Rigorous Characterization of Allosteric Modulation of the Human Metabotropic Glutamate Receptor 1 Reveals Probe- and Assay-Dependent Pharmacology.

Allosteric modulation of metabotropic glutamate receptor subtype 1 (mGlu1) represents a viable therapeutic target for treating numerous central nervous system disorders. Although multiple chemically distinct mGlu1 positive (PAMs) and negative (NAMs) allosteric modulators have been identified, drug discovery paradigms have not included rigorous pharmacological analysis. In the present study, we hypothesized that existing mGlu1 allosteric modulators possess unappreciated probe-dependent or biased pharmacology. Using human embryonic kidney 293 (HEK293A) cells stably expressing human mGlu1, we screened mGlu1 PAMs and NAMs from divergent chemical scaffolds for modulation of different mGlu1 orthosteric agonists in intracellular calcium (iCa2+) mobilization and inositol monophosphate (IP1) accumulation assays. Operational models of agonism and allosterism were used to derive estimates for important pharmacological parameters such as affinity, efficacy, and cooperativity. Modulation of glutamate and quisqualate-mediated iCa2+ mobilization revealed probe dependence at the level of affinity and cooperativity for both mGlu1 PAMs and NAMs. We also identified the previously described mGlu5 selective NAM PF-06462894 as an mGlu1 NAM with a different pharmacological profile from other NAMs. Differential profiles were also observed when comparing ligand pharmacology between iCa2+ mobilization and IP1 accumulation. The PAMs Ro67-4853 and CPPHA displayed apparent negative cooperativity for modulation of quisqualate affinity, and the NAMs CPCCOEt and PF-06462894 had a marked reduction in cooperativity with quisqualate in IP1 accumulation and upon extended incubation in iCa2+ mobilization assays. These data highlight the importance of rigorous assessment of mGlu1 modulator pharmacology to inform future drug discovery programs for mGlu1 allosteric modulators. SIGNIFICANCE STATEMENT: Metabotropic glutamate receptor subtype 1 (mGlu1) positive and negative allosteric modulators have therapeutic potential in multiple central nervous system disorders. We show that chemically distinct modulators display differential pharmacology with different orthosteric ligands and across divergent signaling pathways at human mGlu1. Such complexities in allosteric ligand pharmacology should be considered in future mGlu1 allosteric drug discovery programs.

A Prospective, Multicentre Randomised Controlled Trial Comparing Catheter Ablation Versus Antiarrhythmic Drugs in Patients With Structural Heart Disease Related Ventricular Tachycardia: The CAAD-VT Trial Protocol.

IMPORTANCE: Randomised trials have shown that catheter ablation (CA) is superior to medical therapy for ventricular tachycardia (VT) largely in patients with ischaemic heart disease. Whether this translates to patients with all forms and stages of structural heart disease (SHD-e.g., non-ischaemic heart disease) is unclear. This trial will help clarify whether catheter ablation offers superior outcomes compared to medical therapy for VT in all patients with SHD. OBJECTIVE: To determine in patients with SHD and spontaneous or inducible VT, if catheter ablation is more efficacious than medical therapy in control of VT during follow-up. DESIGN: Randomised controlled trial including 162 patients, with an allocation ratio of 1:1, stratified by left ventricular ejection fraction (LVEF) and geographical region of site, with a median follow-up of 18-months and a minimum follow-up of 1 year. SETTING: Multicentre study performed in centres across Australia. PARTICIPANTS: Structural heart disease patients with sustained VT or inducible VT (n=162). INTERVENTION: Early treatment, within 30 days of randomisation, with catheter ablation (intervention) or initial treatment with antiarrhythmic drugs only (control). MAIN OUTCOMES, MEASURES, AND RESULTS: Primary endpoint will be a composite of recurrent VT, VT storm (≥3 VT episodes in 24 hrs or incessant VT), or death. Secondary outcomes will include each of the individual primary endpoints, VT burden (number of VT episodes in the 6 months preceding intervention compared to the 6 months after intervention), cardiovascular hospitalisation, mortality (including all-cause mortality, cardiac death, and non-cardiac death) and LVEF (assessed by transthoracic echocardiography from baseline to 6-, 12-, 24- and 36-months post intervention). CONCLUSIONS AND RELEVANCE: The Catheter Ablation versus Anti-arrhythmic Drugs for Ventricular Tachycardia (CAAD-VT) trial will help determine whether catheter ablation is superior to antiarrhythmic drug therapy alone, in patients with SHD-related VT. TRIAL REGISTRY: Australian New Zealand Clinical Trials Registry (ANZCTR) TRIAL REGISTRATION ID: ACTRN12620000045910 TRIAL REGISTRATION URL: https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=377617&isReview=true.

