Global Air Pollutant Phenanthrene and Arrhythmic Outcomes in a Mouse Model.

BACKGROUND: The three-ringed polycyclic aromatic hydrocarbon (PAH) phenanthrene (Phe) has been implicated in the cardiotoxicity of petroleum-based pollution in aquatic systems, where it disrupts the contractile and electrical function of the fish heart. Phe is also found adsorbed to particulate matter and in the gas phase of air pollution, but to date, no studies have investigated the impact of Phe on mammalian cardiac function. OBJECTIVES: Our objectives were to determine the arrhythmogenic potential of acute Phe exposure on mammalian cardiac function and define the underlying mechanisms to provide insight into the toxicity risk to humans. METHODS: Ex vivo Langendorff-perfused mouse hearts were used to test the arrhythmogenic potential of Phe on myocardial function, and voltage- and current-clamp recordings were used to define underlying cellular mechanisms in isolated cardiomyocytes. RESULTS: Mouse hearts exposed to ∼8µM Phe for 15-min exhibited a significantly slower heart rate (p=0.0006, N=10 hearts), a prolonged PR interval (p=0.036, N=8 hearts), and a slower conduction velocity (p=0.0143, N=7 hearts). Whole-cell recordings from isolated cardiomyocytes revealed action potential (AP) duration prolongation (at 80% repolarization; p=0.0408, n=9 cells) and inhibition of key murine repolarizing currents-transient outward potassium current (Ito) and ultrarapid potassium current (IKur)-following Phe exposure. A significant reduction in AP upstroke velocity (p=0.0445, n=9 cells) and inhibition of the fast sodium current (INa; p=0.001, n=8 cells) and calcium current (ICa; p=0.0001) were also observed, explaining the slowed conduction velocity in intact hearts. Finally, acute exposure to ∼8µM Phe significantly increased susceptibility to arrhythmias (p=0.0455, N=9 hearts). DISCUSSION: To the best of our knowledge, this is the first evidence of direct inhibitory effects of Phe on mammalian cardiac electrical activity at both the whole-heart and cell levels. This electrical dysfunction manifested as an increase in arrhythmia susceptibility due to impairment of both conduction and repolarization. Similar effects in humans could have serious health consequences, warranting greater regulatory attention and toxicological investigation into this ubiquitous PAH pollutant generated from fossil-fuel combustion. https://doi.org/10.1289/EHP12775.

A Suspected Case of a Neonatal Monkeypox Infection With Ocular Involvement.

Mpox (initially reported as monkeypox virus Clade IIb) ravaged the non-endemic world in 2022 with dermatological and systemic manifestations. The rapid propagation of this virus shed light on the scarcity of information for a virus that was first reported in 1958. We present the first probable neonatal case of mpox with ocular involvement. Ophthalmologists may be the first to diagnose mpox or be a part of the multidisciplinary team required for adequate work-up and treatment to prevent life-long sequelae in the neonatal population.

Effect of Premature Tube Extrusion for Simple Congenital Nasolacrimal Duct Obstruction: A Comparison of Monocanalicular and Bicanalicular Intubation.

ABSTRACT: The objective of this article is to compare the incidence of premature dislocation of silicone tubes and the effect on treatment success between monocanalicular (MCI) and bicanalicular (BCI) intubation in pediatric patients with simple congenital nasolacrimal duct obstruction. Retrospective comparative case series of 108 eyes of 78 pediatric patients with simple congenital nasolacrimal duct obstruction who underwent probing with either BCI (n = 38 eyes) or MCI (n = 70 eyes) from 2017 to 2020. Premature tube extrusion was defined as any tube removed prior to the 3 month postoperative appointment. Success was defined as resolution of tearing 3 months post tube removal. Ages ranged from 10 months to 5.35 years (mean, 1.95 years; Standard deviation (SD), 0.91). Premature tube extrusion occurred in 15 eyes with BCI and 29 eyes with MCI. Success rates were not significantly different regardless of intubation type between the planned tube removal (90.6%) and the premature tube extrusion cohorts (84.1%), P = 0.89. There was no significant difference in treatment success between the planned tube removal (92.7% MCI, 87% BCI) and the premature tube extrusion cohorts (86.2% MCI, 80% BCI). Complications included 2 infections (1 MCI, 1 BCI) and 2 cases of tube related keratopathy (1 MCI, 1 BCI) that all resolved with tube removal. There were 2 BCI patients that presented to the emergency department for premature tube extrusion. Silicone intubation regardless of stent type is an effective treatment for simple congenital nasolacrimal duct obstruction. There was no significant difference in treatment success between tubes that extrude prematurely, and tubes removed at term based on type of intubation.

