Modulation of Slow Desensitization (Tachyphylaxis) of Acid-Sensing Ion Channel (ASIC)1a.

Among the proton-activated channels of the ASIC family, ASIC1a exhibits a specific tachyphylaxis phenomenon in the form of a progressive decrease in the response amplitude during a series of activations. This process is well known, but its mechanism is poorly understood. Here, we demonstrated a partial reversibility of this effect using long-term whole-cell recording of CHO cells transfected with rASIC1a cDNA. Thus, tachyphylaxis represents a slow desensitization of ASIC1a. Prolonged acidifications provided the same recovery from slow desensitization as short acidifications of the same frequency. Slow desensitization and steady-state desensitization are independent processes although the latter attenuates the development of the former. We found that drugs which facilitate ASIC1a activation (e.g., amitriptyline) cause an enhancement of slow desensitization, while inhibition of ASIC1a by 9-aminoacridine attenuates this process. Overall, for a broad variety of exposures, including increased calcium concentration, different pH conditions, and modulating drugs, we found a correlation between their effects on ASIC1a response amplitude and the development of slow desensitization. Thus, our results demonstrate that slow desensitization occurs only when ASIC1a is in the open state.

Analysis of residue-residue interactions in the structures of ASIC1a suggests possible gating mechanisms.

The gating mechanism of acid-sensitive ion channels (ASICs) remains unclear, despite the availability of atomic-scale structures in various functional states. The collapse of the acidic pocket and structural changes in the low-palm region are assumed to trigger activation. For the acidic pocket, protonation of some residues can minimize repulsion in the collapsed conformation. The relationship between low-palm rearrangements and gating is unknown. In this work, we performed a Monte Carlo energy optimization of known ASIC1a structures and determined the residue-residue interactions in different functional states. For rearrangements in the acidic pocket, our results are consistent with previously proposed mechanisms, although significant complexity was revealed for the residue-residue interactions. The data support the proposal of a gating mechanism in the low-palm region, in which residues E80 and E417 share a proton to activate the channel.

Cellular and molecular mechanisms of aflatoxin B1-mediated neurotoxicity: The therapeutic role of natural bioactive compounds.

Aflatoxin B1 (AFB1), a dietary toxin from the mold Aspergillus species, is well acknowledged to elicit extra-hepatic toxicity in both animals and humans. The neurotoxicity of AFB1 has become a global public health concern. Contemporary research on how AFB1 enters the brain to elicit neuronal dysregulation leading to noxious neurological outcomes has increased greatly in recent years. The current review discusses several neurotoxic outcomes and susceptible targets of AFB1 toxicity at cellular, molecular and genetic levels. Specifically, neurotoxicity studies involving the use of brain homogenates, neuroblastoma cell line IMR-32, human brain microvascular endothelial cells, microglial cells, and astrocytes, as well as mammalian and non-mammalian models to unravel the mechanisms associated with AFB1 exposure are highlighted. Further, some naturally occurring bioactive compounds with compelling therapeutic effects on AFB1-induced neurotoxicity are reviewed. In conclusion, available data from literature highlight AFB1 as a neurotoxin and its possible pathological contribution to neurological disorders. Further mechanistic studies aimed at discovering and developing effective therapeutics for AFB1 neurotoxicity is warranted.

Possible Compensatory Role of ASICs in Glutamatergic Synapses.

