The Dysregulated MAD in Mad: A Neuro-theranostic Approach Through the Induction of Autophagic Biomarkers LC3B-II and ATG.

The word mad has historically been associated with the psyche, emotions, and abnormal behavior. Dementia is a common symptom among psychiatric disorders or mad (schizophrenia, depression, bipolar disorder) patients. Autophagy/mitophagy is a protective mechanism used by cells to get rid of dysfunctional cellular organelles or mitochondria. Autophagosome/mitophagosome abundance in autophagy depends on microtubule-associated protein light chain 3B (LC3B-II) and autophagy-triggering gene (ATG) which functions as an autophagic biomarker for phagophore production and quick mRNA disintegration. Defects in either LC3B-II or the ATG lead to dysregulated mitophagy-and-autophagy-linked dementia (MAD). The impaired MAD is closely associated with schizophrenia, depression, and bipolar disorder. The pathomechanism of psychosis is not entirely known, which is the severe limitation of today's antipsychotic drugs. However, the reviewed circuit identifies new insights that may be especially helpful in targeting biomarkers of dementia. Neuro-theranostics can also be achieved by manufacturing either bioengineered bacterial and mammalian cells or nanocarriers (liposomes, polymers, and nanogels) loaded with both imaging and therapeutic materials. The nanocarriers must cross the BBB and should release both diagnostic agents and therapeutic agents in a controlled manner to prove their effectiveness against psychiatric disorders. In this review, we highlighted the potential of microRNAs (miRs) as neuro-theranostics in the treatment of dementia by targeting autophagic biomarkers LC3B-II and ATG. Focus was also placed on the potential for neuro-theranostic nanocells/nanocarriers to traverse the BBB and induce action against psychiatric disorders. The neuro-theranostic approach can provide targeted treatment for mental disorders by creating theranostic nanocarriers.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) causes seizure activity in larval zebrafish via antagonism of γ-aminobutyric acid type A receptor α1ß2γ2.

Hexahydro-1,3,5-trinitro-1,3,5-triazine, or Royal Demolition Explosive (RDX), is a major component of plastic explosives such as C-4. Acute exposures from intentional or accidental ingestion are a documented clinical concern, especially among young male U.S. service members in the armed forces. When ingested in large enough quantity, RDX causes tonic-clonic seizures. Previous in silico and in vitro experiments predict that RDX causes seizures by inhibiting α1ß2γ2 γ-aminobutyric acid type A (GABAA) receptor-mediated chloride currents. To determine whether this mechanism translates in vivo, we established a larval zebrafish model of RDX-induced seizures. After a 3 h of exposure to 300 µM RDX, larval zebrafish exhibited a significant increase in motility in comparison to vehicle controls. Researchers blinded to experimental group manually scored a 20-min segment of video starting at 3.5 h post-exposure and found significant seizure behavior that correlated with automated seizure scores. Midazolam (MDZ), an nonselective GABAAR positive allosteric modulator (PAM), and a combination of Zolpidem (α1 selective PAM) and compound 2-261 (ß2/3-selective PAM) were effective in mitigating RDX-triggered behavioral and electrographic seizures. These findings confirm that RDX induces seizure activity via inhibition of the α1ß2γ2 GABAAR and support the use of GABAAR-targeted anti-seizure drugs for the treatment of RDX-induced seizures.

Riluzole and novel naphthalenyl substituted aminothiazole derivatives prevent acute neural excitotoxic injury in a rat model of temporal lobe epilepsy.

