Our body has an ability to sense quickly environmental insults and to activate cellular defense enzyme genes. Transcription factor NRF2 is essential for the coordinated induction of cellular defense enzymes and protection of tissues. This notion has been supported by experiments using animal models, showing that Nrf2-deficient mice are sensitive to a wide variety of toxic electrophiles and reactive oxygen species (ROS). We found KEAP1 that acts as a subunit of ubiquitin-E3 ligase and degrades NRF2 constitutively. Keap1 also acts as a sensor for electrophilic and oxidative stresses. Covalent modifications of the cysteine residues of KEAP1 abrogate the ubiquitin ligase activity and stabilize NRF2, as if Floodgate opens in response to environmental stresses. This system has been referred to as the Cysteine Code and KEAP1. The two-site recognition / hinge-latch model has also been proposed for the KEAP1-NRF2 system. Disruption of the two-site recognition model explains the mechanism of nuclear accumulation of NRF2 in a Cul3-KEAP1 E3 ubiquitin ligase-dependent manner. We have verified this model through structure biology, mouse genetics, and human disease analyses. Meanwhile, NRF2 inducers are shown to be important for the treatment of stress-based diseases. Especially, NRF2 acts to suppress inflammations through repressing pro-inflammatory cytokine gene expressions and also suppress oxidative tissue damage through inducing a set of antioxidant enzyme genes. Of note, many somatic missense mutations have been identified in KEAP1 and NRF2 genes of human cancers. These mutations disrupt the KEAP1-NRF2 complex and result in constitutive activation of NRF2. Subsequently, elevated expression of NRF2 target genes confers advantages on the growth of cancer cells through the metabolic reprogramming and induction of cellular defense enzymes. Thus, NRF2 inhibitor is now important for the chemo-sensitization therapy of cancers. The KEAP1-NRF2 system opens a new avenue to the understanding of the signal transduction and regulatory processes underlying the stress response and cancer progression.

Liver cancer is the second leading cause of cancer mortality worldwide; up to one-fifth have been estimated to be attributed to aflatoxin exposure. Thus, considerable research effort has focused on prevention strategies to reduce the impact of aflatoxin-induced hepatocarcinogenesis. The synthetic oleanane triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), a powerful activator of Keap1-Nrf2 signaling, protects F344 rats against aflatoxin B1 (AFB1)-induced acute toxicity and preneoplastic lesion formation. In a lifetime cancer bioassay rats were dosed daily with AFB1 for 4 weeks and received either vehicle or CDDO-Im (thrice weekly), one week prior to and throughout the exposure period. Weekly urine and monthly serum samples were collected for biomarker analyses. The comparative response of a toxicogenomic RNA expression signature for AFB1 was examined as were tumor and serum miRNA levels. CDDO-Im completely protected against AFB1-induced liver cancer compared to a 96% incidence observed in the AFB1-only group and, correspondingly, altered the toxicogenomic signature and miRNA trajectories. To ascertain the role of Nrf2, follow-up studies were conducted with two lines of Nrf2 knockout F344 rats. CDDO-Im failed to induce representative Nrf2 target genes in the livers of these animals, further validating the Nrf2 pathway as a key target of CDDO-Im. Moreover, these knockout rats were exceedingly sensitive to the acute toxicity of AFB1. With CDDO-Im treatment, integrated levels of urinary AFB1-N7-guanine, the primary DNA adduct excised from the liver, were significantly reduced and those of aflatoxin-N-acetylcysteine, a glutathione S-transferase (GST)-derived detoxication product, were consistently elevated 3-fold after the first AFB1 dose. Mice are intrinsically resistant to AFB1 hepatotoxicity; however, sensitivity is greatly enhanced in GSTA3 knockout mice. Collectively, these data indicate a key role for the induction of Nrf2-regulated GSTs in the protective effect. Moreover, strategies to elevate NRF2 signaling in populations at high risk for aflatoxin exposures should be included in public health measures to reduce liver cancer burden. Supported by NIH grant R35 CA197222 and MEXT/JSPS KAKENHI.

Nuclear factor erythroid 2-related factor 1 (NFE2L1, also known as NRF1), a ubiquitously expressed CNC-bZIP protein, is an important regulator of the antioxidant response, proteasome homeostasis, genetic stability, mitochondrial respiration, inflammation, lipid metabolism and cell differentiation. Recently, we found that silencing of Nfe2l1 in pancreatic beta-cells, led to a phenotype markedly resembling pre-T2D conditions with disrupted glucose metabolism and impaired insulin secretion. The impaired glucose responsiveness due to Nfe2l1 silencing are likely a result of aberrant expression of a group of glucose metabolic enzymes. The findings demonstrated an important role of NRF1 in regulating glucose metabolism and insulin secretion in pancreatic beta-cells and characterized NRF1 as a key transcription factor that regulates the coupling between glycolysis and mitochondrial metabolism and insulin secretion. In addition, we examined the tumorigenicity of Nfe2l1 deficient insulinoma MIN6 cells (Nfe2l1-KD) and their sensitivity to chemotherapy. We found that Nfe2l1-KD insulinoma cells grew faster and were more aggressive than Scramble cells in vitro. Insulinoma arising from Nfe2l1-KD cells in an allograft transplantation mouse model was more aggressive and chemo-resistant. This conclusion was confirmed by streptozotocin (STZ) administration in an allograft transplantation model in diabetic Akita background mouse. In addition, we found that Nfe2l1-KD cells were resistant to chemotherapeutic drugs STZ and 5-fluorouracil-induced damage, which was linked to binding of hexokinase 1 with mitochondria, enhancing mitochondrial membrane potential and closing mitochondrial potential transition pore. Overall, both in vitro and in vivo data from Nfe2l1-KD MIN6 insulinoma cells indicate a previously un-appreciated action of NRF1 in suppression of tumorigenesis and sensitizes cells and their derivative tumors to chemotherapeutic-induced damage and apoptosis, likely via metabolic reprograming. These data indicate that NRF1 could potentially play an important role in the carcinogenic process and impact chemotherapeutic efficacy, at least within a subset of pancreatic endocrine tumors.

Recent investigations have clarified the importance of mitochondria in various age-related degenerative diseases including late onset Alzheimer´s disease. Mitochondrial functional disturbance is often observed preceding the actual appearance of pathophysiological alterations and contributes to the pathogenesis. The signals from damaged mitochondria are transferred to the nucleus leading to the altered expressions of nuclear-coded genes that includes mitochondrial proteins (i.e. retrograde signaling). Recent observations increasingly show the importance of bZIP transcription factor ATF4 in the mitochondrial retrograde signaling. Nrf2 plays a pivotal role in the coordinated regulation of a battery of oxidative stress-inducible genes such as HO-1 and NQO1 via antioxidant responsive element (ARE). Nrf2 requires an obligatory DNA binding partner small Maf protein to bind to ARE. We previously identified ATF4 as an Nrf2 binding protein through yeast two-hybrid screening. However, in vitro experiments showed that Nrf2-ATF4 heterodimer do not bind to DNA. We then showed that ATF4 and Nrf2 cooperatively regulate the inducible expression of cystine transporter (xCT) gene after proteasome inhibition by making physical contacts on its gene regulatory region. Therefore, we propose that ATF4 is a transcriptional partner in considering Nrf2 targeted therapeutics.

Considering cost-effective prevention strategy against age-related diseases, employment of phytochemicals that activate Nrf2 is a feasible approach. We have previously shown that carnosic acid (CA), a major constituent of the herb rosemary, exerts a neuroprotective effect against brain ischemia-reperfusion injury in mice and that it enhances neurite outgrowh in an Nrf2- and p62-dependent manner in rat PC12h cells. We also showed that CA-quinone binds Keap1 and induces NGF production in human T98G glioblastoma cells and normal human astrocytes in an Nrf2-dependent manner. Using the newly developed Alzheimer´s model mice, we showed that the feeding the mice with rosemary extract diminished the proliferation of activated astrocytes by amyloid-ß accumulation. Further elucidation of transcription factor networks centered on Nrf2 may be useful in the development of a novel neuroprotective strategy.

Membrane proteins play fundamental roles in many physiological processes and also are target proteins for drug development. For better understanding of the functions of the membrane proteins, precise static three-dimensional structures, for example, derived from X-ray crystallography, are very useful without a doubt. We should also note that dynamics of the membrane proteins is important for some functions of the membrane proteins. NMR (nuclear magnetic resonance spectroscopy) basically provide the dynamic of the membrane proteins. However, it is frequently difficult to obtain structural information about the membrane protein dynamics related to the functions, by NMR. This is mainly due to the difficulties in sample preparation: expression and purification of the membrane proteins with the biological activities, and lack of appropriate NMR strategy. In this paper, we will show our recent results of dynamical aspects of G-protein coupled receptors (GPCR) and ion channels, which are responsible for the biological functions.