Phenanthrene alters the electrical activity of atrial and ventricular myocytes of a polar fish, the Navaga cod.

Oil and gas exploration in the Arctic can result in the release of polycyclic aromatic hydrocarbons (PAHs) into relatively pristine environments. Following the recent spill of approximately 17 500 tonnes of diesel fuel in Norilsk, Russia, May 2020, our study focussed on the effects of phenanthrene, a low molecular weight PAH found in diesel and crude oil, on the isolated atrial and ventricular myocytes from the heart of the polar teleost, the Navaga cod (Eleginus nawaga). Acute exposure to phenanthrene in navaga cardiomyocytes caused significant action potential (AP) prolongation, confirming the proarrhythmic effects of this pollutant. We show AP prolongation was due to potent inhibition of the main repolarising current, IKr, with an IC50 value of ~2 µM. We also show a potent inhibitory effect (~55%) of 1 µM phenanthrene on the transient IKr currents that protects the heart from early-after-depolarizations and arrhythmias. These data, along with more minor effects on inward sodium (INa) (~17% inhibition at 10 µM) and calcium (ICa) (~17% inhibition at 30 µM) currents, and no effects on inward rectifier (IK1 and IKAch) currents, demonstrate the cardiotoxic effects exerted by phenanthrene on the atrium and ventricle of navaga cod. Moreover, we report the first data that we are aware of on the impact of phenanthrene on atrial myocyte function in any fish species.

Phenanthrene impacts zebrafish cardiomyocyte excitability by inhibiting IKr and shortening action potential duration.

Air pollution is an environmental hazard that is associated with cardiovascular dysfunction. Phenanthrene is a three-ringed polyaromatic hydrocarbon that is a significant component of air pollution and crude oil and has been shown to cause cardiac dysfunction in marine fishes. We investigated the cardiotoxic effects of phenanthrene in zebrafish (Danio rerio), an animal model relevant to human cardiac electrophysiology, using whole-cell patch-clamp of ventricular cardiomyocytes. First, we show that phenanthrene significantly shortened action potential duration without altering resting membrane potential or upstroke velocity (dV/dt). L-type Ca2+ current was significantly decreased by phenanthrene, consistent with the decrease in action potential duration. Phenanthrene blocked the hERG orthologue (zfERG) native current, IKr, and accelerated IKr deactivation kinetics in a dose-dependent manner. Furthermore, we show that phenanthrene significantly inhibits the protective IKr current envelope, elicited by a paired ventricular AP-like command waveform protocol. Phenanthrene had no effect on other IK. These findings demonstrate that exposure to phenanthrene shortens action potential duration, which may reduce refractoriness and increase susceptibility to certain arrhythmia triggers, such as premature ventricular contractions. These data also reveal a previously unrecognized mechanism of polyaromatic hydrocarbon cardiotoxicity on zfERG by accelerating deactivation and decreasing IKr protective current.

α-Conotoxins active at α3-containing nicotinic acetylcholine receptors and their molecular determinants for selective inhibition.