AGE-breaker ALT711 reverses glycation-mediated cancer cell migration.

Diabetes is associated with increased risk of breast cancer and worse prognoses for cancer patients. Hyperglycemia can result in increased glycation, the process wherein crosslinkages are formed between sugars and extracellular matrix (ECM) proteins through the formation of advanced glycation endproducts (AGEs). Although accumulation of AGEs occurs naturally in vivo over time, it is greatly accelerated by the hyperglycemic environment of diabetic patients. AGE accumulation has been linked to stiffening-related diseases such as hypertension, cancer metastasis, and neurodegenerative disorders. In response, several AGE-inhibiting and AGE-breaking drugs have received significant attention for their ability to reduce AGE accumulation. The resulting effects of these drugs on cell behavior is not well understood. In this study, we measured cancer cell migration in glycated collagen with and without the AGE-breaking drug alagebrium chloride (ALT711) to investigate the drug's ability to disrupt ECM crosslinks and reduce tumor cell spreading, contractility, and migration. The mechanical properties and chemical composition of collagen glycated with increasing concentrations of glucose with and without ALT711 treatment were measured. Increasing glucose concentration resulted in increased AGE accumulation and matrix stiffness as well as increased cancer cell contractility, elongation, and migration. Treatment with ALT711 significantly lowered AGE accumulation within the collagen, decreased collagen stiffness, and reduced cell migration. These findings suggest that while hyperglycemia can increase collagen matrix stiffness, resulting in increased breast cancer cell migration, an AGE-breaker can reverse this phenotype and may be a viable treatment option for reducing cancer cell migration due to glycation.

Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin.

Mupirocin is a clinically important antibiotic produced by Pseudomonas fluorescens NCIMB 10586 that is assembled by a complex trans-AT polyketide synthase. The polyketide fragment, monic acid, is esterified by a 9-hydroxynonanoic acid (9HN) side chain which is essential for biological activity. The ester side chain assembly is initialised from a 3-hydroxypropionate (3HP) starter unit attached to the acyl carrier protein (ACP) MacpD, but the fate of this species is unknown. Herein we report the application of NMR spectroscopy, mass spectrometry, chemical probes and in vitro assays to establish the remaining steps of 9HN biosynthesis. These investigations reveal a complex interplay between a novel iterative or "stuttering" KS-AT didomain (MmpF), the multidomain module MmpB and multiple ACPs. This work has important implications for understanding the late-stage biosynthetic steps of mupirocin and will be important for future engineering of related trans-AT biosynthetic pathways (e.g. thiomarinol).

The evolution of gene-guided management of inherited arrhythmia syndromes: Peering beyond monogenic paradigms towards comprehensive genomic risk scores.

Inherited arrhythmia syndromes have traditionally been viewed as monogenic forms of disease whose pathophysiology is driven by a single highly penetrant rare genetic variant. Although an accurate depiction of a proportion of genetic variants, the variable penetrance frequently noted in genotype positive families and the presence of sporadic genotype negative cases have long highlighted a more nuanced truth being operative. Coupled with our more recent recognition that many rare variants implicated in inherited arrhythmia syndromes possess unexpectedly high allele frequencies within the general population, these observations have contributed to the realization that a spectrum of pathogenicity exists among clinically relevant genetic variants. Notably, variable mutation pathogenicity and corresponding variable degrees of penetrance emphasize a limitation of contemporary guidelines, which attempt to dichotomize genetic variants as pathogenic or benign. Recognition of the existence of low and intermediate penetrant variants insufficient to be causative for disease in isolation has served to emphasize the importance of additional genetic, clinical, and environmental factors in the pathogenesis of rare inherited arrhythmia syndromes. Despite being rare, it has also become increasingly evident that common genetic variants play critical roles in both heritable channelopathies and cardiomyopathies and in aggregate may even be the primary drivers in certain instances, such as genotype negative Brugada syndrome. Our growing realization that the genetic substrates of inherited arrhythmia syndromes have intricacies that extend beyond traditionally perceived monogenic paradigms has highlighted a potential value of leveraging more comprehensive genomic risk scores for predicting disease development and arrhythmic risk.

A simple maneuver to determine if septal accessory pathway ablation requires a left atrial approach.