We proposed a strategy named Drug Rescuing, as a possible solution for an effective drug development. Many lead compounds as well as target proteins have to be thrown into garbage box because of adverse effects. We will be able to rescue the target and compounds as a drug candidate by modifying the ligand chemical on the unrelated parts for pharmacologic action. Structural information of the ligand and membrane protein complex is therefore crucially important for the modification. As an example, based on electron crystallography utilizing a helium cooled cryo-EM [1], we could discriminate 8 water molecules in the water channel AQP4 [2], while their densities were blurred in the higher resolution structure by X-ray crystallography [3]. The counterintuitive notion could be attributed to the difference of surround atmosphere for structure analyses of membrane proteins. The characteristic distribution of the dielectric constants in membrane produces large dipole moment of the two short helices of water channels, whereas the dipole moment without lipid bilayer is very small. In cooperation with the electrostatic field of two short helices, the arrangement of carbonyl groups in the channel act as binding sites in the narrow channel with highly hydrophobic surfaces and lowers the energy barrier for water molecules entering such narrow water channel. These structural studies strongly suggested that electron crystallography is very powerful method for understanding physiological functions of membrane proteins as well as drug development. However, the necessity of crystallization makes this method less popular in the structural biology field. By single particle analysis, many structures have been analyzed. In very short period, we could analyze structure of gap junction channel by the method [5]. Then Drug Rescuing could be realistic method for drug development.

References

[1] Y Fujiyoshi, Adv. Biophys., 35, 25-80 (1998). [2] K Tani et al., J. Mol. Biol., 389, 694-706 (2009). [3] JD Ho et al., Proc. Natl. Acad. Sci. USA, 106, 7437-7442 (2009). [4] M Liao et al., Nature, 504, 107-112 (2013). [5] A Oshima et al., Nature Comms, 7, 13681 (2016).

In the last few years, major technological breakthroughs, particularly the development of new direct electron detection cameras and associated technologies, have enabled single particle cryogenic electron microscopy (cryo-EM) to become the technique of choice for high-resolution structure determination of many challenging biological macromolecules. Atomic structures of many membrane proteins, particularly ion channels, that are refractory to crystallization have now determined by this method, including our own work of determining the atomic structures of TRPV1, TRPA1 and TPRM4. A recent technological development in single particle cryo-EM of integral membrane protein is to enable atomic structure determination of integral membrane proteins in a native or native-like lipid bilayer environment. In many recent cryo-EM studies, integral membrane proteins are reconstituted into native like lipid bilayer environment, using various lipid-protein nanoparticle techniques including lipid nanodisc, saposin based Salipro nanoparticles, or the styrene-maleic acid (SMA) copolymer based native lipid nanodisc. By reconstituting integral membrane proteins into artificial or near native lipid bi-layer environment, it is now feasible to use single particle cryo-EM for studying specific protein - lipid interactions, and to visualize binding of small pharmacological substances in membrane protein in lipid environment.

Recently, we determined atomic structures of nanodisc-embedded TRPV1 in three different conformations. These structures revealed locations of some annular and regulatory lipids that form specific interactions with the channel. Such specific phospholipid interactions enhance binding of a spider toxin to TRPV1 through formation of a tripartite complex. Our structures also reveal that, in the absence of vanilloid agonist, a phosphatidylinositol lipid occupies the capsaicin-binding site of TRPV1, providing important clues about physiological mechanisms of channel regulation.

In my presentation, I will discuss about our recent characterization of an ultrastructurally distinct microglial phenotype that is predominantly associated with pathological states. These cells are rare in steady state conditions, but become prevalent upon chronic stress, aging, and Alzheimer's disease pathology, where they account for two-thirds of the normal microglial population. They exhibit several signs of cellular stress including a condensed, electron-dense cytoplasm and nucleoplasm giving them a 'dark' appearance in electron microscopy, accompanied by endoplasmic reticulum dilation, mitochondrial alterations, and a loss of nuclear heterochromatin pattern. The physiological significance of these dark microglia has yet to be elucidated but they appear extremely active, frequently reaching for synaptic clefts, while extensively encircling axon terminals, dendritic spines, and excitatory synapses with their highly ramified and extremely thin processes. They strongly express CD11b, which forms complement receptor 3 involved in synaptic pruning, specifically in their processes encircling synaptic elements, and myeloid-cell specific TREM2 when associated with amyloid-β plaques. In addition, our recent work revealed the occurrence of these dark microglia in a schizophrenia mouse model induced by prenatal immunological challenge, as well as in early brain development, two conditions where synaptic pruning is exacerbated. These findings indicate that dark microglia could represent a subset of cells that become stressed as a result of their hyperactive involvement with the remodeling of neuronal circuits across development, plasticity, and disease.

Brain is composed of not only neurons but also glia. It has been recently established that in addition to neurons, glial cells including astrocytes can release various transmitters (termed gliotransmitters), such as GABA, glutamate, and d-serine. The functional significance of these gliotransmitters is beginning to unravel as the detailed mechanisms of biosynthesis and release have become available. We have recently demonstrated the role of GABA, synthesized via MAO-B and released through GABA-permeable Best1 channel from reactive astrocytes, in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. During the course of these studies, we have developed various molecular and pharmacological tools and novel animal models to address the etiology of Alzheimer's disease. These newly developed targets and tools have proven to be useful in developing novel therapeutics for Alzheimer's disease that currently has no cure.

Astrocyte derived ATP, together with its degradation product adenosine, is known to regulate synaptic transmission through a presynaptic mechanism. However, little attention has been paid to the direct effect of gliotransmitters on the excitability of neuronal networks beyond synapses. Here, we showed that selective stimulation of astrocytes expressing channelrhodopsin 2 in the CA1 area specifically increased the firing frequency of CCK positive interneurons and decreased the firing rate of pyramidal neurons, but did not affect the activity of parvalbumin positive interneurons, phenomena mimicked by exogenously applied ATP. Further evidence indicated that ATP induced increase and decrease of excitability were caused respectively by P2Y1 receptor mediated inhibition of a TASK3 (tandem pore domain acid sensitive potassium channel 3) like two pore domain potassium channel (K2P) and A1 receptor mediated opening of a G protein coupled inwardly rectifying potassium channel (GIRK). Single cell RT PCR and immunostaining showed that P2Y1 and A1 receptors were differentially expressed on interneurons and pyramidal neurons. Application of antagonists to P2Y1 and A1 receptors disclosed the purine mediated tonic excitation of interneurons and inhibition of pyramidal neurons, respectively. Moreover, activation of ChR2 expressing astrocytes reduced the power of kainate induced hippocampal gamma oscillation. Thus, through distinct receptor subtypes coupled with different K+ channels, astrocyte derived ATP differentially modulates the excitability of different types of neurons and efficiently controls the activity of neuronal network.

Glial cells are very sensitive to environmental changes such as infections, inflammation, neurodegenerative diseases and even psychiatric diseases, and then, change their phenotypes into very different ones. Such a phenotypical change in glial cells is one of the most important and also problematic characteristic features of glia. As for astrocytes, they become ´reactive astrocytes´ and contribute to both beneficial and hazardous brain functions. Here, I talk about reactive astrocyte-mediated network in the somatosensory cortex (S1) and striatum. Peripheral neuropathic pain that includes mechanical allodynia remains poorly treated. While glial activation and altered nociceptive transmission within the spinal cord is associated with the pathogenesis of mechanical allodynia, changes in cortical circuits also accompanies peripheral nerve injury and may represent additional therapeutic targets. Dendritic spine plasticity in the S1 cortex appears within days following peripheral nerve injury, however, its causal relationship to allodynia and the underlying cellular mechanisms remain unsolved. Furthermore, whether glial activation occurs within the S1 cortex following injury and how it contributes to this S1 synaptic plasticity is unknown. Using in vivo two-photon imaging with genetic and pharmacological manipulations, we show that sciatic nerve ligation induces a re-emergence of immature mGluR5 signaling in S1 reactive astrocytes, which elicits spontaneous somatic Ca²⁺ transients, synaptogenic TSP-1 release and synapse formation. Such S1 astrocyte reactivation was evident only during the first week post-injury, correlating with the temporal changes in S1 extracellular glutamate levels and dendritic spine turnover. Blocking this astrocytic signaling pathway suppressed mechanical allodynia, while activating this pathway in the absence of any peripheral injury induced long-lasting (over 1 month) allodynia. We conclude that reactive astrocytes are a key trigger for S1 circuit rewiring and contribute to neuropathic mechanical allodynia. In addition to these, reactive astrocytes cause remodeling of the neuronal network of the ischemic penumbra in the striatum. In this case, astrocytes become rather phagocytic and contribute to the rewiring. We will talk the beneficial roles of reactive astrocytes.