Proton-gated channels of the ASIC family are widely distributed in central neurons, suggesting their role in common neurophysiological functions. They are involved in glutamatergic neurotransmission and synaptic plasticity; however, the exact function of these channels remains unclear. One problem is that acidification of the synaptic cleft due to the acidic content of synaptic vesicles has opposite effects on ionotropic glutamate receptors and ASICs. Thus, the pH values required to activate ASICs strongly inhibit AMPA receptors and almost completely inhibit NMDA receptors. This, in turn, suggests that ASICs can provide compensation for post-synaptic responses in the case of significant acidifications. We tested this hypothesis by patch-clamp recordings of rat brain neuron responses to acidifications and glutamate receptor agonists at different pH values. Hippocampal pyramidal neurons have much lower ASICs than glutamate receptor responses, whereas striatal interneurons show the opposite ratio. Cortical pyramidal neurons and hippocampal interneurons show similar amplitudes in their responses to acidification and glutamate. Consequently, the total response to glutamate agonists at different pH levels remains rather stable up to pH 6.2. Besides these pH effects, the relationship between the responses mediated by glutamate receptors and ASICs depends on the presence of Mg2+ and the membrane voltage. Together, these factors create a complex picture that provides a framework for understanding the role of ASICs in synaptic transmission and synaptic plasticity.

Unlocking the PIP-box: A peptide library reveals interactions that drive high-affinity binding to human PCNA.

The human sliding clamp, Proliferating Cell Nuclear Antigen (hPCNA), interacts with over 200 proteins through a conserved binding motif, the PIP-box, to orchestrate DNA replication and repair. It is not clear how changes to the features of a PIP-box modulate protein binding and thus how they fine-tune downstream processes. Here, we present a systematic study of each position within the PIP-box to reveal how hPCNA-interacting peptides bind with drastically varied affinities. We synthesized a series of 27 peptides derived from the native protein p21 with small PIP-box modifications and another series of 19 peptides containing PIP-box binding motifs from other proteins. The hPCNA-binding affinity of all peptides, characterized as KD values determined by surface plasmon resonance, spanned a 4000-fold range, from 1.83 nM to 7.59 µM. The hPCNA-bound peptide structures determined by X-ray crystallography and modeled computationally revealed intermolecular and intramolecular interaction networks that correlate with high hPCNA affinity. These data informed rational design of three new PIP-box sequences, testing of which revealed the highest affinity hPCNA-binding partner to date, with a KD value of 1.12 nM, from a peptide with PIP-box QTRITEYF. This work showcases the sequence-specific nuances within the PIP-box that are responsible for high-affinity hPCNA binding, which underpins our understanding of how nature tunes hPCNA affinity to regulate DNA replication and repair processes. In addition, these insights will be useful to future design of hPCNA inhibitors.

Towards Modeling Anhedonia and Its Treatment in Zebrafish.

Mood disorders, especially depression, are a major cause of human disability. The loss of pleasure (anhedonia) is a common, severely debilitating symptom of clinical depression. Experimental animal models are widely used to better understand depression pathogenesis and to develop novel antidepressant therapies. In rodents, various experimental models of anhedonia have already been developed and extensively validated. Complementing rodent studies, the zebrafish (Danio rerio) is emerging as a powerful model organism to assess pathobiological mechanisms of affective disorders, including depression. Here, we critically discuss the potential of zebrafish for modeling anhedonia and studying its molecular mechanisms and translational implications.

Induction of aggression and anxiety-like responses by perfluorooctanoic acid is accompanied by modulation of cholinergic- and purinergic signaling-related parameters in adult zebrafish.

Perfluorooctanoic acid (PFOA) is a contaminant of global concern owing to its prevalent occurrence in aquatic and terrestrial environments with potential hazardous impact on living organisms. Here, we investigated the influence of realistic environmental concentrations of PFOA (0, 0.25, 0.5, or 1.0 mg/L) on relevant behaviors of adult zebrafish (Danio rerio) (e.g., exploration to novelty, social preference, and aggression) and the possible role of PFOA in modulating cholinergic and purinergic signaling in the brain after exposure for 7 consecutive days. PFOA significantly increased geotaxis as well as reduced vertical exploration (a behavioral endpoint for anxiety), and increased the frequency and duration of aggressive episodes without affecting their social preference. Exposure to PFOA did not affect ADP hydrolysis, whereas ATP and AMP hydrolysis were significantly increased at the highest concentration tested. However, AChE activity was markedly decreased in all PFOA-exposed groups when compared with control. In conclusion, PFOA induces aggression and anxiety-like behavior in adult zebrafish and modulates both cholinergic and purinergic signaling biomarkers. These novel data can provide valuable insights into possible health threats related to human activities, demonstrating the utility of adult zebrafish to elucidate how PFOA affects neurobehavioral responses in aquatic organisms.