Neuronal α3-containing nicotinic acetylcholine receptors (nAChRs) in the peripheral nervous system (PNS) and non-neuronal tissues are implicated in a number of severe disease conditions ranging from cancer to cardiovascular diseases and chronic pain. However, despite the physiological characterization of mouse models and cell lines, the precise pathophysiology of nAChRs outside the CNS remains not well understood, in part because there is a lack of subtype-selective antagonists. α-Conotoxins isolated from cone snail venom exhibit characteristic individual selectivity profiles for nAChRs and, therefore, are excellent tools to study the determinants for nAChR-antagonist interactions. Given that human α3ß4 subtype selective α-conotoxins are scarce and this is a major nAChR subtype in the PNS, the design of new peptides targeting this nAChR subtype is desirable. Recent studies using α-conotoxins RegIIA and AuIB, in combination with nAChR site-directed mutagenesis and computational modelling, have shed light onto specific nAChR residues, which determine the selectivity of the α-conotoxins for the human α3ß2 and α3ß4 subtypes. Publications describing the selectivity profile and binding sites of other α-conotoxins confirm that subtype-selective nAChR antagonists often work through common mechanisms by interacting with the same structural components and sites on the receptor. LINKED ARTICLES: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

Key Structural Determinants in the Agonist Binding Loops of Human ß2 and ß4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3ß4 Subtype Selectivity of α-Conotoxins.

α-Conotoxins represent a large group of pharmacologically active peptides that antagonize nicotinic acetylcholine receptors (nAChRs). The α3ß4 nAChR, a predominant subtype in the peripheral nervous system, has been implicated in various pathophysiological conditions. As many α-conotoxins have multiple pharmacological targets, compounds specifically targeting individual nAChR subtypes are needed. In this study, we performed mutational analyses to evaluate the key structural components of human ß2 and ß4 nAChR subunits that determine α-conotoxin selectivity for α3ß4 nAChR. α-Conotoxin RegIIA was used to evaluate the impact of non-conserved human ß2 and ß4 residues on peptide affinity. Two mutations, α3ß2[T59K] and α3ß2[S113R], strongly enhanced RegIIA affinity compared with wild-type α3ß2, as seen by substantially increased inhibitory potency and slower off-rate kinetics. Opposite point mutations in α3ß4 had the contrary effect, emphasizing the importance of loop D residue 59 and loop E residue 113 as determinants for RegIIA affinity. Molecular dynamics simulation revealed the side chains of ß4 Lys59 and ß4 Arg113 formed hydrogen bonds with RegIIA loop 2 atoms, whereas the ß2 Thr59 and ß2 Ser113 side chains were not long enough to form such interactions. Residue ß4 Arg113 has been identified for the first time as a crucial component facilitating antagonist binding. Another α-conotoxin, AuIB, exhibited low activity at human α3ß2 and α3ß4 nAChRs. Molecular dynamics simulation indicated the key interactions with the ß subunit are different to RegIIA. Taken together, these data elucidate the interactions with specific individual ß subunit residues that critically determine affinity and pharmacological activity of α-conotoxins RegIIA and AuIB at human nAChRs.

Molecular Basis for Differential Sensitivity of α-Conotoxin RegIIA at Rat and Human Neuronal Nicotinic Acetylcholine Receptors.

α-Conotoxins, as nicotinic acetylcholine receptor (nAChR) antagonists, are powerful tools for dissecting biologic processes and guiding drug development. The α3ß2 and α3ß4 nAChR subtypes are expressed in the central and peripheral nervous systems and play a critical role in various pathophysiological conditions ranging from nicotine addiction to the development and progression of lung cancer. Here we used the α4/7-conotoxin RegIIA, a disulfide-bonded peptide from the venom of Conus regius, and its analog [N11A,N12A]RegIIA to probe the specific pharmacological properties of rat and human nAChR subtypes. nAChR subtypes were heterologously expressed in Xenopus oocytes and two-electrode voltage clamp recordings used to investigate the effects of the peptides on nAChR activity. RegIIA potently inhibited currents evoked by acetylcholine (ACh) at rat α3ß2 (IC50 = 10.7 nM), whereas a 70-fold lower potency was observed at human α3ß2 nAChR (IC50 = 704.1 nM). Conversely, there were no species-specific differences in sensitivity to RegIIA at the α3ß4 nAChR. Receptor mutagenesis and molecular dynamics studies revealed that this difference can be attributed primarily to a single amino acid change: Glu198 on the rat α3 subunit corresponding to a proline on the human subunit. These findings reveal a novel species- and subunit-specific receptor-antagonist interaction.