Many epidemiologic and clinical studies have shown that attention-deficit/hyperactivity disorder (ADHD) is strongly associated with an increased risk of substance use disorder (SUD). The relationship between ADHD and SUD is discussed sometimes in the context of adolescents' delinquency or crimes. Conduct disorder (CD), one of the typical externalizing psychiatric disorders, plays an important role on leading ADHD patients to SUD. On the other hand, dysfunction of the brain reward system inherent in ADHD patients makes them have no confidence in themselves, which lead them to depressive disorder, one of the typical internalizing disorders. In such case, ADHD patients are likely to indulge in substance to relieve their depressive or anxiety symptoms, sometimes resulting in SUD. In sum, CD and depression play a mediating role to lead ADHD patients to SUD, while the direct association between CD or depression and SUD is much stronger than ADHD. Once ADHD is comorbid with SUD, patients suffering from both ADHD and SUD often confront catastrophic crisis such as suicide or homicide. Because ADHD is characterized by inattentiveness and impulsivity, any kinds of substances, regardless of legal or illegal, can deteriorate ADHD symptoms, leading ADHD patients to such catastrophic events. Some epidemiological evidence showed that ADHD patients with comorbid SUD suffered from synergistically increased risk of suicide. From a view point of suicide prevention, CD or mood disorders, as mediating factors from ADHD to SUD, should be carefully monitored and treated among ADHD patients.

Attention deficit-hyperactivity disorder (ADHD) is a neuropsychiatric disorder with an average heritability estimated around 76%. Individuals whose ADHD persists into adulthood have approximately 50% of the lifetime risk to develop any SUD co-morbidity and patients with SUD present a higher prevalence of ADHD than general population. This link between ADHD and SUDs has been documented in family members of both adults and children with ADHD. Despite this high incidence of SUD in subjects with ADHD and the studies that consistently show genetic basis for the overlap, little is known about the specific genetic factors that could be involved in the development of both disorders. Until today, research has mainly focused on candidate genes of the dopamine, serotonin, norepinephrin neurotransmitter systems, or SNARE complexe. Among the most vulnerable patients with ADHD to develop SUD later in life are those who have co-morbid CD or ODD and evidence suggest that co-occurrence of symptoms for ADHD and CD contribute in an epistatic manner to a more severe form of SUD than individuals with ADHD or CD alone. Most recently, there is growing interest in studying the potential role of post-transcriptional regulatory elements in the susceptibility to different complex disorders. It will be presented the results of the first case-control association study in ADHD that evaluate the role of SNPs within miRNAs or miRNA target sites and, thus, with a potential involvement in the modulation of the miRNA-mediated gene expression. Finally, new data about the genetic relationship between ADHD and cannabis dependence will be presented.

ADHD and substance use disorders (SUDs) are highly comorbid with 20-25% of all SUD patients also meeting criteria for adult ADHD (van Emmerik-van Oortmerssen et al., 2012) and about 50% of the patients with childhood ADHD developing SUD (Manuzza et al., 2008). This high level of concurrent and prospective comorbidity is largely explained by genetic overlap (Derks et al., 2014). Moreover, prospective clinical and retrospective register studies suggest that early treatment of ADHD with stimulants may prevent or postpone the development of SUD in children with ADHD (Groenman et al., 2013; Zheng Chang et al., 2014). Together these findings suggest that ADHD and SUD share neurobiological mechanisms.

In this presentation, we explore the neurobiological overlap between ADHD and SUD using data from cross-sectional and prospective neurocognitive and neuroimaging studies. The neurobiology of both ADHD and SUD can be summarized as a combination of an impaired motivational and reward system and a deficient cognitive controle system, including impairments of delay discounting and error monitoring (Frodl, 2010; moline & Pelham, 2014; Vitria et al., 2017; . In addition, patients with both disorders show reduced dopamine transporter binding of methylphenidate (Crunelle et al., 2013) causing reduced efficacy of standard doses of methylphenidate for the treatment of ADHD patients with comorbid SUD and the need for high dose stimulant treatment in these comorbid patients (Carpentier & levin, 2017).

Pharmacotherapy is a potent intervention in childhood and adult ADHD, but the effectiveness of pharmacological treatment is diminished in ADHD adults with comorbid substance use disorders. Until recently randomised controlled trials had difficulty demonstrating any positive effect of several medications (methylphenidate, amphetamines and atomoxetine) in the treatment of ADHD in this patient group. This lecture will focus on contributing factors and possible explanations, reviewing all randomized placebo-controlled pharmacotherapy trials. The number of studies is limited, and several studies are hampered by qualitative flaws. The results in general are inconclusive for most medications studied, but more recent trials using psychostimulants in robust dosing have demonstrated significantly positive results.

Possible explanations relating to the particular characteristics and problems of this complex patient group will be discussed. Several factors seem to influence the effectiveness of medication, including ADHD symptom severity, psychiatric comorbidity, persistent drug use, choice of medication and concomitant psychosocial intervention. Special attention will be given to the determining role of abstinence in ensuring a positive effect.

In most SUD patients several of these contributing factors will be present, impeding treatment and negatively influencing prognosis. However, identifying and addressing these factors is a first step in optimising treatment results. Taking these factors into account will contribute to an appropriate use of pharmacotherapy (drug selection, dosage, conditions) in clinical practice, and may improve the likelihood of detecting significant effects in future research, as the recent positive trials have indicated.

For reliable transmission at chemical synapses, neurotransmitters must be released dynamically in response to neuronal activity in the form of action potentials. Stable synaptic transmission is dependent on the efficacy of transmitter release and the rate of resupplying synaptic vesicles to their release sites. Accurate regulation is conferred by proteins sensing Ca²⁺ entering through voltage-gated Ca²⁺ channels opened by an action potential. Presynaptic Ca²⁺ concentration changes are dynamic functions in space and time, with wide fluctuations associated with different rates of neuronal activity. Thus, regulation of transmitter release includes reactions involving multiple Ca²⁺-dependent proteins, each operating over a specific time window. Classically, studies of presynaptic proteins function favored large invertebrate presynaptic terminals. I have established a useful mammalian synapse model based on sympathetic neurons in culture. Using this model synapse to study the roles of presynaptic proteins in neuronal activity for the control of transmitter release efficacy and synaptic vesicle recycling, I summarize that neuronal activity-dependent millisecond Ca²⁺ dynamics activate multiple protein cascades for synaptic vesicle control.

Voltage-gated calcium channels are important for the transmission of pain signals. N-type calcium channels are expressed in the synaptic terminals of primary afferent fibers where they control the release of neurotransmitters such as substance P and CGRP. Consequently, inhibiting N-type calcium channels mediates analgesia. In this regard, activation of opioid receptors mediates a G-protein dependent inhibition of N-type calcium channels, thus increasing pain thresholds. N-type calcium channels can also be directly targeted pharmacologically via peptide toxins such as Prialt which physically prevent calcium permeation. Finally, they are regulated by gabapentinoids that interact with the ancillary calcium channel alpha2-delta subunit and reduce the trafficking of the channels to the synapse. On the other hand, upregulation of alpha2-delta expression following nerve injury increases N-type calcium channel density and thus contributes to neuropathic pain. In this context, I will present new data that concern cellular mechanisms that regulate alpha2-delta subunit function and how this can potentially be exploited for the development of new analgesics. I will also present findings concerning opioid receptor regulation of N-type channels and its relevance for morphine tolerance.