Epileptogenic seizures, or status epilepticus (SE), leads to excitotoxic injury in hippocampal and limbic neurons in the kainic acid (KA) animal model of temporal lobe epilepsy (TLE). Here, we have further characterized neural activity regulated methylaminoisobutryic acid (MeAIB)/glutamine transport activity in mature rat hippocampal neurons in vitro that is inhibited by riluzole (IC50 = 1 µM), an anti-convulsant benzothiazole agent. We screened a library of riluzole derivatives and identified SKA-41 followed by a second screen and synthesized several novel chlorinated aminothiazoles (SKA-377, SKA-378, SKA-379) that are also potent MeAIB transport inhibitors in vitro, and brain penetrant following systemic administration. When administered before KA, SKA-378 did not prevent seizures but still protected the hippocampus and several other limbic areas against SE-induced neurodegeneration at 3d. When SKA-377 - 379, (30 mg/kg) were administered after KA-induced SE, acute neural injury in the CA3, CA1 and CA4/hilus was also largely attenuated. Riluzole (10 mg/kg) blocks acute neural injury. Kinetic analysis of SKA-378 and riluzoles' blockade of Ca2+-regulated MeAIB transport in neurons in vitro indicates that inhibition occurs via a non-competitive, indirect mechanism. Sodium channel NaV1.6 antagonism blocks neural activity regulated MeAIB/Gln transport in vitro (IC50 = 60 nM) and SKA-378 is the most potent inhibitor of NaV1.6 (IC50 = 28 µM) compared to NaV1.2 (IC50 = 118 µM) in heterologous cells. However, pharmacokinetic analysis suggests that sodium channel blockade may not be the predominant mechanism of neuroprotection here. Riluzole and our novel aminothiazoles are agents that attenuate acute neural hippocampal injury following KA-induced SE and may help to understand mechanisms involved in the progression of epileptic disease.

Computational design of peptides to target NaV1.7 channel with high potency and selectivity for the treatment of pain.

The voltage-gated sodium NaV1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human NaV1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human NaV1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC50s of 7 nM and 4 nM for hNaV1.7 and more than 1000-fold selectivity over human NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.8, and NaV1.9 channels. PTx2-3127 inhibits NaV1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally designed peptide inhibitors of human NaV1.7 channels have transformative potential to define a new class of biologics to treat pain.

A metasurface-based electronically steerable compact antenna system with reconfigurable artificial magnetic conductor reflector elements.

Beyond 5G networks would require newer technologies to deliver a smarter network. In accordance with these requirements, an electronically steerable compact antenna system capable of beam-switching in the azimuth plane is proposed. The design uses a monopole antenna as the main radiator surrounded by metasurface-based electronically reconfigurable reflector elements designed for the sub-6GHz range. The reflector elements use a reconfigurable capacitively loaded loop (CLL) which can be electronically activated to work as an artificial magnetic conductor (AMC). The design offers a digitally controllable directional radiation pattern covering all 360° in the azimuth plane with a step-size of 30°, a directional gain of ≥ 4.98 dBi and a high front-to-back lobe ratio (FBR) of ≥ 14.9 dB. The compact and modular nature of the design combined with the use of commercial off-the-shelf (COTS) components and 3D-printing makes the design low-cost and easier to integrate with various internet of thing (IoT) applications.

Identification of a TNF-TNFR-like system in malaria vectors (Anopheles stephensi) likely to influence Plasmodium resistance.