Evaluating the Implementation of a Digital Diabetes Prevention Program in an Integrated Health Care Delivery System Among Older Adults: Results of a Natural Experiment.

The purpose of this natural experiment study was to assess the effectiveness of a 12-month digital Diabetes Prevention Program (DPP) for adults aged 65-75 years with prediabetes and obesity within a large, integrated health care system. Adjusting for propensity scores and covariates, patients who enrolled and participated in the digital DPP had a mean weight loss of 8.6 lb over 12 months and 5.7 lb by 24 months, compared with a steady, minimal weight loss of 1.3 lb over 12 months and 2.8 lb by 24 months among patients not enrolled. There was a significant difference in mean change in A1C between enrolled and nonenrolled patients over 12 months (-0.10%), but not by 24 months (-0.06%). Digital DPP appears to be an effective weight loss option and potential diabetes prevention intervention for older adults at high risk for type 2 diabetes.

Lessons from bright-spots for advancing knowledge exchange at the interface of marine science and policy.

Evidence-informed decision-making is in increasing demand given growing pressures on marine environments. A way to facilitate this is by knowledge exchange among marine scientists and decision-makers. While many barriers are reported in the literature, there are also examples whereby research has successfully informed marine decision-making (i.e., 'bright-spots'). Here, we identify and analyze 25 bright-spots from a wide range of marine fields, contexts, and locations to provide insights into how to improve knowledge exchange at the interface of marine science and policy. Through qualitative surveys we investigate what initiated the bright-spots, their goals, and approaches to knowledge exchange. We also seek to identify what outcomes/impacts have been achieved, the enablers of success, and what lessons can be learnt to guide future knowledge exchange efforts. Results show that a diversity of approaches were used for knowledge exchange, from consultative engagement to genuine knowledge co-production. We show that diverse successes at the interface of marine science and policy are achievable and include impacts on policy, people, and governance. Such successes were enabled by factors related to the actors, processes, support, context, and timing. For example, the importance of involving diverse actors and managing positive relationships is a key lesson for success. However, enabling routine success will require: 1) transforming the ways in which we train scientists to include a greater focus on interpersonal skills, 2) institutionalizing and supporting knowledge exchange activities in organizational agendas, 3) conceptualizing and implementing broader research impact metrics, and 4) transforming funding mechanisms to focus on need-based interventions, impact planning, and an acknowledgement of the required time and effort that underpin knowledge exchange activities.

Randomized Controlled Trial of Advance Notification Phone Calls vs Text Messages Prior to Mailed Fecal Test Outreach.

BACKGROUND & AIMS: Mailing fecal immunochemical test (FITs) to individuals who are due for screening (mailed FIT outreach) increases colorectal cancer (CRC) screening. Little is known about how phone-based advance notifications (primers) affect the effectiveness of mailed FIT outreach programs. METHODS: We performed a prospective study of patients at a large urban health center, 50-75 years old and due for screening, with no record of a prior FIT. Participants were randomly assigned to groups that received a live phone call primer (n = 1203) or a text message primer (n = 1622), from June through December 2018. The participants were then mailed a FIT kit, followed by 2 automated calls, and live reminder calls delivered by the care team. The main outcome was completion of FIT within 3 months of assignment to the live phone call or text message group. RESULTS: Participants had a FIT completion rate of 16.8%, a mean age of 58 years, and 80% were Latino. In adjusted intention to treat analyses (n = 2825), FIT completion rates were higher in the patients assigned to receive a live phone call vs text message primer (percentage point difference, 3.3%; 95% CI, 0.4%-6.2%). Between-group differences increased to 7.3% points (95% CI, 3.6%-11.0%) in the per-protocol analysis of 2144 participants reached by the text message (1320/1622, 81%), live call (438/1203, 36%), or voice message (386/1203, 32%). This rate increased to 14.9% points (95% CI; 9.6%-20.1%) in the per-protocol analysis of 1758 participants reached by the text message or reached by the live call. CONCLUSIONS: In a randomized trial, advance notification live phone calls outperformed text messages in prompting health center patients who had not previously completed a FIT to complete a mailed FIT. Clinicaltrials.gov no: NCT03167125.