TRP (transient receptor potential) channels are non-selective cation channels having relatively high CaCa²⁺ permeability, and comprise six related protein families (TRPC, TRPV, TRPM, TRPA, TRPML, TRPP) in mammals. One subunit of the TRP channel is composed of six transmembrane domains and a pore region with both amino and carboxyl termini on the cytosolic side. Among the huge TRP super family of ion channels, some have been proven to be involved in thermosensation detecting ambient temperatures from cold to hot. There are now eleven thermosensitive TRP channels (TRPV1, TRPV2, TRPV3, TRPV4, TRPM2, TRPM3, TRPM4, TRPM5, TRPM8, TRPA1 and TRPC5) with distinct temperature thresholds for their activation. Structures of TRPV1, TRPV2, TRPM4, TRPM8 and TRPA1 have been clarified in the last several years. We found that some thermosensitive TRP channels make a complex with CaCa²⁺-activated chloride channel, anoctamin1 (ANO1). Interaction between TRPV4 and ANO1 is involved in water efflux in choroid plexus, salivary gland and lacrimal grand epithelial cells. And TRPV1 (TRPA1)/ANO1 interaction was found to be involved in the enhancement of nociceptive signals through further depolarization upon chloride efflux in peripheral sensory neurons. This interaction could be one of the reasons why TRP channels have high CaCa²⁺ permeability.

Voltage-gated calcium channels of the CaV2 sub-family are involved in neurotransmitter release in the central and peripheral nervous systems. CaV2.2 channels (N-type) are of particular importance in neurotransmission in primary afferent terminals mediating pain transmission. These channels are heteromeric. The alpha1 pore-forming subunit is associated with auxiliary beta and alpha2delta subunits which increase the functional expression and modify channel properties. alpha2delta-1 plays a key role in rodent models of neuropathic pain and in development of neuropathic mechanical hypersensitivity and allodynia. The mRNA and protein for alpha2delta-1 is strongly up-regulated in injured dorsal root ganglion neurons and such injuries lead to a chronic neuropathic pain-state (1,2).

We have studied the role of the auxiliary alpha2delta subunits in CaV2.2 (N-type) calcium channel trafficking and function (3) and the key functions of their Von-Willebrand factor domain in this process (3-5). Furthermore our recent work shows that proteolytic cleavage of alpha2delta subunits into alpha2 and delta is essential for most of their effects (6). I will describe studies which increase our understanding of the roles of alpha2delta subunits using knock-in mice containing tagged calcium channels.

References

1 Bauer C. S. et al. The increased trafficking of the calcium channel subunit a2d-1 to presynaptic terminals in neuropathic pain is inhibited by the alpha2delta ligand pregabalin. Journal of Neuroscience 29 4076-4088 (2009).

2 Patel R. et al. alpha2delta-1 gene deletion affects somatosensory neuron function and delays mechanical hypersensitivity in response to peripheral nerve damage. J Neurosci 33 16412-16426 (2013).

3 Cassidy J. S et al. Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary alpha2delta-1 subunits. Proc. Natl. Acad. Sci. U. S. A 111 8979-8984 (2014).

4 Canti C. et al. The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of alpha2delta subunits is key to trafficking voltage-gated Ca2+ channels. Proc. Natl. Acad. Sci. USA 102 11230-11235 (2005).

5 Hoppa M. B., et al. alpha2delta expression sets presynaptic calcium channel abundance and release probability. Nature 486 122-125 (2012).

6 Kadurin I. et al. Proteolytic maturation of alpha2delta represents a checkpoint for activation and neuronal trafficking of latent calcium channels ELife 5 e21143 (2016).

The D2 dopamine (DA) receptor (D2R) signals through multiple second messenger pathways making it difficult to discern which are linked to specific effects of D2R-targeted drugs; however, this complexity provides an opportunity to develop pathway-selective therapeutics. Structure-activity analyses and molecular modeling using the D2R G protein-biased agonist, MLS1547, led to a structural model for biased signaling that entails a hydrophobic binding pocket formed by residues I184, F189, and V190 at the interface between the fifth transmembrane segment (TM5) and the second extracellular loop of the D2R. To investigate the role of these residues in regulating signaling by the D2-like receptors (D2R, D3R, and D4R), and the β2-adrenergic receptor (B2R), we constructed point mutations at I184, V190 and F189 of the D2R, and at the aligned residues for F189 (i.e., position 5.38) within the D3R, D4R, and B2R, and studied their effects on G protein-mediated signaling, β-arrestin recruitment, and GRK2 interactions using BRET-based biosensors and in vitro signaling assays. The D2R point mutations I184A and V190A produced a small change in the potency of DA for stimulating β-arrestin recruitment or G protein activation. Strikingly, the F189A mutation ablated the ability of DA and other D2R agonists to recruit β-arrestin while G protein-signaling efficacy was maintained. Further, the D2R F189A mutant was unable to directly interact with GRK2 and failed to internalize from the cell surface following DA stimulation. In addition, we found that mutating the residues in the D3R, D4R, and B2R at position 5.38 to Ala resulted in parallel findings (i.e., loss of agonist-stimulated β-arrestin recruitment, but minimal to no effects on G protein-mediated signaling). These data demonstrate that the D2R F189A mutant, and similarly mutated D3R, D4R, and B2R, are highly biased towards G protein-mediated signaling and suggest that the presence of a Phe or Tyr residue at this position (5.38) is important for stabilizing an activated state for recruiting β-arrestin. Conformational changes propagated through TM5 might thus act as a molecular switch for receptor signaling via β-arrestin recruitment. These results may have implications for the design of novel signaling-biased compounds for the treatment of GPCR-related disorders.

The dopamine D3 receptor (D3R) is a target of pharmacotherapeutic interest in a variety of neuropsychiatric disorders including schizophrenia, substance use and bipolar disorders. Determination of the high-resolution crystal structure of the D3R has invigorated structure-based drug design, providing refinements to the molecular dynamic models and testable predictions about receptor-ligand interactions. The recent and precipitous increase in opioid analgesic abuse and overdose has inspired investigation of the D3R as a target for therapeutic intervention. We have recently discovered novel and D3R crystal structure-guided 4-phenylpiperazines with exceptionally high D3R affinities and/or selectivities with varying efficacies. VK4-116 was selected as a lead compound based on its in vitro profile: D3R Ki = 6.84 nM, 1700 fold D3R versus D2R binding selectivity and its metabolic stability in mouse, rat and monkey liver microsomes. VK4-116 (5-15 mg/kg, i.p.) dose-dependently blocks acquisition of oxycodone self-administration (5-15 mg/kg/infusion) and inhibits drug taking and seeking in rats trained to self-administer oxycodone. VK4-116 pretreatment (5-15 mg/kg) also blocks oxycodone-induced (1 mg/kg, i.p.) reinstatement of drug seeking in previously oxycodone trained, and subsequently behaviorally extinguished rats. Interestingly, VK4-116 (5-15 mg/kg) dose-dependently blocks naloxone-precipitated (1 mg/kg) conditioned place aversion in rats chronically treated with oxycodone (3 mg/kg, i.p.; twice daily for 7 days). Moreover, VK4-116 pretreatment shows little effect on sucrose self-administration or on the antinociceptive effects of oxycodone, tested in the hot-plate assay. Translational studies in cynomolgus monkeys are underway. Taken together, these findings suggest a crucial role for central D3R in opioid reward and dependence. Importantly, VK4-116 may serve as an effective agent for mitigating the development of prescription opioid addiction, reducing the severity of withdrawal and preventing relapse.

The dopamine D2-like receptors (D2-likeR) are targets for the treatment of Parkinson's disease, schizophrenia and drug abuse. To date, drug discovery efforts have focused on targeting the orthosteric site of the D2-likeR that binds dopamine. Orthosteric D2R agonists are employed for the treatment of PD but display a limited duration of efficacy and have significant drawbacks, including induction of psychosis and compulsive behaviours. Similarly, while orthosteric D2-likeR antagonism is sufficient for antipsychotic activity, it can be associated with extrapyramidal side effects and tardive dyskinesia. Such side-effects can be attributed, in part, to a lack of target specificity and/or the inability of orthosteric drugs to replicate the spatiotemporal pattern of endogenous dopamine signalling in the brain. Drugs that bind topographically distinct allosteric sites can display greater subtype-selectivity and, because they still allow the endogenous agonist to bind, maintain endogenous spatiotemporal patterns of signalling. Allosteric drugs that target the D2-likeR and act to tune up (Positive Allosteric Modulator, PAM) or tune down (Negative Allosteric Modulator, NAM) the action of dopamine may present a safer, more effective therapeutic therapeutic approach. While this approach has yet to be therapeutically exploited, recent years have seen the identification of novel allosteric scaffolds for the dopamine receptors. I will present our findings regarding the binding mode and mechanism of action of novel NAMs and PAMs of the D2-likeRs. In particular we have explored the relationship between interactions with distinct allosteric binding pockets and patterns of modulatory effects. We reveal effects upon both orthosteric agonist affinity and efficacy, modulatory effects that are biased towards particular signalling pathways and unique patterns of subtype selectivity. We also explore the pharmacology of bitopic (dual allosteric/orthosteric) ligands and how the pharmacology of clinically-used antipsychotics might be derived from such a binding mode. In summary, our studies of D2-likeR allosteric modulators illustrate how the complex pharmacology of such ligands can sculpt dopamine receptor responses in a manner that cannot be achieved through the action of orthosteric drugs. Understanding the mechanism of action of such allosteric ligands can provide a platform for the development of improved therapeutics that target these receptors.