INTRODUCTION: Septal accessory pathway (AP) ablation can be challenging due to the complex anatomy of the septal region. The decision to access the left atrium (LA) is often made after failure of ablation from the right. We sought to establish whether the difference between ventriculo-atrial (VA) time during right ventricular (RV) apical pacing versus the VA during tachycardia would help establish the successful site for ablation of septal APs. METHODS: Intracardiac electrograms of patients with orthodromic reciprocating tachycardia (ORT) using a septal AP with successful catheter ablation were reviewed. The ∆VA was the difference between the VA interval during RV apical pacing and the VA interval during ORT. The difference in the VA interval during right ventricular entrainment and ORT (StimA-VA) was also measured. RESULTS: The median ∆VA time was significantly less in patients with a septal AP ablated on the right side compared with patients with a septal AP ablated on the left side (12 ± 19 vs. 56 ± 10 ms, p < .001). The StimA-VA was significantly different between the two groups (22 ± 14 vs. 53 ± 9 ms, p < .001). The ∆VA and StimA-VA were always ≤ 40 ms in patients with non-decremental septal APs ablated from the right side and always greater than 40 ms in those with septal APs ablated from the left. CONCLUSION: ΔVA and StimA-VA values identified with RV apical pacing in the setting of ORT involving a septal AP predict when left atrial access will be necessary for successful ablation.

Elective Cardioversion in the Era of Novel Oral Anticoagulants - Does a Nurse Administered Verbal Questionnaire for Compliance Negate the Need for Routine Transoesophageal Echocardiography?

BACKGROUND: Anticoagulation prior to elective external direct current cardioversion (EDCCV) is mandatory. The inability to monitor compliance with novel oral anticoagulants (NOACs) raises a potential safety issue. We aimed to evaluate whether a structured, nurse-led assessment of compliance prior to EDCCV ensures safety without the need for routine transoesophageal echocardiography (TOE). METHODS: Data was prospectively collected on consecutive patients undergoing EDCCV during 2014-2015. All procedures were supervised by an electrophysiology clinical nurse consultant (EPCNC). Drug compliance was verbally assessed using a standardised questionnaire by the EPCNC. Novel oral anticoagulants compliance was required for a continuous period of 3 weeks prior to EDCCV; otherwise a TOE-guided EDDCV was performed. All patients had follow-up 30 days post-procedure. RESULTS: Three hundred and eleven cardioversions were performed on 256 patients in whom 154 (49.5%) were prescribed a NOAC (rivaroxaban (n=105; 68.2%), dabigatran (n=38; 24.7%), apixaban (n=11; 7.1%)). Median age was 63 years (24-94 yrs), mean CHADS2-Vasc score was 2.0±1.5 and 138 (89.6%) were outpatients. One hundred and twenty-nine (83.8%) EDCCV were for atrial fibrillation and 25 (16.2%) for atrial flutter. Sinus rhythm was achieved in 90.3% of cases. Fourteen patients (9%) assessed as non-compliant underwent TOE. 129 (83.8%) EDCCV were performed without prior TOE. No stroke or systemic embolism was identified in any patient treated with either warfarin or a NOAC. CONCLUSIONS: A standardised, verbal questionnaire can be administered to detect NOAC non-compliance in patients undergoing EDCCV. With appropriate compliance assessment a nurse-led EDCCV without routine TOE did not significantly compromise safety in this study group.

Using the Surface ECG to Identify Right Ventricular Pacing Lead Position: A Cautionary Tale.

Chronic right ventricular (RV) apical pacing may lead to the development of heart failure in some patients. Although pacing of the RV septum has been proposed as an alternative, positioning a lead in the true septum has proven challenging. In addition to fluoroscopy at implant, it has been suggested that 12-lead surface electrocardiogram (ECG) can be used to determine septal lead position; however, studies show this may be inaccurate. We present a case where a change in the ECG QRS axis late after pacemaker insertion with an active fixation lead highlights the difficulties of ECG localization of pacing leads.

Surface ECG and Fluoroscopy are Not Predictive of Right Ventricular Septal Lead Position Compared to Cardiac CT.