Approaches to Introduce Helical Structure in Cysteine-Containing Peptides with a Bimane Group.

An i-i+4 or i-i+3 bimane-containing linker was introduced into a peptide known to target Estrogen Receptor alpha (ERα), in order to stabilise an α-helical geometry. These macrocycles were studied by CD and NMR to reveal the i-i+4 constrained peptide adopts a 310 -helical structure in solution, and an α-helical conformation on interaction with the ERα coactivator recruitment surface in silico. An acyclic bimane-modified peptide is also helical, when it includes a tryptophan or tyrosine residue; but is significantly less helical with a phenylalanine or alanine residue, which indicates such a bimane modification influences peptide structure in a sequence dependent manner. The fluorescence intensity of the bimane appears influenced by peptide conformation, where helical peptides displayed a fluorescence increase when TFE was added to phosphate buffer, compared to a decrease for less helical peptides. This study presents the bimane as a useful modification to influence peptide structure as an acyclic peptide modification, or as a side-chain constraint to give a macrocycle.

Implementing the National Heart, Lung, and Blood Institute's Strategic Vision in the Division of Cardiovascular Sciences.

As we commemorate the 70th Anniversary of the National Heart, Lung, and Blood Institute (NHLBI) and celebrate important milestones that have been achieved by the Division of Cardiovascular Sciences (DCVS), it is imperative that DCVS and the Extramural Research community at-large continue to address critical public health challenges that persist within the area of Cardiovascular Diseases (CVD). The NHLBI's Strategic Vision, developed with extensive input from the extramural research community and published in 2016, included overarching goals and strategic objectives that serve to provide a general blueprint for sustaining the legacy of the Institute by leveraging opportunities in emerging scientific areas (e.g., regenerative medicine, omics technology, data science, precision medicine, and mobile health), finding new ways to address enduring challenges (e.g., social determinants of health, health inequities, prevention, and health promotion), and training the next generation of heart, lung, blood, and sleep researchers. DCVS has developed a strategic vision implementation plan to provide a cardiovascular framing for the pursuit of the Institute's overarching goals and strategic objectives garnered from the input of the broader NHLBI community. This plan highlights six scientific focus areas that demonstrate a cross-cutting and multifaceted approach to addressing cardiovascular sciences, including 1) addressing social determinants of cardiovascular health (CVH) and health inequities, 2) enhancing resilience, 3) promoting CVH and preventing CVD Across the lifespan, 4) eliminating hypertension-related CVD, 5) reducing the burden of heart failure, and 6) preventing vascular dementia. These priorities will guide our efforts in Institute-driven activities in the coming years but will not exclude development of other novel ideas or the support of investigator-initiated grant awards. The DCVS Strategic Vision implementation plan is a living document that will evolve with iterative dialogue with the NHLBI community and adapt as the dynamic scientific landscape changes to seize emerging opportunities.

Stress increases susceptibility to pentylenetetrazole-induced seizures in adult zebrafish.