In this talk I will show how recent high resolution D2-family receptor structures have facilitated the design and validation of novel chemical probes for D2 and D4 dopamine receptors.

Lung cancer is one of the leading causes of human death worldwide. Despite advances in therapeutic regimens, the efficacy of currently available lung cancer therapies is still limited, emphasizing the necessity to develop novel strategies for lung cancer prevention and therapy. Many previous studies have demonstrated the crucial role of the insulin-like growth factor receptor (IGF1R) signaling pathway in the cancer development and progression and anticancer drug resistance in several types of cancer including lung cancer. In our recent studies, we observed elevated expression of IGFs along with IGF-1R activation in early stage of human lung carcinogenesis. Tobacco carcinogens and stress hormone induced a rapid IGF2 secretion and IGF-1R activation via voltage-dependent calcium channel (VDCC)-intervened calcium influx in airway epithelial cells, ultimately leading to lung tumor formation. Moreover, blockade of the VDCC-mediated Ca2+ influx by treatment with clinically available antihypertensive agents, such as voltage-dependent calcium channel blockers, significantly suppressed tobacco carcinogens- and chronic stress-induced lung carcinogenesis. Publicly available database revealed inverse correlation between use of calcium channel blockers and lung cancer diagnosis. These results clearly show the potential of the IGF axis as a target in lung cancer chemoprevention. However, clinical studies utilizing IGF-1R-targeted agents have shown disappointing results. We have demonstrated that targeting the IGF signaling leads to activation of bypass signaling via cancer cell reprogramming and the communication with stroma cells, eventually leading to tumor relapse and metastatic tumor growth. In this presentation, I will discuss the role of the IGF signaling in lung cancer development and progression, the mechanisms underlying resistance to IGF1R-targeted therapies, and novel alternative approaches to block the IGF signaling activation for lung cancer prevention and therapy.

Stem cell markers such as Nanog have been implicated in various cancer, but whether they are functionally contributing to cancer pathogenesis has remained unclear. Tumor-initiating stem-like cells (TICs) were isolated from mouse hepatocellular carcinoma (HCC) and human HCC patients. Using knowledge gained from the oncogenic signaling of TICs, we addressed new mechanistic hypotheses based on the novel discovery that the pluripotent transcription factor, NANOG, can reprogram the metabolism of HCC-derived TICs. These studies will motivate novel strategies for targeting and removing TICs and suggest new avenues to treat malignancies associated with human HCV and alcoholism and/or obesity. We determined novel targets of NANOG in TICs from patient and mouse models of HCC using genome-wide NANOG-binding site analysis (ChIP-seq) and how Nanog is regulated at the transcriptional to promote oncogenesis and self-renewal in TICs. NANOG-mediated metabolic reprogramming through suppression of mitochondria function in both experimental and clinical HCC downstream of TLR4/NANOG generates TICs and drives liver tumorigenesis. The p53 tumor suppressor acts as a barrier against stem cell proliferation, and inactivation of p53, or its stabilizing partner NUMB (cell fate decision molecule), leads to expansion of TICs. Although treatment efficacy of HCV has been dramatically improved in recent years, the incidence of HCV-associated HCC keeps rising due to the prevalence of obesity, alcoholism and illicit drug usage in HCV patients. RNA-binding protein MSI2 is elevated in several cancers and is linked to poor prognosis. We also performed RIP-seq using anti-MSI2 antibody in TICs and subsequent validation by qPCR analysis. Among the MSI2-bound RNAs, MYC mRNA and long noncoding RNA (LncRNA) were identified by RIP-qPCR analysis. MSI2 promotes liver tumorigenesis by maintenance of MYC expression by inhibition of LncRNA processing. This newly discovered mechanistic framework for TIC proliferation represents a key innovation and holds significant potential as a therapeutic target.

Cancer stem cells (CSCs) are known to express high levels of antioxidant proteins and drug efflux transporters, and are therefore believed to be responsible for tumor recurrence after chemotherapy. Several CSC markers such as cluster of differentiation 44 (CD44) and aldehyde dehydrogenase (ALDH) have been used to isolate CSC population from cancer cell lines and tumor masses. In this study, we investigated the role of NF-E2-related factor 2 (NFE2L2; NRF2), a key regulator of the expression of antioxidant and detoxifying genes, in CSCs resistance. First, we isolated the CD44-high cell population (ADR44P) from doxorubicin-resistance breast cancer MCF7 and utilized it as a CSC-like cell line system. The ADR44P cells expressed the standard isoform of CD44 (CD44s) and displayed relatively high resistance to doxorubicin and oxidative stress compared to the CD44-low MCF7. Particularly, NRF2 and its target genes expression were substantially high in ADR44P. The treatment of ADR44P cells with hyaluronic acid (HA), a CD44 ligand, further activated NRF2 expression, whereas the inhibition of HA synthesis attenuated NRF2 activation, implying the direct activating role of CD44 in NRF2 signaling. In line with these, overexpression of CD44s in MCF7 led to NRF2 activation. As an underlying mechanism of CD44s-meidated NRF2 activation, it was observed that p62 levels were increased and its suppression resulted in the blockade of NRF2 activation in ADR44P cells. As functional implications of NRF2 activation in CD44+ CSC-like cells, the silencing of NRF2 rendered ADR44P cells sensitive to anticancer drugs, and NRF2-knockdown ADR44P cells displayed a significant tumor growth retardation in mouse xenografts. In addition to CD44, we observed that the aldehyde dehydrogenase (ALDH)-enriched ovarian cancer stem-like cells expressed high levels of NRF2 and its target genes, and stable silencing of NRF2 led to the reductions in CSC markers expression, colony formation, sphere formation, doxorubicin resistance, and tumor growth in xenografts model. Similarly to CD44+ CSCs-like cells, ALDH+ CSC-like cells showed p62 elevation, and the inhibition of p62 suppressed NRF2 signaling. Collectively, these results showed that CSCs-like cells activated NRF2 signaling, which can endow CSCs with stress resistance, aggressive phenotypes, and tumor growth.

The KEAP1-NRF2 system, originally identified as a critical defense mechanism against oxidative stress, is often dysregulated in various human cancers forming solid tumors, resulting in the aberrant activation of NRF2. Increased accumulation of NRF2 in cancers is strongly associated with the poor prognoses of cancer patients, including those with lung and breast cancers. Multiple lines of evidence suggest that aberrantly activated NRF2 in cancer cells drives their malignant progression and that the cancer cells consequently develop NRF2 addiction. We recently found a significant correlation between NRF2 and IL-11 status in breast cancer patients. Based on a previous report demonstrating that IL-11 is induced downstream of NRF2, we examined the significance of IL-11 in NRF2-driven tumorigenesis with a newly established NRF2 addiction cancer model. Expression of Il11 was elevated during the tumorigenesis of the NRF2 addiction cancer model, but intriguingly, it was hardly detected when the cancer model cells were cultured in vitro. These results imply that a signal originating from the microenvironment cooperates with NRF2 to activate Il11. To the best of our knowledge, this is the first report showing the influence of the microenvironment on the NRF2 pathway in cancer cells and the contribution of NRF2 to the secretory phenotypes of cancers. Disruption of Il11 in the NRF2 addiction cancer model remarkably inhibited the tumorigenesis, suggesting an essential role of IL-11 in NRF2-driven tumorigenesis. Thus, this study suggests that IL-11 is a potential therapeutic target for NRF2-addicted breast cancers.

Cancer immunotherapy, particularly immune checkpoint blockade resurges the effector function of tumor-infiltrating T cells and provides clinical success in various types of cancers. Yet, more than half of treated patients fail to respond to immune checkpoint blockade therapy, even in combination. It is therefore required to develop more effective cancer immunotherapies and define biomarkers to properly evaluate immune responses in cancer patients for detecting responders.

