic steatohepatitis (NASH), inflammatory bowel disease, cardiovascular diseases, allergy, chronic kidney disease, etc.

In this presentation, we show the outline of various systemic diseases caused by LGS, especially focused on multi-organ relationships such as "Gut-liver axis" and "Oral-gut axis". In addition, we show the effect of improving LGS status on various systemic diseases and the possibility of LGS as a target for new drug discovery.

Pluripotent stem cells can be used for both regenerative medicine and in vitro modeling for diseases and drug screening. For regenerative medicine, pluripotent stem cells need to differentiate into a specific cell type that is required for transplantation therapy. Cell products for transplantation therapy have lot-to-lot variations in quality, which affect their efficacy and side effects as well as the cost of the products. Quality evaluation of cell products requires non-invasive methods although well-used methods, such as expression analysis of genes and proteins, deconstruct cell products. To evaluate the quality of cell products in a non-deconstructive way, we developed a machine learning model that predicts the function of cell products using label-free images of cell morphology. Next, for in vitro modeling, we sought to recapitulate the interaction of brain regions by fusing brain-region-specific organoids. Imaging and viral vectors allowed for the evaluation of in vitro interaction of brain regions. In this talk, we will introduce our recent work with multidisciplinary approaches.

The ultimate goals of neuroscience are to understand human brain structures and functions, and to utilize them to overcome neurological disorders. The research on the human brain had been limited to non-invasive MRI or PET studies, pathological analysis with postmortem brains or in silico genomic analyses. Although animal or cellular models had been developed for investigating neurological disorders as alternatives to living human brain tissues, many of them did not represent correct human pathological phenotypes. In these circumstances, emergence of iPSCs and organoid culture systems provides us a novel way to investigate the development and dysfunction of the human brain tissues in vitro. We developed self-organizing 3D organoid culture of iPSCs for construction of human brain tissues. Combining 3D brain organoid cultures of human PSCs, 4D imaging and image analysis, we establish efficient methods to faithfully recapitulate and to quantitatively analyze the ontogenetic formation of human brain tissues. We induce degeneration of the formed tissues to construct human neurological disease models. We further explore the ways to prevent or restore the degeneration for clinical treatment and drug discovery.

The platform based on the techniques for iPSC generation and self-organizing 3D culture will become a powerful tool for investigating human brain development and neurological diseases.

The brain is a well-organized organ consisting of telencephalon, diencephalon, mesencephalon, and metencephalon. Each brain region has specific functions and interacts with other regions by neuronal axons and synaptic connections. Thus, region-specific neuronal differentiation from human pluripotent stem cells would help for better understanding human brain development and the pathogenesis of various neurological diseases, and for use in drug development, disease modeling and regenerative therapies.

Recently, we analyzed gene expression profile of human developing ventral midbrain by single-cell RNA-sequencing and established new protocol for generation of midbrain dopaminergic (DA) neurons from human embryonic stem cells by modification of crucial morphogenic factors and transcription factors. In addition, we generated brain organoid that is co-cultured with human induced pluripotent stem cells (hiPSCs)-derived neurons and microglial progenitor cells. We also generated multiregional assembloid that mimics the neural network formation of nigrostriatal pathway and the pathogenesis of Parkinson's disease (PD). using region-specific differentiation technology. Furthermore, we will introduce new therapeutic advantage of medicine for promoting synaptic formation of grafted hiPSC-derived DA neurons towards more beneficial cell transplantation therapy for PD.

To overcome coronavirus disease 2019 (COVID-19), it is essential to develop a therapeutic drug. For that purpose, an excellent in vitro model that can be used for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is needed. It is desired to use a physiologically relevant model rather than a cancer cell line such as Vero cell, which is widely used in SARS-CoV-2 research. We have conducted SARS-CoV-2 research using (1) organoids and (2) organ-on-a-chips. (1) We are trying to clarify the mechanism of SARS-CoV-2 infection using airway organoids. Among various airway epithelial cells, ciliated cells can be easily infected. On the other hand, basal cells, which are stem cells in the airway, cannot be infected with SARS-CoV-2. We confirmed that basal cells that survive after the viral infection can repair damaged airways. (2) Using an organ-on-a-chip that can reproduce the epithelial-endothelial barrier, we are investigating the mechanism by which SARS-CoV-2 destroys the biological barrier. SARS-CoV-2 infects epithelial cells but not endothelial cells. We confirmed that infected epithelial cells secrete interferons (IFN), and then enhance the vascular permeability. Currently, we are trying to develop a therapeutic drug using our organoids and organ-on-a-chip.

The skin forms a barrier between the body and the environment, preventing water loss and penetration of exogenous substances, and upholds homeostasis. The prevention of exogenous substance penetration a priori indicates that a passage of either toxic or medicinal compounds through the skin represents very important problem for topical drug development and application. The topical drugs are developed to deliver an active molecule either to a certain compartment in the skin or through the skin to peripheral blood circulation. In both cases, the pharmacological activity is determined by drug concentration, either in skin or in blood. 

The efficiency and efficacy of topical drug delivery to peripheral blood circulation is judged by the blood concentration of a drug. Moreover, the blood concentration-time course can be predicted using skin permeation parameters obtained from the in vitro skin permeation experiments. Thus, skin permeation experiments are very important to assess systemic exposure of chemicals. Synthetic membranes and three-dimensional cultured human skin models have been utilized for in vitro skin permeation experiments as alternative membranes, especially in the development of cosmetic products. Furthermore, recently, in silico perdiction of absorbed drug after topical application have been developed. In this presentation, usefulness of estimation of skin absorption with synthetic menbrane as well as in silico model would be introduced. I hope this presentation would be helpful for estimation of skin absorption of chemicals to assess systemic exposure.

A well-functioning immune system is essential for maintaining the integrity of an organism. Immune cells are an integral part of other systems including the respiratory, dermal, gastrointestinal, neurological, cardiovascular, reproductive, hepatic, musculoskeletal system, and endocrine systems. As a consequence, exposure to immunotoxic compounds can have serious adverse health consequences affecting responses to both communicable and non-communicable diseases. It is therefore important to understand the immunotoxic potential of xenobiotics and the risk they pose to humans.

Developing novel test methods to evaluating the immunotoxic potential of xenobiotics, Japan has developed five Adverse Outcome Pathways (AOPs) for immunotoxicity in the Organisation for Economic Co-operation and Development (OECD) project.  Based on the AOPs, Japan is on-going a Detailed Review Paper (DRP) for In vitro tests addressing immunotoxicity with a focus on immunosuppression. The DRP aims to present and discuss the application and interpretation of in vitro immunotoxicity assays, mainly covering immunosuppression, and to define a tiered approach to testing and assessment. 　After accepting the above documents, Japan is planning to develop test guidelines for in vitro immunotoxicity to OECD.