Identification of Plasmodium-resistance genes in malaria vectors remains an elusive goal despite the recent availability of high-quality genomes of several mosquito vectors. Anopheles stephensi, with its three distinctly-identifiable forms at the egg stage, correlating with varying vector competence, offers an ideal species to discover functional mosquito genes implicated in Plasmodium resistance. Recently, the genomes of several strains of An. stephensi of the type-form, known to display high vectorial capacity, were reported. Here, we report a chromosomal-level assembly of an intermediate-form of An. stephensi strain (IndInt), shown to have reduced vectorial capacity relative to a strain of type-form (IndCh). The contig level assembly with a L50 of 4 was scaffolded into chromosomes by using the genome of IndCh as the reference. The final assembly shows a heterozygous paracentric inversion, 3Li, involving 8 Mbp, which is syntenic to the extensively-studied 2La inversion implicated in Plasmodium resistance in An. gambiae involving 21 Mbp. Deep annotation of genes within the 3Li region in the IndInt assembly using the state-of-the-art protein-fold prediction and other annotation tools reveals the presence of a tumor necrosis factor-alpha (TNF-alpha) like gene, which is the homolog of the Eiger gene in Drosophila. Subsequent chromosome-wide searches revealed homologs of Wengen (Wgn) and Grindelwald (Grnd) genes, which are known to be the receptors for Eiger in Drosophila. We have identified all the genes in IndInt required for Eiger-mediated signaling by analogy to the TNF-alpha system, suggesting the presence of a functionally-active Eiger signaling pathway in IndInt. Comparative genomics of the three type-forms with that of IndInt, reveals structurally disruptive mutations in Eiger gene in all three strains of the type-form, suggesting compromised innate immunity in the type-form as the likely cause of high vectorial capacity in these strains. This is the first report of the presence of a homolog of Eiger in malaria vectors, known to be involved in cell death in Drosophila, within an inversion region in IndInt syntenic to an inversion associated with Plasmodium resistance in An. gambiae.

A Randomized Controlled Trial of Intravenous Scopolamine Versus Active-Placebo Glycopyrrolate in Patients With Major Depressive Disorder.

Objective: To investigate scopolamine's rapid-acting antidepressant effects using an active placebo comparator. Most prior intravenous scopolamine studies reduced depressive symptomatologies compared to saline placebo infusions within 3 days. However, the confounding effect of placebo is unknown given that only saline placebo has been used in prior studies.Methods: In this trial, 40 patients with major depressive disorder were randomized to receive single intravenous doses of either scopolamine hydrobromide (4-6 µg/kg) or glycopyrronium bromide (4 µg/kg) between August 2019 and April 2021 in Auckland, New Zealand. Glycopyrronium was chosen as the active placebo due to its similar antimuscarinic properties to scopolamine but inability to cross the blood-brain barrier. The primary mood outcome measure was the Montgomery-Åsberg Depression Rating Scale (MADRS) administered pre-infusion and 1, 3, 7, 14, 28, and 42 days post-infusion.Results: Per protocol, this trial was abandoned for futility at n = 40. While scopolamine reduced MADRS scores by 12.6 (± 8.7 SD) points at day 3, glycopyrronium showed similar reductions (11.2 ± 9.6 SD). Frequentist linear mixed models showed no antidepressant effects of scopolamine versus placebo (d = 0.17), and Bayesian mixed effect models showed moderate evidence in favor of the null hypothesis at day 3 (Bayes factor = 0.32). Participants remained well-blinded to drug allocation, with 50% of participants correctly guessing their allocation.Conclusions: The observed MADRS improvement was larger than in prior studies, but no antidepressant effects were observed. This study using an active placebo confirms recent studies demonstrating the lack of antidepressant efficacy of scopolamine.Trial Registration: Australian New Zealand Clinical Trials Registry identifier: ACTRN12619000569101.

GM1 ganglioside modifies microglial and neuroinflammatory responses to α-synuclein in the rat AAV-A53T α-synuclein model of Parkinson's disease.

Among the pathological events associated with the dopaminergic neurodegeneration characteristic of Parkinson's disease (PD) are the accumulation of toxic forms of α-synuclein and microglial activation associated with neuroinflammation. Although numerous other processes may participate in the pathogenesis of PD, the two factors mentioned above may play critical roles in the initiation and progression of dopamine neuron degeneration in PD. In this study, we employed a slowly progressing model of PD using adeno-associated virus-mediated expression of human A53T α-synuclein into the substantia nigra on one side of the brain and examined the microglial response in the striatum on the injected side compared to the non-injected (control) side. We further examined the extent to which administration of the neuroprotective ganglioside GM1 influenced α-synuclein-induced glial responses. Changes in a number of microglial morphological measures (i.e., process length, number of endpoints, fractal dimension, lacunarity, density, and cell perimeter) were indicative of the presence of activated microglial and an inflammatory response on the injected side of the brain, compared to the control side. In GM1-treated animals, no significant differences in microglial morphology were observed between the injected and control striata. Follow-up studies showed that mRNA expression for several inflammation-related genes was increased on the A53T α-synuclein injected side vs. the non-injected side in saline-treated animals and that such changes were not observed in GM1-treated animals. These data show that inhibition of microglial activation and potentially damaging neuroinflammation by GM1 ganglioside administration may be among the many factors that contribute to the neuroprotective effects of GM1 in this model and possibly in human PD.