Conotoxin αD-GeXXA utilizes a novel strategy to antagonize nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) play essential roles in transmitting acetylcholine-mediated neural signals across synapses and neuromuscular junctions, and are also closely linked to various diseases and clinical conditions. Therefore, novel nAChR-specific compounds have great potential for both neuroscience research and clinical applications. Conotoxins, the peptide neurotoxins produced by cone snails, are a rich reservoir of novel ligands that target receptors, ion channels and transporters in the nervous system. From the venom of Conus generalis, we identified a novel dimeric nAChR-inhibiting αD-conotoxin GeXXA. By solving the crystal structure and performing structure-guided dissection of this toxin, we demonstrated that the monomeric C-terminal domain of αD-GeXXA, GeXXA-CTD, retains inhibitory activity against the α9α10 nAChR subtype. Furthermore, we identified that His7 of the rat α10 nAChR subunit determines the species preference of αD-GeXXA, and is probably part of the binding site of this toxin. These results together suggest that αD-GeXXA cooperatively binds to two inter-subunit interfaces on the top surface of nAChR, thus allosterically disturbing the opening of the receptor. The novel antagonistic mechanism of αD-GeXXA via a new binding site on nAChRs provides a valuable basis for the rational design of new nAChR-targeting compounds.

Alanine scan of α-conotoxin RegIIA reveals a selective α3ß4 nicotinic acetylcholine receptor antagonist.

Activation of the α3ß4 nicotinic acetylcholine receptor (nAChR) subtype has recently been implicated in the pathophysiology of various conditions, including development and progression of lung cancer and in nicotine addiction. As selective α3ß4 nAChR antagonists, α-conotoxins are valuable tools to evaluate the functional roles of this receptor subtype. We previously reported the discovery of a new α4/7-conotoxin, RegIIA. RegIIA was isolated from Conus regius and inhibits acetylcholine (ACh)-evoked currents mediated by α3ß4, α3ß2, and α7 nAChR subtypes. The current study used alanine scanning mutagenesis to understand the selectivity profile of RegIIA at the α3ß4 nAChR subtype. [N11A] and [N12A] RegIIA analogs exhibited 3-fold more selectivity for the α3ß4 than the α3ß2 nAChR subtype. We also report synthesis of [N11A,N12A]RegIIA, a selective α3ß4 nAChR antagonist (IC50 of 370 nM) that could potentially be used in the treatment of lung cancer and nicotine addiction. Molecular dynamics simulations of RegIIA and [N11A,N12A]RegIIA bound to α3ß4 and α3ß2 suggest that destabilization of toxin contacts with residues at the principal and complementary faces of α3ß2 (α3-Tyr(92), Ser(149), Tyr(189), Cys(192), and Tyr(196); ß2-Trp(57), Arg(81), and Phe(119)) may form the molecular basis for the selectivity shift.

Dicarba analogues of α-conotoxin RgIA. Structure, stability, and activity at potential pain targets.

α-Conotoxin RgIA is both an antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype and an inhibitor of high-voltage-activated N-type calcium channel currents. RgIA has therapeutic potential for the treatment of pain, but reduction of the disulfide bond framework under physiological conditions represents a potential liability for clinical applications. We synthesized four RgIA analogues that replaced native disulfide pairs with nonreducible dicarba bridges. Solution structures were determined by NMR, activity assessed against biological targets, and stability evaluated in human serum. [3,12]-Dicarba analogues retained inhibition of ACh-evoked currents at α9α10 nAChRs but not N-type calcium channel currents, whereas [2,8]-dicarba analogues displayed the opposite pattern of selectivity. The [2,8]-dicarba RgIA analogues were effective in HEK293 cells stably expressing human Cav2.2 channels and transfected with human GABAB receptors. The analogues also exhibited improved serum stability over the native peptide. These selectively acting dicarba analogues may represent mechanistic probes to explore analgesia-related biological receptors.