BACKGROUND: Controversy exists regarding the optimal lead position for chronic right ventricular (RV) pacing. Placing a lead at the RV septum relies upon fluoroscopy assisted by a surface 12-lead electrocardiogram (ECG). We compared the postimplant lead position determined by ECG-gated multidetector contrast-enhanced computed tomography (MDCT) with the position derived from the surface 12-lead ECG. METHODS: Eighteen patients with permanent RV leads were prospectively enrolled. Leads were placed in the RV septum (RVS) in 10 and the RV apex (RVA) in eight using fluoroscopy with anteroposterior and left anterior oblique 30° views. All patients underwent MDCT imaging and paced ECG analysis. ECG criteria were: QRS duration; QRS axis; positive or negative net QRS amplitude in leads I, aVL, V1, and V6; presence of notching in the inferior leads; and transition point in precordial leads at or after V4. RESULTS: Of the 10 leads implanted in the RVS, computed tomography (CT) imaging revealed seven to be at the anterior RV wall, two at the anteroseptal junction, and one in the true septum. For the eight RVA leads, four were anterior, two septal, and two anteroseptal. All leads implanted in the RVS met at least one ECG criteria (median 3, range 1-6). However, no criteria were specific for septal position as judged by MDCT. Mean QRS duration was 160 ± 24 ms in the RVS group compared with 168 ± 14 ms for RVA pacing (P = 0.38). CONCLUSIONS: We conclude that the surface ECG is not sufficiently accurate to determine RV septal lead tip position compared to cardiac CT.

Cardiac Sarcoidosis, Left Ventricular Impairment and Chronic Right Ventricular Pacing: Pacing or Pathology?

BACKGROUND: Cardiac sarcoidosis (CS) is an uncommon and under-recognised disease which most frequently presents with atrioventricular (AV) block and may also present with ventricular arrhythmias and left ventricular (LV) systolic dysfunction. Because of its protean clinical manifestations, confirming a diagnosis of CS is often challenging. METHODS: We report two cases where patients presented with atrioventricular (AV) block without evidence of underlying myocardial disease, underwent chronic dual-chamber pacing, and presented several years later with severe LV systolic dysfunction. RESULTS: Both patients were referred for assessment of pacing-induced cardiomyopathy with a view to upgrading their device to cardiac resynchronisation therapy (CRT). Subsequent investigation revealed features consistent with CS and appropriate immunosuppressive therapy resulted in improvement in LV function avoiding the requirement for CRT. CONCLUSION: We present a review of the diagnosis of cardiac sarcoidosis, the importance of imaging modalities and current treatment recommendations.

Neuropeptides and polypeptide hormones in echinoderms: new insights from analysis of the transcriptome of the sea cucumber Apostichopus japonicus.

Echinoderms are of special interest for studies in comparative endocrinology because of their phylogenetic position in the animal kingdom as deuterostomian invertebrates. Furthermore, their pentaradial symmetry as adult animals provides a unique context for analysis of the physiological and behavioral roles of peptide signaling systems. Here we report the first extensive survey of neuropeptide and peptide hormone precursors in a species belonging to the class Holothuroidea. Transcriptome sequence data obtained from the sea cucumber Apostichopus japonicus were analyzed to identify homologs of precursor proteins that have recently been identified in the sea urchin Strongylocentrotus purpuratus (class Echinoidea). A total of 17 precursor proteins have been identified in A. japonicus, including precursors of peptides related to thyrotropin-releasing hormone, pedal peptide/orcokinin-type peptides, AN peptides/tachykinins, luqins, corticotropin-releasing hormone (CRH), GPA2-type glycoprotein hormone subunits and bursicon. In addition, an unusual finding was an A. japonicus calcitonin-type precursor protein (AjCTLPP), the first to be discovered that comprises two calcitonin-like peptides; this contrasts with the products of the alternatively-spliced calcitonin/CGRP gene in vertebrates, which comprise either calcitonin or CGRP. Collectively, the data obtained provide new insights on the evolution and diversity of neuropeptides and polypeptide hormones. Furthermore, because A. japonicus is one of several sea cucumber species that are used for human consumption, our findings may have practical and economic impact by providing a basis for neuroendocrine-based strategies to improve methods of aquaculture.

Aortic stenosis: Update in monitoring and management.

BACKGROUND: Aortic stenosis (AS) remains one of the most commonly encountered valvular pathologies. Medical management does not alter the progression of the disease, making assessment of severity and timing of referral for valve replacement the most important aspects of caring for patients with AS. OBJECTIVE: To review the contemporary management of AS, including signs and symptoms, echocardiography and decision making in management. DISCUSSION: Severe symptomatic AS is frequently accompanied by dyspnoea, chest pain or syncope and a physical examination might reveal the presence of an ejection systolic murmur. Echocardiography is the first and most useful investigation to stratify risk and determine requirement for valve replacement by assessing valve gradients and left ventricular function. Surgical and transcatheter options now exist for treatment of AS and decision making is usually multidisciplinary and based on individual patient parameters.

