Expression and Functional Analysis of Ctenophore Glutamate Receptor Genes.

The functional analysis of ctenophore neurotransmitter receptors, transporters, and ion channels can be greatly simplified by use of heterologous expression systems. Heterologous expression allows the characterization of individual membrane proteins, expressed at high levels in cells, where background activity by endogenous ion channels and transporters is with few exceptions minimal. The goal of such experiments is to gain an in-depth understanding of the behavior and regulation of individual molecular species, which is challenging in native tissue, but especially so in the case of ctenophores and other marine organisms. Coupled with transcriptome analysis, and immunohistochemical studies of receptor expression in vivo, experiments with heterologous expression systems can provide valuable insight into cellular activity, prior to more challenging functional studies on native tissues.

Dynamic Regulatory Assessment: Unpacking the Process Reveals Readiness to Pilot for Europe-An EFPIA View.

The medicines regulatory network of the European Economic Area comprises 30 countries, their National Competent Authorities (NCA), and the European Medicines Agency (EMA). The NCAs and EMA are involved at different stages of the medicine life cycle; not all are engaged in a particular medicine's development discussions. As a result, knowledge management (ie, acquisition and transfer between medicine developer and the NCAs) is fragmented and inefficient. Dynamic regulatory assessment (DRA), a regulatory science concept developed by the European Federation of Pharmaceutical Industries and Associations (EFPIA), could drive increased connectedness supporting more continuous knowledge building. DRA works via iterative release and assessment of discrete data packets (DDPs) at mutually agreed milestones during development, culminating in more efficient development and faster authorization. This commentary seeks to build on an earlier article by unpacking the DRA concept, with a particular focus on DDPs. Its aim is to show how DDPs can support efficient and predictable release of data to encourage development and assessment of promising medicines, and it makes the case for piloting the DRA concept with European regulators now.

Dynamic Regulatory Assessment: evolving the European Regulatory Framework for the Benefit of Patients and Public Health-an EFPIA View.

The European Union regulatory framework supports development, review, authorization, and maintenance of medicines to benefit public health; however, many elements are 2 decades old and undergoing review. Scrutiny was triggered by the coronavirus disease 2019 pandemic, the need to support future innovative medicines, the digital transformation of data exchange, and the need to address efficiency and capacity limitations. There are also ongoing evolutions in regulatory science for medicines (eg, cell and gene therapies), medical device combinations, and software, as well as the need to fully leverage contemporary information technology (IT). Important initiatives to address these challenges include the European Medicines Agency (EMA) Regulatory Science Strategy,1 the EU Regulatory Network Strategy,2 and the Big Data Steering Group,3 alongside European Commission-led initiatives such as the Pharmaceutical Strategy.4 Dynamic regulatory assessment (DRA) is a concept that seeks to integrate these various elements to re-imagine regulatory review interactions across the product life cycle. DRA calls for iterative regulatory dialogue, data submission, and evidence assessment, enabled by contemporary IT. DRA will facilitate iterative interaction and data assessment as it accumulates over a product's life cycle, bringing significant efficiencies for all product types. The DRA concept primarily evolved through dialogue within working groups of the European Federation of Pharmaceutical Industries and Associations. This article describes the long-term vision of the European Federation of Pharmaceutical Industries and Associations and outlines important strategic elements of progress, including: aligning on a multi-stakeholder vision for DRA in the European Union and across regions; leveraging learnings from ongoing initiatives; and advancing IT, governance, and standards considerations. Ultimately, DRA should consider outcomes that deliver optimal benefits for patients in the European Union and worldwide.

21st century excitatory amino acid research: A Q & A with Jeff Watkins and Dick Evans.

In 1981 Jeff Watkins and Dick Evans wrote what was to become a seminal review on excitatory amino acids (EAAs) and their receptors (Watkins and Evans, 1981). Bringing together various lines of evidence dating back over several decades on: the distribution in the nervous system of putative amino acid neurotransmitters; enzymes involved in their production and metabolism; the uptake and release of amino acids; binding of EAAs to membranes; the pharmacological action of endogenous excitatory amino acids and their synthetic analogues, and notably the actions of antagonists for the excitations caused by both nerve stimulation and exogenous agonists, often using pharmacological tools developed by Jeff and his colleagues, they provided a compelling account for EAAs, especially l-glutamate, as a bona fide neurotransmitter in the nervous system. The rest, as they say, is history, but far from being consigned to history, EAA research is in rude health well into the 21st Century as this series of Special Issues of Neuropharmacology exemplifies. With EAAs and their receptors flourishing across a wide range of disciplines and clinical conditions, we enter into a dialogue with two of the most prominent and influential figures in the early days of EAA research: Jeff Watkins and Dick Evans.

Partial agonists go molecular.

Single-channel analysis previously revealed a key role for a short-lived 'flipped' state during glycine receptor activation by partial agonists. Structures solved by Yu and colleagues now reveal a surprising mechanism involving a partially activated agonist-bound closed state that is too long-lived to be considered the flipped state.

Structural biology of kainate receptors.