Stress is the body's reaction to any change that requires adaptive responses. In various organisms, stress is a seizure-related comorbidity. Despite the exposure to stressors eliciting aversive behaviors in zebrafish, there are no data showing whether stress potentiates epileptic seizures in this species. Here, we investigated whether a previous exposure to an intense acute stressor positively modulates the susceptibility to seizures in pentylenetetrazole (PTZ)-challenged zebrafish. The conspecific alarm substance (CAS) was used to elicit aversive responses (3.5 mL/L for 5 min), observed by increased bottom dwelling and erratic movements. Then, fish were immediately exposed to 7.5 mM PTZ for 10 min to induce seizure-like behaviors. Stress increased the seizure intensity, the number of clonic-like seizure behaviors (score 4), as well as facilitated the occurrence of score 4 episodes by decreasing the latency in which fish reached the score 4. Moreover, fish with heightened anxiety showed increased susceptibility to PTZ, since positive correlations between anxiety- and seizure-like behaviors were found. Overall, since CAS also increased whole-body cortisol levels in zebrafish, our novel findings show a prominent response to PTZ-induced seizures in previously stressed zebrafish. Moreover, we reinforce the growing utility of zebrafish models to assess seizure-related comorbidities aiming to elucidate how stress can affect epileptic seizures in vertebrates.

Computational Analysis of the Crystal and Cryo-EM Structures of P-Loop Channels with Drugs.

The superfamily of P-loop channels includes various potassium channels, voltage-gated sodium and calcium channels, transient receptor potential channels, and ionotropic glutamate receptors. Despite huge structural and functional diversity of the channels, their pore-forming domain has a conserved folding. In the past two decades, scores of atomic-scale structures of P-loop channels with medically important drugs in the inner pore have been published. High structural diversity of these complexes complicates the comparative analysis of these structures. Here we 3D-aligned structures of drug-bound P-loop channels, compared their geometric characteristics, and analyzed the energetics of ligand-channel interactions. In the superimposed structures drugs occupy most of the sterically available space in the inner pore and subunit/repeat interfaces. Cationic groups of some drugs occupy vacant binding sites of permeant ions in the inner pore and selectivity-filter region. Various electroneutral drugs, lipids, and detergent molecules are seen in the interfaces between subunits/repeats. In many structures the drugs strongly interact with lipid and detergent molecules, but physiological relevance of such interactions is unclear. Some eukaryotic sodium and calcium channels have state-dependent or drug-induced π-bulges in the inner helices, which would be difficult to predict. The drug-induced π-bulges may represent a novel mechanism of gating modulation.

A Bimane-Based Peptide Staple for Combined Helical Induction and Fluorescent Imaging.

The thiol-selective fluorescent imaging agent, dibromobimane, has been repurposed to crosslink cysteine- and homocysteine-containing peptides, with the resulting bimane linker acting as both a structural constraint and a fluorescent tag. Macrocyclisation was conducted on nine short peptides containing two cysteines and/or homocysteines, both on-resin and in buffered aqueous solution, to give macrocycles ranging in size from 16 (i,i+2) to 31 (i,i+7) atoms. The structures were defined by CD, NMR structure calculations by using Xplor-NIH, NMR secondary shift and JHαNH analyses to reveal helical structure in the i,i+4 (1, 2), and i,i+3 (5) constrained peptides. Cellular-uptake studies were conducted with three of the macrocycles. Subsequent confocal imaging revealed punctate fluorescence within the cytosol indicative of peptides trapped in endocytic vesicles. These studies demonstrate that dibromobimane is an effective tool for defining secondary structure within short peptides, whilst simultaneously introducing a fluorescent tag suitable for common cell-based experiments.

Optical control of L-type Ca2+ channels using a diltiazem photoswitch.

L-type Ca2+ channels (LTCCs) play a crucial role in excitation-contraction coupling and release of hormones from secretory cells. They are targets of antihypertensive and antiarrhythmic drugs such as diltiazem. Here, we present a photoswitchable diltiazem, FHU-779, which can be used to reversibly block endogenous LTCCs by light. FHU-779 is as potent as diltiazem and can be used to place pancreatic ß-cell function and cardiac activity under optical control.

Phenylalkylamines in calcium channels: computational analysis of experimental structures.