Cancer cells harboring inherent genetic instability form new antigens (so called neoantigens), stemming from gene mutations, which have not been previously recognized by the immune system. Therefore, there are two types of tumor antigens: tumor-specific antigens (TSAs), which are either oncogenic viral proteins or abnormal proteins stemming from somatic mutations (neoantigens), and tumor-associated antigens (TAAs), which are highly or aberrantly expressed normal proteins. It is not yet determined how CD8+ T cells specific for each type of antigen contribute to clinical tumor regression and whether activation of these self vs non-self antigen specific CD8+ T cells are controlled differently. FoxP3+CD4+ regulatory T cells (Tregs) inhibit anti-tumor immune responses. To address the differences of Treg suppression to these antigens, Melan-A (a representative TAA)-, and Flu and CMV (surrogate TSA)-specific CD8+ T cells were stimulated in the presence of Tregs. Tregs rendered Melan-A-specific T cells anergic (i.e., hypo-proliferative and cytokine hypo-producing upon antigen re-stimulation). In contrast, while CMV-specific CD8+ T cells proliferated in the presence of Tregs, they highly expressed co-inhibitory molecules such as PD-1, suggesting the importance of anti-PD-1 mAb for unleashing anti-tumor activity by neoantigen-specific CD8+ T cells. Thus, developing novel strategies to control Tregs, particularly in cancers that do not contain large numbers of TSAs (neoantigens) could be important for effective cancer immunotherapies.

As a resistant mechanism to immune checkpoint blockade therapy in the context of EGFR-mutated non-small cell lung cancers (NSCLCs), EGFR mutations promoted Treg infiltration and inhibited effector T-cell infiltration into tumor tissues by directly targeting chemokine expression. Thus, integrated analyses of immunological and genome assays revealed the comprehensive immune network in the tumor microenvironment, leading to the development of novel cancer immunotherapies.

Molecular profiling is a key component of precision medicine for cancer, as it provides actionable information on targetable genes or pathways for personalized treatments. Clinical tests based on panel sequencing platform are accumulating with clinical data of cancer patients. Systematic approaches to integrate clinical and genomic data could uncover the relationship between genomic alterations and clinical outcome. We have established genomic data warehouse with a unique cohort of 5,000 cancer patients with colon, stomach, lung and breast cancer, which is linked to clinical data warehouse in the hospital. Statistical analysis enabled us to extract genomic variants related to patient survival in each cancer type. The prognostic models using both clinical and genomic features outperformed clinical information-only method. The model was capable of predicting patient survival in an independent cohort. Our study provides a valuable resource to realize precision oncology as well as translational research on drug development.

Abstract not yet available.

Highly sensitive methods have been developed to obtain circulating tumor cells (CTCs) from the peripheral blood. CTC enumeration and characterization with certified systems provides reliable information on prognosis and may serve as liquid biopsy to identify therapeutic targets or mechanisms of resistance on metastatic cells. Metastatic cells might have unique characteristics that can differ from the bulk of cancer cells in the primary tumor currently used for stratification of patients to systemic therapy. Moreover, monitoring of CTCs before, during and after systemic therapy (e.g., chemotherapy, hormonal therapy, antibody therapy) might provide unique information for the future clinical management of the individual cancer patient and might serve as surrogate marker for response to therapy. In the context of recent success in antibody-mediated blockade of immune checkpoint control molecules, expression of the PD-L1 on CTCs might be of interest as potential predictive marker. Functional characterization using specialized in vitro and in vivo test systems has started, which might provide novel insights into the biology of CTCs and serve as models for drug testing.

Emerging evidence regarding the value of healthcare delivered via effective interprofessional teams has created new priorities for interprofessional education (IPE). The WHO in its Framework for Action on Interprofessional Education and Collaborative Practice proposes that an interprofessional collaborative practice-ready healthcare workforce can be achieved through IPE. The WHO defines IPE as occurring "when students from two or more professions learn about, from and with each other to enable effective collaboration and improve health outcomes." Increasingly, health professions schools are placing students together in settings to promote their interprofessional identity. IPE is a global phenomenon. In the United States, the Interprofessional Education Collaborative published core competencies for interprofessional collaborative practice bringing attention to the need for IPE within health professions training in the US. The field has advanced in other global regions as well including, for example, the Centre for the Advancement of Interprofessional Education in the UK, the Australasian Interprofessional Practice and Education Network, the European Interprofessional Practice and Education Network, and the Canadian Interprofessional Health Collaborative. Various models for organizing competencies for interprofessional collaborative practice have been developed to guide development of curricula aimed at student achievement of these competencies. Moreover, accrediting bodies mandate IPE in health professions programs in the US. Integration of IPE into the curricula of health professions schools is challenging. To facilitate integration, one widely used systematic approach to curriculum development is the six-step model. This model provides a framework for developing, implementing, evaluating, and continually improving IPE experiences. Relative to the six steps, the needs for IPE are clear, to enhance patient outcomes and safety. The goals and objectives derived can be aligned with those of professional organizations. Education methods and strategies, and evaluation tools can be developed new or adapted from external resources such as MedEdPORTAL and the National Center for Interprofessional Practice and Education. Finally, faculty may not be prepared to teach in an interprofessional setting, and faculty development thus becomes a necessity. Although challenging, incorporating IPE into our students' education will provide our learners with the training they need to become part of the collaborative practice-ready healthcare workforce.

Six-year system in Japanese pharmacy education started from 2007. The reason why we chose the big change is that pharmacists should focus on more clinical issue than material objects associated with needs of medical front. Especially, Japanese pharmacy education lacked humanism education, and pharmaceutical students are not generally very good at communication. From the reasons, the School of Pharmacy, Chiba University stated IPE with Medical School and School of Nursing.

　The purpose of our IPE is to develop autonomous healthcare professionals who provide patient and service user-centric care. Our IPE is 5 stages, and the last step, called Step 5, Clinical IPE, is making a treatment plan and discharge planning of actual patients in University Hospital. Students have already learned a holistic perspective though IPE Sterp1～4. Pharmacy students make a proposal not only general drug therapy but also approach individualized medicine. However, they can't decide which is the best for the patient because pharmacy students are insufficient training to get patient's information widely and consider deeply. While advancing a discussion, pharmacy students consider only medicine, spontaneously or they think only patient's information. However, in Clinical IPE, pharmacy students can lead better choice for the patient by discussion with other students considering with patient's opinions. From above, pharmacy students leaned the real collaboration with other profession and the meaning of patient and service user-centric care.

The Inter-Professional Education (IPE) program at Universidad Europea (Madrid, Spain) started 4 years ago and currently involves 6 health degrees and more than 1000 students every year. We are institutional members of the British IPE society (CAIPE) and members of the editorial board of the Journal of Inter-professional Education, as well as collaborators in the IPE development in Latinoamerica and Caribbean led by the Panamerican Health Organization (PAHO/WHO).

In the School of Biomedical and Health Sciences we have developed and organized activities that aim to create a collaborative and synergic environment among students of different degrees (Medicine, Nursing, Psychology, Pharmacy, Dentistry, and Biotecnology). This plan is the higher education answer to the new necessities and challenges that our healthcare practitioners are facing in their daily practice and professional development in Spain.

As part of our ongoing IPE program we focus the attention on those situations in which this multidisciplinary approach have a major impact. We found that medication errors are good examples of such situations as they can be related with the misunderstandings, distrust or simply unawareness among different health care professionals.

In the past two years, simulated interprofessional training related to medication errors have been carried out in a high fidelity environment. Our objective is to sensitize future health care professionals about events that can cause or lead to inappropriate medication use or patient harm. The practice includes an initial simulated situation followed by debriefing for analysis and comprehension of the key points: decision making, conflict solution and responsibility.

Anonymous questionnaires, taken in order to control acceptance, learning satisfaction and suggestions, indicate a strong and enthusiast support of this IPE program. The results obtained and the acquired experience, are driving us to further develop new interprofessional activities in the near future as well as introducing IPE activities in new educational areas.

In 2013, a group of scientists from Nigeria, Zimbabwe, Kenya, Uganda, Ethiopia, Sweden and South Africa, engaged a European funded e-infrastructure project to set up a facility for the sharing of research tools in pharmacology called the Pharmacology Science Gateway (PSG), http://sgw.africa-grid.org/pharmacology-science-gateway. The main purpose of the platform being to provide research resources such as open source software, access to high performance computing to process data from clinical and biomedical research in genomics, platforms for sharing and discussing research results or/and solving laboratory problem such as bioanalytical protocols.