This review summarizes structural studies on kainate receptors that explain unique functional properties of this receptor family. A large number of structures have been solved for ligand binding domain dimer assemblies, giving insight into the subtype selective pharmacology of agonists, antagonists, and allosteric modulators. Structures and biochemical studies on the amino terminal domain reveal mechanisms that play a key role in assembly of heteromeric receptors. Surprisingly, structures of full length homomeric GluK2, GluK3 and heteromeric GluK2/GluK5, receptors reveal a novel structure for the desensitized state that is strikingly different from that for AMPA receptors.

Glutamate receptors from diverse animal species exhibit unexpected structural and functional diversity.

The identification of AMPA, kainate and NMDA glutamate receptor subtypes by Watkins and colleagues underlies much of our understanding of excitatory synaptic transmission in the central nervous system of animals. Ongoing large scale genome sequencing projects in species for which physiological analysis of receptor function is challenging are resulting in identification of numerous eukaryotic glutamate receptor ion channels in the animal kingdom of life. On the basis of sequence similarity, these are frequently classified into the three vertebrate subtypes, initially identified using subtype selective ligands. Recent work reveals unexpected ligand binding profiles for these newly identified glutamate receptors, for example, kainate receptors on which NMDA acts as a competitive antagonist, and high affinity homomeric glycine activated glutamate receptors. Structural studies reveal that only subtle changes in the ligand binding domain, often identified only in retrospect, underlie different patterns of ligand binding, and that the biology of glutamate receptors is more complex than first anticipated.

Residence time determines invasiveness and performance of garlic mustard (Alliaria petiolata) in North America.

While biological invasions have the potential for large negative impacts on local communities and ecological interactions, increasing evidence suggests that species once considered major problems can decline over time. Declines often appear driven by natural enemies, diseases or evolutionary adaptations that selectively reduce populations of naturalised species and their impacts. Using permanent long-term monitoring locations, we document declines of Alliaria petiolata (garlic mustard) in eastern North America with distinct local and regional dynamics as a function of patch residence time. Projected site-specific population growth rates initially indicated expanding populations, but projected population growth rates significantly decreased over time and at the majority of sites fell below 1, indicating declining populations. Negative soil feedback provides a potential mechanism for the reported disappearance of ecological dominance of A. petiolata in eastern North America.

Structural biology of glutamate receptor ion channels: towards an understanding of mechanism.

Ionotropic glutamate receptors (iGluRs) are tetrameric ion channels that mediate signal transmission at neuronal synapses, where they contribute centrally to the postsynaptic plasticity that underlies learning and memory. Receptor activation by l-glutamate triggers complex allosteric cascades that are transmitted through the layered and highly flexible receptor assembly culminating in opening a cation-selective pore. This process is shaped by the arrangement of the four core subunits as well as the presence of various auxiliary subunits, and is subject to regulation by an array of small molecule modulators targeting a number of sites throughout the complex. Here, we discuss recent structures of iGluR homomers and heteromers illuminating the organization and subunit arrangement of the core tetramer, co-assembled with auxiliary subunits and in complex with allosteric modulators.

Medicines Adaptive Pathways to Patients: Why, When, and How to Engage?

Medicines Adaptive Pathways to Patients (MAPPs) seeks to foster access to novel beneficial treatments for the right patient groups at the earliest appropriate time in the product life-span, in a sustainable fashion. We summarize the MAPPs engagement process and critical questions to be asked at each milestone of the product life-span. These considerations are of relevance for regulatory and access pathways that strive to address the "evidence vs. access" conundrum.

Potential Use of Artificial Intelligence for Regulatory Intelligence: Biopharmaceutical Industry's Views.

BACKGROUND: Pharmaceutical companies rely on regulatory intelligence (RI) to analyze information from internal and external sources. To facilitate RI activities, companies are seeking ways to harness technology to optimize their capabilities. Specifically, there is a growing interest for artificial intelligence (AI) to enhance RI activities. However, exploration of the potential utility of AI and related technologies will be key to begin unlocking these tools for the regulatory community. METHODS: To identify potential development paths for these technologies, we interviewed over 30 global regulatory leaders at 22 pharmaceutical companies and 3 leading companies in RI and AI technologies. Thirteen of the 22 pharmaceutical companies also provided responses to a subsequent informational survey. RESULTS: This study elucidated potential value proposition, barriers, and risks to integrating AI into the RI field. Twenty of the 22 participating companies consider that AI offers significant opportunity for RI activities of data processing (mining, searching, monitoring, alerting). Thirty-two percent of companies envisage use in data synthesis (combining different types of information across formats), 36% in data analysis (trends, patterns, predictive analytics), and 23% in decision making. Additionally, results of this research provided insights about the potential role of precompetitive consortia, which may enhance future actualization. CONCLUSIONS: Opportunity presents for AI to enhance quality, speed, and efficiency of RI activities. This assessment of the current technology landscape revealed a lack of fully developed AI tools; however, the RI community demand is beginning to be recognized. Therefore, now more than ever, the timing to advance AI within the RI field is right.