Cardiac implantable electronic devices: An update.

BACKGROUND: Cardiac implantable electronic devices (CIEDs) are a core component in the management of heart rhythm disorders. The complexity of devices, the information gathered and therapy delivered by CIEDs continues to advance at pace. OBJECTIVE: The aim of this paper is to provide an update on advances in CIED technology and how this applies to managing patients with CIEDs in general practice. DISCUSSION: In recent years, there have been notable advances in CIED technology. These include widespread magnetic resonance imaging compatibility and automated algorithms to assist in the clinical management of patients. There is the ability for clinicians and pacemaker clinics to monitor devices remotely, avoiding in-clinic visits. Options are now available for leadless pacemakers and subcutaneous defibrillators as an alternative to indwelling leads and associated infection and vascular issues. Techniques have been developed to allow leads to capture the native conduction system, providing physiological cardiac activation (conduction system pacing) for treatment and prevention of heart failure.

Left atrial volume index: A predictor of atrial fibrillation recurrence following direct current cardioversion - A systematic review and meta-analysis.

This systematic review and meta-analysis was conducted to determine the clinical relevance of echocardiographically measured left atrial (LA) size to predict the recurrence of atrial fibrillation (AF) after direct current cardioversion (DCCV). A search was performed on Medline (Ovid), Embase (Elsevier), Cochrane Central Register of Controlled Trials (CENTRAL) in Cochrane Library, Wiley and Web of Science (Clarivate) to identify relevant studies. Amongst the initial 4066 citations identified, 31 fulfilled the criteria for inclusion in the data analysis incorporating 2725 patients with a mean follow-up period of 6.5 months. The weighted mean left atrial volume index (LAVI) was 40.56 ml/m2 (95 %CI:37.24-43.88) in the sinus rhythm (SR) maintenance group versus 48.69 ml/m2 (95 % CI: 44.42-52.97) in the AF recurrence group with P value of < 0.001, left atrial diameter (LAD) was 42.06 mm (95 %CI: 41.08-43.05) in the SR maintenance group versus 45.13 mm (95 %CI: 44.09-46.16) in the AF recurrence group, P value < 0.001. Effect size analysis of LAVI showed that each unit increase in LAVI resulted in an increase in the risk of AF recurrence by 6 % (95 % CI: 3 %-10 %). Age and AF duration were also statistically significant between the two groups however comorbidities, use of beta blockers or amiodarone were not significantly different. This meta-analysis shows that AF duration, LAVI, LAD and age predict the risk of recurrence of atrial fibrillation post electrical cardioversion with LAVI being the most clinically relevant echocardiographic feature.

Use of copeptin in interpretation of the water deprivation test.

INTRODUCTION: Currently, the water deprivation test remains the standard method for distinguishing primary polydipsia (PP) from cranial diabetes insipidus (cDI) and nephrogenic diabetes insipidus (nDI). There is increasing interest in a direct estimate of antidiuretic hormone using plasma copeptin as a stable and reliable surrogate marker. We present our experience of measuring copeptin during the water deprivation test. METHODS: Forty-seven people (17 men) underwent a standard water deprivation test between 2013 and 2021. Plasma copeptin was measured at the start of the test and at the end of the period of water deprivation (maximum osmotic stimulation). Results were classified using prespecified diagnostic criteria. As it is known that a significant proportion of tests will reveal indeterminate results, a final diagnosis was obtained by including relevant pre- and post-test clinical criteria. This diagnosis was then used to plan individual treatment. RESULTS: Basal and stimulated copeptin were significantly higher in the nephrogenic DI group than other categories (p < .001). There was no significant difference in basal or stimulated copeptin between PP, cDI or partial DI. Nine results were indeterminate where the serum and urine osmolality did not give a unified diagnosis. Stimulated copeptin was helpful in reclassifying these patients into the final diagnostic groups. CONCLUSION: Plasma copeptin has additional clinical utility in interpretation of the water deprivation test and may continue to have a place alongside newer stimulation tests.

Matrix stiffness regulates tumor cell intravasation through expression and ESRP1-mediated alternative splicing of MENA.