An Integrative Multi-Omics Analysis Reveals MicroRNA-143 as a Potential Therapeutic to Attenuate Retinal Angiogenesis.

Retinal neovascularization is a severe complication of proliferative diabetic retinopathy (PDR). MicroRNAs (miRNAs) are master regulators of gene expression that play an important role in retinal neovascularization. In this study, we show that miR-143-3p is significantly downregulated in the retina of a rat model of oxygen-induced retinopathy (OIR) by miRNA-sequencing. Intravitreal injection of synthetic miR-143 mimics significantly ameliorate retinal neovascularization in OIR rats. miR-143 is identified to be highly expressed in the neural retina particularly in the ganglion cell layer and retinal vasculature. In miR-143 treated cells, the functional evaluation showed a decrease in cell migration and delayed endothelial vessel-like tube remodeling. The multiomics analysis suggests that miR-143 negatively impacts endothelial cell activity through regulating cell-matrix adhesion and mediating hypoxia-inducible factor-1 signaling. We predict hub genes regulated by miR-143 that may be involved in mediating endothelial cell function by cytoHubba. We also demonstrate that the retinal neovascular membranes in patients with PDR principally consist of endothelial cells by CIBERSORTx. We then identify 2 hub genes, thrombospondin 1 and plasminogen activator inhibitor, direct targets of miR-143, that significantly altered in the PDR patients. These findings suggest that miR-143 appears to be essential for limiting endothelial cell-matrix adhesion, thus suppressing retinal neovascularization.

The seizure-inducing plastic explosive RDX inhibits the α1ß2γ2 GABAA receptor.

OBJECTIVE: Royal demolition explosive (RDX) can induce seizures in wildlife and humans following release into the environment or after voluntary consumption. During the Vietnam War, RDX intoxication was the most common cause of generalized seizures in US service personnel, and in some sections of the armed forces, eating of RDX has continued as "a dare" to this day. After its mechanism of action was long unknown, RDX was recently shown to be a GABAA receptor antagonist. We here determined the GABAA receptor subtype-selectivity of RDX and mapped its functional binding site. METHODS: We used whole-cell patch-clamp to determine the potency of RDX on 10 recombinantly expressed GABAA receptors and mapped the RDX binding site using a combination of Rosetta molecular modeling and site-directed mutagenesis. RESULTS: RDX was found to reversibly inhibit the α1ß2γ2 GABAA receptor with an IC50 of 23 µmol/L (95% CI 15.1-33.3 µmol/L), whereas α4 and α6 containing GABAA receptor combinations were 4-10-fold less sensitive. RDX is binding to the noncompetitive antagonist (NCA) site in the pore. In a molecular model based on the cryo-EM structure of the resting state of the α1ß2γ2 receptor, RDX forms two hydrogen bonds with the threonines at the T6' ring and makes hydrophobic interactions with the valine and alanine in 2' position of the α1 or ß2 subunits. INTERPRETATION: Our findings characterize the mechanism of action of RDX at the atomistic level and suggest that RDX-induced seizures should be susceptible to treatment with GABAA modulating drugs such as benzodiazepines, barbiturates, propofol, or neurosteroids.

Mapping molecular subtype specific alterations in breast cancer brain metastases identifies clinically relevant vulnerabilities.