The justification for this initiative being the apparent lack of continuity of mentorship and training of pharmacologists after their completion of PhD studies locally in Africa or abroad, a situation which has resulted in their failing to develop to competent research leaders who can (a) continue to conduct research at a high level as in the laboratories they graduated from, (b) develop and write competitive research proposals to attract both joint and individual research grants, and (c) establish research groups that can in turn produce the next generation of competent researchers. We believe that this is a loss on investment in the training of pharmacologists who should be conducting research to address the healthcare burden Africa faces and that one way of addressing this challenge is to establish a mentoring and training program which targets postdoctoral fellows and mid-career pharmacologists for development with respect to (a) ability to access and use some of the technical and scientific resources they might not have at their research institutes using both e-platform shared resources & network based access to laboratories with such resources, (b) to train on materials developed by world leaders in the various selected fields of pharmacology through both face to face training and/or e-learning platforms, and (c) identifying world leading experts in the various fields of pharmacology to act as mentors to individuals or groups of young pharmacologists on research question development, research grant proposal writing and research group leadership. This presentation will highlight the current status of this PSG and prospects of scaling up the trainingg of laboraotry pharmacologists in Africa.

If Nigeria can be example of Sub-Saharan Africa; it has 40 federal universities, 46 state universities, 69 private universities (according to the Nigerian Universities Commission) for a population of 193 million Nigerians (Worldometer). Out of the federal universities, one was founded in 1960 and three were founded in 1962, more than half a century ago. For long, scholarship in Nigerian institutions adopted the three-legged stool paradigm: teaching, research, and service. The outcome on Nigerian development and economy over half a century includes massive brain drain, infrastructural and functional challenges, importation of most of the everyday needs of Nigerians especially drugs and medical supplies and equipment, expensive medical tourism abroad, and underutilization of all kinds of raw materials, including crude oil, tree barks, and other sources of drugs and health care delivery resources. Clearly, the three-legged stool cannot comfortably seat Africa.

In 'Scholarship Reconsidered: Priorities of the Professoriate' (1990), Boyer suggested a broadening of scholarship to include: the scholarship of discovery, the scholarship of teaching, the scholarship of integration, and the scholarship of application.

Africa is traditionally a patriarchal society of people led by age and authority (who wait for change to come from above) and which fails to underscore the contribution of the individual initiative. With the advent of the Internet, this tradition is giving way to a culture of holistic human development fed by information technology, self-directed learning, life-long learning, and self-motivation. Nigeria, and indeed much of Africa, is forced into a new paradigm of scholarship.

The TRES Paradigm for African Academia encourages scientific education that is useful and utilized based on established facts that theory and skills, heads and hands, and individual initiatives together generate development. The TRES paradigm includes enterprising - theory and skills - as a cardinal goal in order to enable utilization of knowledge for services and production of goods and gain. The African PhD Pharmacology cannot be for just the bookshelf but should result in enterprising: science-based improvement of society. Diversification of reward from academic promotion (through publication of scientific papers per se) to include wealth and national honours is necessary amongst Africans.

How to promote pharmacology in resource-strained countries? First by freely-available web-based databases and pharmacology-learning facilities. IUPHAR with 68 member societies (～35,000 pharmacologists), is an NGO to WHO, and has set up freely-available web-based databases on drug targets and education, in conjunction with a strong alliance with the British Pharmacological society (BPS), producing the IUPHAR/BPS guidetopharmacology.org, and the Concise Guide to Pharmacology. The nomenclature committee of IUPHAR (NC-IUPHAR) ensures that this guide to drug targets is supported by >90 subcommittees of ～860 expert scientists, in a unique cooperative international initiative. The receptor reviews are highly cited (h-index 84). IUIS (immunology) and IUPHAR are collaborating on inflammation and immune system research as immunopharmacological drug targets are crucial for new drug discovery, and the new database guide to immunopharmacology is now freely available. We are endeavouring to raise funds for a major initiative to supply simple immunopharmacological protocols which can be used for scientists in developing countries. We have now created an alliance with the Medicines for Malaria Venture to create a freely available website on drug targets for malaria research.

Natural product research is frequently a major issue in resource-strained companies and IUPHAR brings together two different worlds by creating synergies between them, rather than independent research, and increasing mechanistic background via databases. Scientific education to the developing (and developed) world via our publicly available web sites (Pharmacology Education Project) is a major issue for IUPHAR. We have set up six clinical mentoring centres which provide free advice for clinical scientists in developing countries. A series of clinical workshops on regulatory and other issues have also been held.

IUPHAR, through its IOSP initiative, has been running short courses in in-vivo pharmacology, predominately in Africa, since 2007, via David Lewis (Chair, IUPHAR IOSP) - these have now been extended via the Chinese Pharmacological Society, in two Chinese Institutions, promoting good practice in animal welfare, ethics and the 3Rs, but also pharmacology education. We wish to lever central research funding in India, China, South America, Africa, via the pharmacological societies, with European or US funding to create multiple synergies for pharmacology research and education.

Abstract not yet available.

Abstract not yet available.

Retinal photoreceptor cells, composed of rod and cone photoreceptor cells, work as light detectors through phototransduction. Both rod and cone photoreceptor cells develop cell type-specific unique structures, including ribbon synapses and photosensitive outer segments. These elaborate structures are essential for proper photoreception, phototransduction, and signal transmission to secondary neurons in the retina. Degeneration of photoreceptor cells in human is characteristic in several inherited retinal diseases such as retinitis pigmentosa, macular degeneration, and Usher syndrome. Understanding of molecular mechanisms underlying photoreceptor cell development and maintenance is important for developing treatment for retinal degeneration diseases. We have been investigating molecular mechanisms of photoreceptor cell differentiation, maturation, function, and survival by mainly using mouse molecular genetics. Based on these studies, we are currently attempting to develop a novel neuroprotection method using mouse photoreceptor degeneration models. We will present our recent findings on function and possible neuroprotection of photoreceptor cells.

Everyone wants to stay healthy. Humans have put great effort into researching and developing better medicines and treatment methods in order to promote and maintain their health.

To develop more effective and safer drugs, in recent years pharmaceutical development has been shifting toward the discovery of biodrugs (generated in the human body and possessing effective functions) and away from finding compound drugs among candidate molecules synthesized from chemical compounds. A representative example of biodrugs is one that uses antibodies (proteins), which provide an immunological function.

The iPSC regenerative medicine products that Healios is developing use cells which are the smallest unit of life, to develop biodrugs. We expect such biodrugs to serve as breakthrough drugs for chronic diseases caused by aging or hereditary conditions that result in cell degeneration.

Healios believes such products will be part of a major paradigm shift in the life science arena that will lead the biorevolution of the 21st century.

The first in man application of iPS-derived cells started in September 2014, targeted age-related macular degeneration (AMD). AMD is caused by the senescence of retinal pigment epithelium (RPE), so that we aimed to replace damaged RPE with normal, young RPE made from iPS cells. We judged the outcome 1 year after the surgery. Primary endpoint was the safety, mainly the tumor formation and immune rejection. The grafted RPE cell sheet was not rejected nor made tumor after two years. The patient's visual acuity stabilized after the surgery whereas it deteriorated before surgery in spite of 13 times injection of anti-VEGF in the eye.

　Although autologous RPE sheet transplantation is scientifically best approach, it is time consuming and expensive and it is necessary to prepare allogeneic transplantation to establish a standard treatment. RPE cells are suitable for allogeneic transplantation because they suppress the activation of the T-cell. From in vitro and in vivo study, it is possible that the rejection is considerably suppressed by using the iPS cell with matched HLA. Our new protocol has accepted by ministry in Feb 2017. We are planning transplantation using allogeneic iPS-RPE cell suspension & sheet, and also autologous iPS-RPE. For the cell suspension transplantation we will not combine CNV removal and apply to milder cases than sheet transplantation.

　In Japan, pharmaceutical law has been changed and a new chapter for regenerative medicine was created for clinical trial. Also the separate law for safety of regenerative medicine for clinical research (study) was enforced in 2015. These laws made the suitable condition for the brand new field of regenerative medicine. We are making regenerative medicine in co-operation with ministry & academia.

The Focus on Pharmacology project was an ambitious programme of work supported by the British Pharmacological Society aiming to deepen understanding of pharmacology education, employment and impact. The project was a response to changes in the scientific and pharmaceutical landscape, such as a greater focus on interdisciplinary collaboration and an increased role for academia and contract research organisations in drug discovery and development respectively. Focus asked fundamental questions about the pharmacology education and employment landscape, about the needs of educators and employers and developed tools and campaigns to support it within the education, life sciences and healthcare sectors. This talk will explore the challenges for UK pharmacology in the context of a global pharmacology community.