Experimental 3D structures of calcium channels with phenylalkylamines (PAAs) provide basis for further analysis of atomic mechanisms of these important cardiovascular drugs. In the crystal structure of the engineered calcium channel CavAb with Br-verapamil and in the cryo-EM structure of the Cav1.1 channel with verapamil, the ligands bind in the inner pore. However, there are significant differences between these structures. In the crystal structure the ligand ammonium group is much closer to the ion in the selectivity-filter region Site 3, which is most proximal to the inner pore, than in the cryo-EM structure. Here we used Monte Carlo energy minimizations to dock PAAs in calcium channels. Our computations suggest that in the crystal structure Site 3 is occupied by a water molecule rather than by a calcium ion. Analysis of the published electron density map does not rule out this possibility. In the cryo-EM structures the ammonium group of verapamil is shifted from the calcium ion in Site 3 either along the pore axis, towards the cytoplasm or away from the axis. Our unbiased docking reproduced these binding modes. However, in the cryo-EM structures detergent and lipid molecules interact with verapamil. When we removed these molecules, the nitrile group of verapamil bound to the calcium ion in Site 3. Models of Cav1.2 with different PAAs suggest similar binding modes and direct contacts of the ligands electronegative atoms with the calcium ion in Site 3. Such interactions explain paradoxes in structure-activity relationships of PAAs.

Taurine modulates the stress response in zebrafish.

The zebrafish (Danio rerio) is used as an emergent model organism to investigate the behavioral and physiological responses to stress. The anxiolytic-like effects of taurine in zebrafish support the existence of different mechanisms of action, which can play a role in preventing stress-related disorders (i.e., modulation of GABAA, strychnine-sensitive glycine, and NMDA receptors, as well as antioxidant properties). Herein, we investigate whether taurine modulates some behavioral and biochemical responses in zebrafish acutely submitted to chemical and mechanical stressors. We pretreated zebrafish for 1 h in beakers at 42, 150, and 400 mg/L taurine. Fish were later acutely exposed to a chemical stressor (conspecific alarm substance) or to a mechanical stressor (net chasing), which elicits escaping responses and aversive behaviors. Locomotion, exploration, and defensive-like behaviors were measured using the novel tank and the light-dark tests. Biochemical (brain oxidative stress-related parameters) and whole-body cortisol levels were also quantified. We showed that taurine prevents anxiety/fear-like behaviors and protein carbonylation and dampens the cortisol response following acute stress in zebrafish. In summary, our results demonstrate a protective role of taurine against stress-induced behavioral and biochemical changes, thereby reinforcing the growing utility of zebrafish models to investigate the neuroprotective actions of taurine in vertebrates.

Extremely Potent Block of Bacterial Voltage-Gated Sodium Channels by µ-Conotoxin PIIIA.

µ-Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide µ-conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10-12 to 10-5 M), we estimated an IC50 = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.

Hydrophobic Amines and Their Guanidine Analogues Modulate Activation and Desensitization of ASIC3.

Acid-sensing ion channel 3 (ASIC3) is an important member of the acid-sensing ion channels family, which is widely expressed in the peripheral nervous system and contributes to pain sensation. ASICs are targeted by various drugs and toxins. However, mechanisms and structural determinants of ligands' action on ASIC3 are not completely understood. In the present work we studied ASIC3 modulation by a series of "hydrophobic monoamines" and their guanidine analogs, which were previously characterized to affect other ASIC channels via multiple mechanisms. Electrophysiological analysis of action via whole-cell patch clamp method was performed using rat ASIC3 expressed in Chinese hamster ovary (CHO) cells. We found that the compounds studied inhibited ASIC3 activation by inducing acidic shift of proton sensitivity and slowed channel desensitization, which was accompanied by a decrease of the equilibrium desensitization level. The total effect of the drugs on the sustained ASIC3-mediated currents was the sum of these opposite effects. It is demonstrated that drugs' action on activation and desensitization differed in their structural requirements, kinetics of action, and concentration and state dependencies. Taken together, these findings suggest that effects on activation and desensitization are independent and are likely mediated by drugs binding to distinct sites in ASIC3.