The molecular events and transcriptional plasticity driving brain metastasis in clinically relevant breast tumor subtypes has not been determined. Here we comprehensively dissect genomic, transcriptomic and clinical data in patient-matched longitudinal tumor samples, and unravel distinct transcriptional programs enriched in brain metastasis. We report on subtype specific hub genes and functional processes, central to disease-affected networks in brain metastasis. Importantly, in luminal brain metastases we identify homologous recombination deficiency operative in transcriptomic and genomic data with recurrent breast mutational signatures A, F and K, associated with mismatch repair defects, TP53 mutations and homologous recombination deficiency (HRD) respectively. Utilizing PARP inhibition in patient-derived brain metastatic tumor explants we functionally validate HRD as a key vulnerability. Here, we demonstrate a functionally relevant HRD evident at genomic and transcriptomic levels pointing to genomic instability in breast cancer brain metastasis which is of potential translational significance.

Integrating Genetic Structural Variations and Whole-Genome Sequencing Into Clinical Neurology.

Advances in genome sequencing technologies have unlocked new possibilities in identifying disease-associated and causative genetic markers, which may in turn enhance disease diagnosis and improve prognostication and management strategies. With the capability of examining genetic variations ranging from single-nucleotide mutations to large structural variants, whole-genome sequencing (WGS) is an increasingly adopted approach to dissect the complex genetic architecture of neurologic diseases. There is emerging evidence for different structural variants and their roles in major neurologic and neurodevelopmental diseases. This review first describes different structural variants and their implicated roles in major neurologic and neurodevelopmental diseases, and then discusses the clinical relevance of WGS applications in neurology. Notably, WGS-based detection of structural variants has shown promising potential in enhancing diagnostic power of genetic tests in clinical settings. Ongoing WGS-based research in structural variations and quantifying mutational constraints can also yield clinical benefits by improving variant interpretation and disease diagnosis, while supporting biomarker discovery and therapeutic development. As a result, wider integration of WGS technologies into health care will likely increase diagnostic yields in difficult-to-diagnose conditions and define potential therapeutic targets or intervention points for genome-editing strategies.

Cryptography in Hierarchical Coded Caching: System Model and Cost Analysis.

The idea behind network caching is to reduce network traffic during peak hours via transmitting frequently-requested content items to end users during off-peak hours. However, due to limited cache sizes and unpredictable access patterns, this might not totally eliminate the need for data transmission during peak hours. Coded caching was introduced to further reduce the peak hour traffic. The idea of coded caching is based on sending coded content which can be decoded in different ways by different users. This allows the server to service multiple requests by transmitting a single content item. Research works regarding coded caching traditionally adopt a simple network topology consisting of a single server, a single hub, a shared link connecting the server to the hub, and private links which connect the users to the hub. Building on the results of Sengupta et al. (IEEE Trans. Inf. Forensics Secur., 2015), we propose and evaluate a yet more complex system model that takes into consideration both throughput and security via combining the mentioned ideas. It is demonstrated that the achievable rates in the proposed model are within a constant multiplicative and additive gap with the minimum secure rates.

Artificial Intelligence-Based Portable Bioelectronics Platform for SARS-CoV-2 Diagnosis with Multi-nucleotide Probe Assay for Clinical Decisions.