Focus on Pharmacology projects were developed from a set of research questions covering areas such as where pharmacology is taught, where pharmacologists work, how the discipline and related sciences should be taught in the modern world and how should graduates and trainees be supported, understanding the needs of educators and the contribution of pharmacology to the life sciences and healthcare. The work-streams were developed in parallel as external commissions, in house work by the British Pharmacological Society and external research partnerships and designed to feed into the development of the new Society strategy for 2018 onwards. This talk will give an overview of project highlights such as an audit of the education and employment landscape, development of strategic skills initiatives for in vivo sciences and clinical pharmacology and an analysis of pharmacology's role in drug discovery and development.

The British Pharmacological Society has developed a new core curriculum for undergraduate pharmacology degrees. To do this, we used a modification of the Delphi Process. We began with a pharmacology educator workshop to explore the core attributes expected of pharmacology graduates. We then developed these discussions into knowledge, skills and attitudes statements and sent them, in the form of a questionnaire, to our Expert Group, which included pharmacology professionals from across academia and industry. In an iterative process, we asked the Expert Group to rank each statement according to how much they agreed it was a core graduate attribute. Where there was disagreement, we modified statements according to feedback. After 3 rounds of questionnaires, we had a draft core curriculum which was then finalised through a discussion workshop with the education community. In this workshop, we also discussed practical aspects of curriculum implementation and the potential for the Society to develop resources to support it.

In the future, the Society will be considering how, in addition to pharmacology programmes, the curriculum can be used to inform degrees in the biomedical sciences, as well as clinical and post-graduate pharmacology training provision. We have also used the Delphi method to develop a curriculum for the use of research animals.

Focus on Pharmacology was a research-led approach aiming to help us understand and support the discipline. The approach of Society driven research has 'proved concept' in that the wealth of information has helped set, or strengthen our trajectory into the future through its influence on our next long-term strategy. The common findings arising from this programme of work showed that we should have confidence in the discipline, embrace collaborative science and healthcare practice, and that we should use the Society to build community and visibility for pharmacology. These principles are embedded in our vision and mission, and have already informed ongoing projects and strategic commitments. The Society will build on the legacy of Focus through setting the agenda in education and skills, for students, professionals and educators alike. Similarly, 'focusing' on strategic skills areas such as clinical pharmacology has enabled us to build internal capacity and expertise - and our standing with policy and decision makers. This talk will explore the impact of Focus from the UK perspective, but open this up into a conversation about the role of research and collaboration for the advancement of pharmacology on a global scale.

Background: The term druggable genome was coined in 2002 for the estimate of 10% of the human proteome likely to bind small molecules with lead-like chemical properties and sufficient binding affinity for activity modulation. A number of databases now provide protein target lists based largely on chemistry-to-protein mappings extracted from the literature. The IUPHAR/BPS Guide to PHARMACOLOGY (GtoPdb) contains expert curated target sets at a high stringency and updated approximately bi-monthly [1]. This work introduces the statistics of GtoPdb proteome coverage and compares this with other established sources.

Methods: The first step was to review the statistics of GtoPdb for proteins with ligand interactions and the subset where those interactions have quantitative binding data. We then turned to UniProt where we have submitted cross-references. We also used chemistry cross-reference in UniProt to extract target counts for other sources. The intersects and differences were analysed by Venn diagrams, Gene Ontology distributions and other types of cross-reference counts.

Results: GtoPdb includes 1684 human protein targets with ligand interactions with 1489 having quantitative binding data for 6716 ligands. Many of these could provide plausible starting points for drug research and our data show 2089 ligands have clinical data. However, only 313 proteins are the primary targets of 1233 approved drugs, reflecting the validation bottleneck for new targets. We retrieved the following counts for human target counts in other UniProt sources for DrugBank 2376, ChEMBL 3310 and BindingDB 2474. The intersect between all four was only 517. Gene Ontology functional categories, target class content, 3D structure, pathways, disease genes were analysed for the GtoPdb and the consensus sets

Conclusion: This work presents a concise data-supported druggable proteome snapshot from GtoPdb and comparative coverage for three other resources. These four show surprisingly divergent curatorial selectivity. However, the consensus is increasing slowly and the union covers nearly 20% of the proteome. The utility of data-supported druggable proteome lists is high, particularly when genetic data points to new targets likely to have at least some chemical starting points for validation experiments.

References: [1]] Harding et al. (2018). Nucl. Acids Res. 45 (Database Issue)

The G protein-coupled receptor database, GPCRdb

G protein-coupled receptors (GPCRs) are the most abundant mediators of both human signalling processes and therapeutic effects. The GPCR database, GPCRdb comprises reference data, online analysis tools and interactive visualisation (www.gpcrdb.org) (1).

New GPCRdb sections that will be presented:

1. Drugs: drug statistics, target mapping and browsing of drugs, targets, indications, clinical progression. Based on our analysis of the new trends for GPCR drugs, targets and indications covering FDA-approved drugs and agents in clinical trials (2).

2. Signal Proteins: facilitates investigation of GPCR-G protein coupling profiles, interfaces and in vitro mutations; and was added as part of a landmark study on GPCR-G protein selectivity (3).

3. Genetic variation: features variation statistics, browsing and an Estimated economic burden caused by genetic variation; and was added as part of a comprehensive report on pharmacogenomics of GPCR drug targets (4).

4. GPCRome-wide homology models: models of unprecedented quality are provided for inactive, intermediate and active states - except for classes C and F that only have inactive templates (1).

5. Ligand database with biological activities and commercial availability for 150,000 GPCR ligands from ChEMBL (1).

6. Crystallisation construct design tool based on all available GPCR structures (5)

7. Sequence signatures tool to elucidate functional determinants in evolutionary and functionally related receptors.

8. Residue contact networks data and tools to elucidate e.g. determinants of specific receptor conformational and activity states.

9. Biased agonist database. Preliminary results will be shared for a forthcoming resource of ligands with a proposed signal pathway-bias annotated from publications and patents.

References

1 Pandy-Szekeres, G. et al. GPCRdb in 2018: adding GPCR structure models and ligands. Nucleic Acids Res, 2017

2 Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schioth, H. B. & Gloriam, D. E. Trends in GPCR drug discovery: new agents, targets and indications. Nature reviews. Drug discovery, 2017

3 Flock, T. et al. Selectivity determinants of GPCR-G-protein binding. Nature, 2017

4 Hauser, A. S. et al. Pharmacogenomics of GPCR Drug Targets. Cell 2017

5 Munk, C. et al. GPCR crystallisation constructs and conditions - In-depth analysis and public resource. Manuscript.

More than 500 protein kinases are encoded in the human genome. Although several kinases have been well-characterized and demonstrated to be physiologically important, the functions of a number of kinases remain to be elucidated. To identify the specific substrates for the specific kinases, we have recently developed an in vitro approach termed the kinase-interacting substrate screening (KISS) method (Amano et al. J Cell Biol 2015) and an in vivo approach termed kinase-oriented substrate screening (KIOSS) method (Nagai et al. Neuron 2016; Nagai et al. Trend Pharmacol Sci 2016). By using these methods, we identified the PKA substrates downstream of dopamine from mouse striatum, and found more than one hundred candidate substrates of PKA, including Rap1 GEF (Rasgrp2). We also found that PKA phosphorylation of Rasgrp2 activated its guanine nucleotide exchange activity on Rap1. Cocaine exposure activated Rap1 in nucleus accumbense in mice through dopamine. The expression of constitutively active PKA or Rap1 in accumbal D1R-expressing medium spiny neurons (D1R-MSNs) enhanced neuronal firing rates and behavioral responses to cocaine exposure through MAPK. These findings demonstrate a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors. We also constructed an on-line database system, named KANPHOS (Kinase-Associated Neural PHOspho-Signaling), that provides the phosphorylation signals identified by our methods as well as those previously reported in the literature. We here discuss the phospho-proteomic approach and the database.

G-protein coupled receptors (GPCRs) represent the single largest class of druggable targets in the human genome. Of the 390 or so druggable and non-olfactory human GPCRs there exist many which are orphan and/or understudied; we refer to these as "oGPCRs". Here I will show how we plan to illuminate the pharmacology, signaling pathways, chemical biology, distribution and function of 143 oGPCRs. This project seeks to discover and develop specific, community accessible tools, chemical probe molecules and engineered animals that enable investigators to interrogate the biological functions of oGPCRs. Given the central importance of GPCRs for all areas of biomedical research, illuminating the pharmacology, function, signaling and chemical biology of these oGPCRs will have far-reaching impact for both therapeutics and basic biomedical science.