Isolation and characterization of α-conotoxin LsIA with potent activity at nicotinic acetylcholine receptors.

A new α-conotoxin LsIA was isolated from the crude venom of Conus limpusi using assay-guided RP-HPLC fractionation. Synthetic LsIA was a potent antagonist of α3ß2, α3α5ß2 and α7 nAChRs, with half-maximal inhibitory concentrations of 10, 31 and 10 nM, respectively. The structure of LsIA determined by NMR spectroscopy comprised a characteristic disulfide bond-stabilized α-helical structure and disordered N-terminal region. Potency reductions of up to 9-fold were observed for N-terminally truncated analogues of LsIA at α7 and α3ß2 nAChRs, whereas C-terminal carboxylation enhanced potency 3-fold at α3ß2 nAChRs but reduced potency 3-fold at α7 nAChRs. This study gives further insight into α-conotoxin pharmacology and the molecular basis of nAChR selectivity, highlighting the influence of N-terminal residues and C-terminal amidation on conotoxin pharmacology.

Dicarba α-conotoxin Vc1.1 analogues with differential selectivity for nicotinic acetylcholine and GABAB receptors.

Conotoxins have emerged as useful leads for the development of novel therapeutic analgesics. These peptides, isolated from marine molluscs of the genus Conus, have evolved exquisite selectivity for receptors and ion channels of excitable tissue. One such peptide, α-conotoxin Vc1.1, is a 16-mer possessing an interlocked disulfide framework. Despite its emergence as a potent analgesic lead, the molecular target and mechanism of action of Vc1.1 have not been elucidated to date. In this paper we describe the regioselective synthesis of dicarba analogues of Vc1.1 using olefin metathesis. The ability of these peptides to inhibit acetylcholine-evoked current at rat α9α10 and α3ß4 nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes has been assessed in addition to their ability to inhibit high voltage-activated (HVA) calcium channel current in isolated rat DRG neurons. Their solution structures were determined by NMR spectroscopy. Significantly, we have found that regioselective replacement of the native cystine framework with a dicarba bridge can be used to selectively tune the cyclic peptide's innate biological activity for one receptor over another. The 2,8-dicarba Vc1.1 isomer retains activity at γ-aminobutyric acid (GABAB) G protein-coupled receptors, whereas the isomeric 3,16-dicarba Vc1.1 peptide retains activity at the α9α10 nAChR subtype. These singularly acting analogues will enable the elucidation of the biological target responsible for the peptide's potent analgesic activity.

Determination of the α-conotoxin Vc1.1 binding site on the α9α10 nicotinic acetylcholine receptor.

α-Conotoxin Vc1.1 specifically and potently inhibits the nicotinic acetylcholine receptor subtype α9α10 (α9α10 nAChR) and is a potential novel treatment for neuropathic pain. Here, we used a combination of computational modeling and electrophysiology experiments to determine the Vc1.1 binding site on the α9α10 nAChR. Interactions of Vc1.1 with two probable binding sites, α9α10 and α10α9, were modeled. Mutational energies calculated by assuming specific interactions in the α10α9 binding site correlated better with electrophysiological recordings than those assuming interactions with the α9α10 binding site. Two novel Vc1.1 analogues, [N9F]Vc1.1 and [N9W]Vc1.1, were predicted to have large differences in affinity between the two binding sites. Data from functional studies were consistent with computational predictions that assumed preferred binding of Vc1.1 to the α10α9 pocket. Moreover, our modeling study suggested that a single hydrogen bond formed between Vc1.1 and position 59 of the α10α9 pocket confers specificity to rat versus human α9α10 nAChRs.