Family matters.

Genome sequence data from a range of animal species are raising questions about the origins of glutamate receptors.

Impact of a brief faculty training to improve patient-centered communication while using electronic health records.

OBJECTIVE: Despite rapid EHR adoption, few faculty receive training in how to implement patient-centered communication skills while using computers in exam rooms. We piloted a patient-centered EHR use training to address this issue. METHODS: Faculty received four hours of training at Cleveland Clinic and a condensed 90-minute version at the University of Chicago. Both included a lecture and a Group-Objective Structured Clinical Exam (GOSCE) experience. Direct observations of 10 faculty in their clinical practices were performed pre- and post-workshop. RESULTS: Thirty participants (94%) completed a post-workshop evaluation assessing knowledge, attitude, and skills. Faculty reported that training was important, relevant, and should be required for all providers; no differences were found between longer versus shorter training. Participants in the longer training reported higher GOSCE efficacy, however shorter workshop participants agreed more with the statement that they had gained new knowledge. Faculty improved their patient-centered EHR use skills in clinical practice on post- versus pre-workshop ratings using a validated direct-observation rating tool. CONCLUSION: A brief lecture and GOSCE can be effective in training busy faculty on patient-centered EHR use skills. PRACTICE IMPLICATIONS: Faculty training on patient-centered EHR skills can enhance patient-doctor communication and promotes positive role modeling of these skills to learners.

Preferential assembly of heteromeric kainate and AMPA receptor amino terminal domains.

Ion conductivity and the gating characteristics of tetrameric glutamate receptor ion channels are determined by their subunit composition. Competitive homo- and hetero-dimerization of their amino-terminal domains (ATDs) is a key step controlling assembly. Here we measured systematically the thermodynamic stabilities of homodimers and heterodimers of kainate and AMPA receptors using fluorescence-detected sedimentation velocity analytical ultracentrifugation. Measured affinities span many orders of magnitude, and complexes show large differences in kinetic stabilities. The association of kainate receptor ATD dimers is generally weaker than the association of AMPA receptor ATD dimers, but both show a general pattern of increased heterodimer stability as compared to the homodimers of their constituents, matching well physiologically observed receptor combinations. The free energy maps of AMPA and kainate receptor ATD dimers provide a framework for the interpretation of observed receptor subtype combinations and possible assembly pathways.

The Challenge of Interpreting Glutamate-Receptor Ion-Channel Structures.

Ion channels activated by glutamate mediate excitatory synaptic transmission in the central nervous system. Similar to other ligand-gated ion channels, their gating cycle begins with transitions from a ligand-free closed state to glutamate-bound active and desensitized states. In an attempt to reveal the molecular mechanisms underlying gating, numerous structures for glutamate receptors have been solved in complexes with agonists, antagonists, allosteric modulators, and auxiliary proteins. The embarrassingly rich library of structures emerging from this work reveals very dynamic molecules with a more complex conformational spectrum than anticipated from functional studies. Unanticipated conformations solved for complexes with competitive antagonists and a lack of understanding of the structural basis for ion channel subconductance states further highlight challenges that have yet to be addressed.

The structure and function of glutamate receptors: Mg2+ block to X-ray diffraction.

Experiments on the action of glutamate on mammalian and amphibian nervous systems started back in the 1950s but decades passed before it became widely accepted that glutamate was the major excitatory neurotransmitter in the CNS. The pace of research greatly accelerated in the 1980s when selective ligands that identified glutamate receptor subtypes became widely available, and voltage clamp techniques, coupled with rapid perfusion, began to resolve the unique functional properties of what cloning subsequently revealed to be a large family of receptors with numerous subtypes. More recently the power of X-ray crystallography and cryo-EM has been applied to the study of glutamate receptors, revealing their atomic structures, and the conformational changes that underlie their gating. In this review I summarize the history of this field, viewed through the lens of a career in which I spent 3 decades working on the structure and function of glutamate receptor ion channels. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

Novel Functional Properties of Drosophila CNS Glutamate Receptors.

Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation. VIDEO ABSTRACT.

Molecular lock regulates binding of glycine to a primitive NMDA receptor.

The earliest metazoan ancestors of humans include the ctenophore Mnemiopsis leidyi The genome of this comb jelly encodes homologs of vertebrate ionotropic glutamate receptors (iGluRs) that are distantly related to glycine-activated NMDA receptors and that bind glycine with unusually high affinity. Using ligand-binding domain (LBD) mutants for electrophysiological analysis, we demonstrate that perturbing a ctenophore-specific interdomain Arg-Glu salt bridge that is notably absent from vertebrate AMPA, kainate, and NMDA iGluRs greatly increases the rate of recovery from desensitization, while biochemical analysis reveals a large decrease in affinity for glycine. X-ray crystallographic analysis details rearrangements in the binding pocket stemming from the mutations, and molecular dynamics simulations suggest that the interdomain salt bridge acts as a steric barrier regulating ligand binding and that the free energy required to access open conformations in the glycine-bound LBD is largely responsible for differences in ligand affinity among the LBD variants.