In the context of the recent pandemic, the necessity of inexpensive and easily accessible rapid-test kits is well understood and need not be stressed further. In light of this, we report a multi-nucleotide probe-based diagnosis of SARS-CoV-2 using a bioelectronics platform, comprising low-cost chemiresistive biochips, a portable electronic readout, and an Android application for data acquisition with machine-learning-based decision making. The platform performs the desired diagnosis from standard nasopharyngeal and/or oral swabs (both on extracted and non-extracted RNA samples) without amplifying the viral load. Being a reverse transcription polymerase chain reaction-free hybridization assay, the proposed approach offers inexpensive, fast (time-to-result: ≤ 30 min), and early diagnosis, as opposed to most of the existing SARS-CoV-2 diagnosis protocols recommended by the WHO. For the extracted RNA samples, the assay accounts for 87 and 95.2% test accuracies, using a heuristic approach and a machine-learning-based classification method, respectively. In case of the non-extracted RNA samples, 95.6% decision accuracy is achieved using the heuristic approach, with the machine-learning-based best-fit model producing 100% accuracy. Furthermore, the availability of the handheld readout and the Android application-based simple user interface facilitates easy accessibility and portable applications. Besides, by eliminating viral RNA extraction from samples as a pre-requisite for specific detection, the proposed approach presents itself as an ideal candidate for point-of-care SARS-CoV-2 diagnosis.

Molecular determinants of pro-arrhythmia proclivity of d- and l-sotalol via a multi-scale modeling pipeline.

Drug isomers may differ in their proarrhythmia risk. An interesting example is the drug sotalol, an antiarrhythmic drug comprising d- and l- enantiomers that both block the hERG cardiac potassium channel and confer differing degrees of proarrhythmic risk. We developed a multi-scale in silico pipeline focusing on hERG channel - drug interactions and used it to probe and predict the mechanisms of pro-arrhythmia risks of the two enantiomers of sotalol. Molecular dynamics (MD) simulations predicted comparable hERG channel binding affinities for d- and l-sotalol, which were validated with electrophysiology experiments. MD derived thermodynamic and kinetic parameters were used to build multi-scale functional computational models of cardiac electrophysiology at the cell and tissue scales. Functional models were used to predict inactivated state binding affinities to recapitulate electrocardiogram (ECG) QT interval prolongation observed in clinical data. Our study demonstrates how modeling and simulation can be applied to predict drug effects from the atom to the rhythm for dl-sotalol and also increased proarrhythmia proclivity of d- vs. l-sotalol when accounting for stereospecific beta-adrenergic receptor blocking.

Towards point-of-care diagnosis of Alzheimer's disease: Multi-analyte based portable chemiresistive platform for simultaneous detection of ß-amyloid (1-40) and (1-42) in plasma.

Label-free simultaneous detection of Alzheimer's disease (AD) specific biomarkers Aß40 and Aß42 peptides on a single platform using polypyrrole nanoparticle-based chemiresistive biosensors is reported here. The proposed interdigitated-microelectrode based inexpensive multisensor-platform can concurrently detect Aß40 and Aß42 in spiked-plasma in the range of 10-14 - 10-6 g/mL (with LoDs being 5.71 and 9.09 fg/mL, respectively), enabling the estimation of diagnostically significant Aß42/Aß40 ratio. A detailed study has been undertaken here to record the individual sensor responses against spiked-plasma samples with varying amounts and proportions of the two target peptides, towards enabling disease-progression monitoring using the Aß-ratio. As compared to the existing cost-ineffective brain-imaging techniques such as PET and MRI, and the high-risk CSF based invasive AD biomarkers detecting procedures, the proposed approach offers a viable alternative for affordable point-of-care AD diagnostics, with possible usage in performance evaluation of therapeutic drugs. Towards point-of-care applications, the portable readout used in this work was conjugated with an android-based mobile app for data-acquisition and analysis.

Novel wideband circularly polarized DRA with squint-free radiation characteristics.