RegIIA: an α4/7-conotoxin from the venom of Conus regius that potently blocks α3ß4 nAChRs.

Neuronal nicotinic acetylcholine receptors (nAChRs) play pivotal roles in the central and peripheral nervous systems. They are implicated in disease states such as Parkinson's disease and schizophrenia, as well as addictive processes for nicotine and other drugs of abuse. Modulation of specific nAChRs is essential to understand their role in the CNS. α-Conotoxins, disulfide-constrained peptides isolated from the venom of cone snails, potently inhibit nAChRs. Their selectivity varies markedly depending upon the specific nAChR subtype/α-conotoxin pair under consideration. Thus, α-conotoxins are excellent probes to evaluate the functional roles of nAChRs subtypes. We isolated an α4/7-conotoxin (RegIIA) from the venom of Conus regius. Its sequence was determined by Edman degradation and confirmed by sequencing the cDNA of the protein precursor. RegIIA was synthesized using solid phase methods and native and synthetic RegIIA were functionally tested using two-electrode voltage clamp recording on nAChRs expressed in Xenopus laevis oocytes. RegIIA is among the most potent antagonist of the α3ß4 nAChRs found to date and is also active at α3ß2 and α7 nAChRs. The 3D structure of RegIIA reveals the typical folding of most α4/7-conotoxins. Thus, while structurally related to other α4/7 conotoxins, RegIIA has an exquisite balance of shape, charge, and polarity exposed in its structure to potently block the α3ß4 nAChRs.

Prevalence of sub-clinical vitamin A deficiency in 2-5-year-old children in Tehran.

To determine vitamin A status using conjunctival impression cytology (CIC) in children aged 2-5 years, we assessed 1257 randomly selected children in urban and rural areas of Tehran. History of using supplemental vitamin A, respiratory or diarrhoeal infection in the previous 6 months, residential location, parents' education, family economic status, and child's age, sex and weight were recorded. Sub-clinical vitamin A deficiency (defined as abnormal CIC) was found in 23.6% of the sample, a rate classified as a moderate public health problem. There was a statistically significant relationship between sex and age and abnormal CIC (P < 0.05).

Prevalence of sub-clinical vitamin A deficiency in 2-5-year-old children in Tehran

To determine vitamin A status using conjunctival impression cytology [CIC] in children aged 2- 5 years, we assessed 1257 randomly selected children in urban and rural areas of Tehran. History of using supplemental vitamin A, respiratory or diarrhoeal infection in the previous 6 months, residential location, parents' education, family economic status, and child's age, sex and weight were recorded. Sub- clinical vitamin A deficiency [defined as abnormal CIC] was found in 23.6% of the sample, a rate classified as a moderate public health problem. There was a statistically significant relationship, between sex and age and abnormal CIC [P < 0.05]

Retinal degeneration but not obesity is observed in null mutants of the tubby-like protein 1 gene.

The tub gene is a member of a small, well conserved neuronal gene family of unknown function. Mutations within this gene lead to early-onset blindness and deafness, as well as late-onset obesity and insulin resistance. To test the hypothesis that mutations within other members of this gene family would lead to similar phenotypes as observed in tubby mice, and hence have similar functional properties, we have generated null mutants of the tubby-like protein ( Tulp ) 1 gene by homologous recombination. Similarly to tubby mice, Tulp1 (-/-)mice exhibit an early-onset retinal degeneration with a progressive, rapid loss of photoreceptors, further supporting the notion that previously identified mutations within the human TULP1 gene are indeed causative of retinitis pigmentosa. However, in contrast to tubby mice, Tulp1 (-/-)mice exhibited normal hearing ability and, surprisingly, normal body weight despite the fact that both TUB and TULP1 are expressed in the same neurons within the hypothalamus in areas known to be involved in feeding behavior and energy homeo stasis. However, TUB and TULP1 show a distinctly different staining pattern in the nucleus of these neurons, perhaps explaining the difference in body weight between the Tulp1 (-/-)and tubby mutant mice.