A new single-fed circularly polarized dielectric resonator antenna (CP-DRA) without beam squint is presented. The DRA comprises an S-shaped dielectric resonator (SDR) with a metalized edge and two rectangular dielectric resonators (RDRs) blocks. Horizontal extension section is applied as an extension of the SDR, and a vertical-section is placed in parallel to the metallic edge. A vertical coaxial probe is used to excite the SDR and the vertical RDR blocks through an S-shaped metal element and a small rectangular metal strip. The two added RDRs that form an L-shaped DR improve the radiation characteristics and compensate for the beam squint errors. A wideband CP performance is achieved due to the excitation of several orthogonal modes such as [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text]. The experimental results demonstrate an impedance bandwidth of approximately [Formula: see text] (3.71-7.45 GHz) and a 3-dB axial-ratio (AR) bandwidth of about [Formula: see text] (3.72-6.53 GHz) with a stable broadside beam achieving a measured peak gain of about [Formula: see text]. Furthermore, a 100% correction in beam squint value from [Formula: see text] to [Formula: see text] with respect to the antenna boresight is achieved.

Investigation of NH3 as a selective thyroid hormone receptor modulator in larval zebrafish (Danio rerio).

Thyroid hormones (THs) are essential for normal vertebrate development and diverse environmental chemicals are hypothesized to cause developmental toxicity by disrupting TH-mediated signaling. The larval zebrafish (Danio rerio) is an emerging in vivo model of developmental TH disruption; however, the effects of TR antagonism have not yet been studied in zebrafish. NH3, generally considered a potent and specific thyroid hormone receptor (TR) antagonist, has been used in rodents and Xenopus laevis to characterize phenotypes of TR antagonism. The objective of this study is to determine the effects of NH3 on endpoints previously determined to be TH-sensitive in larval zebrafish, specifically teratology and mortality, photomotor behavior, and mRNA expression of TH signaling genes. Zebrafish embryos were exposed to NH3 via static waterborne exposure at concentrations ranging from 0.001 to 10 µM beginning at 6 h post-fertilization (hpf) through 5 days post fertilization (dpf). Significant mortality and teratogenesis was observed at 3, 4, and 5 dpf in zebrafish exposed to NH3 at 10 µM. At concentrations that did not cause significant mortality, NH3 did not exert a consistent antagonistic effect on photomotor behavior assays or mRNA expression when administered alone or in the presence of exogenous T4. Rather, depending on the NH3 concentration and larval age NH3 decreased or increased swimming triggered by transition from light to dark. Similarly, inconsistent antagonistic and agonistic effects on mRNA expression of TH signaling genes were noted following treatment with NH3 alone. NH3 did inhibit T4 (30 nM)-induced gene expression; however, this was only consistently observed at a concentration of NH3 (10 µM) that also caused significant mortality. Collectively, these results suggest that NH3 does not act solely as a TR antagonist in larval zebrafish, but instead exhibits complex modulatory effects on TR activity. These data support the hypothesis that NH3 is a selective thyroid hormone receptor modulator. Further studies of NH3 interactions with the zebrafish thyroid hormone receptor are required to characterize the activity of NH3 in target tissues of the larval zebrafish at the molecular level, highlighting the importance of characterizing NH3 effects in specific models of TH-disruption to better interpret its actions in mechanistic screens of environmental chemicals for TH action.

New capsaicin analogs as molecular rulers to define the permissive conformation of the mouse TRPV1 ligand-binding pocket.

The capsaicin receptor TRPV1 is an outstanding representative of ligand-gated ion channels in ligand selectivity and sensitivity. However, molecular interactions that stabilize the ligand-binding pocket in its permissive conformation, and how many permissive conformations the ligand-binding pocket may adopt, remain unclear. To answer these questions, we designed a pair of novel capsaicin analogs to increase or decrease the ligand size by about 1.5 Å without altering ligand chemistry. Together with capsaicin, these ligands form a set of molecular rulers for investigating ligand-induced conformational changes. Computational modeling and functional tests revealed that structurally these ligands alternate between drastically different binding poses but stabilize the ligand-binding pocket in nearly identical permissive conformations; functionally, they all yielded a stable open state despite varying potencies. Our study suggests the existence of an optimal ligand-binding pocket conformation for capsaicin-mediated TRPV1 activation gating, and reveals multiple ligand-channel interactions that stabilize this permissive conformation.