During intravasation, cancer cells cross the endothelial barrier and enter the circulation. Extracellular matrix stiffening has been correlated with tumor metastatic potential; however, little is known about the effects of matrix stiffness on intravasation. Here, we utilize in vitro systems, a mouse model, specimens from patients with breast cancer, and RNA expression profiles from The Cancer Genome Atlas Program (TCGA) to investigate the molecular mechanism by which matrix stiffening promotes tumor cell intravasation. Our data show that heightened matrix stiffness increases MENA expression, which promotes contractility and intravasation through focal adhesion kinase activity. Further, matrix stiffening decreases epithelial splicing regulatory protein 1 (ESRP1) expression, which triggers alternative splicing of MENA, decreases the expression of MENA11a, and enhances contractility and intravasation. Altogether, our data indicate that matrix stiffness regulates tumor cell intravasation through enhanced expression and ESRP1-mediated alternative splicing of MENA, providing a mechanism by which matrix stiffness regulates tumor cell intravasation.

Beta-blockade enhances anthracycline control of metastasis in triple-negative breast cancer.

Beta-adrenergic blockade has been associated with improved cancer survival in patients with triple-negative breast cancer (TNBC), but the mechanisms of these effects remain unclear. In clinical epidemiological analyses, we identified a relationship between beta-blocker use and anthracycline chemotherapy in protecting against TNBC progression, disease recurrence, and mortality. We recapitulated the effect of beta-blockade on anthracycline efficacy in xenograft mouse models of TNBC. In metastatic 4T1.2 and MDA-MB-231 mouse models of TNBC, beta-blockade improved the efficacy of the anthracycline doxorubicin by reducing metastatic development. We found that anthracycline chemotherapy alone, in the absence of beta-blockade, increased sympathetic nerve fiber activity and norepinephrine concentration in mammary tumors through the induction of nerve growth factor (NGF) by tumor cells. Moreover, using preclinical models and clinical samples, we found that anthracycline chemotherapy up-regulated ß2-adrenoceptor expression and amplified receptor signaling in tumor cells. Neurotoxin inhibition of sympathetic neural signaling in mammary tumors using 6-hydroxydopamine or genetic deletion of NGF or ß2-adrenoceptor in tumor cells enhanced the therapeutic effect of anthracycline chemotherapy by reducing metastasis in xenograft mouse models. These findings reveal a neuromodulatory effect of anthracycline chemotherapy that undermines its potential therapeutic impact, which can be overcome by inhibiting ß2-adrenergic signaling in the tumor microenvironment. Supplementing anthracycline chemotherapy with adjunctive ß2-adrenergic antagonists represents a potential therapeutic strategy for enhancing the clinical management of TNBC.

The neuropeptide transcriptome of a model echinoderm, the sea urchin Strongylocentrotus purpuratus.

Neuronal secretion of peptide signaling molecules (neuropeptides) is an evolutionarily ancient feature of nervous systems. Here we report the identification of 20 cDNAs encoding putative neuropeptide precursors in the sea urchin Strongylocentrotus purpuratus (Phylum Echinodermata), providing new insights on the evolution and diversity of neuropeptides. Identification of a gonadotropin-releasing hormone-like peptide precursor (SpGnRHP) is consistent with the widespread phylogenetic distribution of GnRH-type neuropeptides in the bilateria. A protein (SpTRHLP) comprising multiple copies of peptides that share structural similarity with thyrotropin-releasing hormone (TRH) is the first TRH-like precursor to be identified in an invertebrate. SpCTLP is the first calcitonin-like peptide with two N-terminally located cysteine residues to be found in a non-chordate species. Discovery of two proteins (SpPPLNP1, SpPPLNP2) comprising homologs of molluscan pedal peptides and arthropod orcokinins indicates the existence of a bilaterian family of pedal peptide/orcokinin-type neuropeptides. Other proteins identified contain peptides that do not share apparent sequence similarity with known neuropeptides. These include Spnp5, which comprises multiple copies of C-terminally amidated peptides that have an N-terminal Ala-Asn motif (AN peptides), and Spnp9, Spnp10 and Spnp12, which contain putative neuropeptides with a C-terminal Phe-amide, Ser-amide or Pro-amide, respectively. Several proteins (Spnp11, 14, 15, 16, 17, 18, 19 and 20) contain putative neuropeptides with multiple cysteine residues (2, 6 or 8), which may mediate formation of intramolecular or intermolecular disulphide bridges. Looking ahead, the identification of these neuropeptide precursors in S. purpuratus has provided a strong basis for a comprehensive analysis of neuropeptide function in this model echinoderm species.

Ablation of typical atrial flutter using mini electrode measurements for maximum voltage-guided ablation: A randomized, controlled trial.

BACKGROUND: Novel ablation catheters with mini electrode (ME) sensing have become available but their utility is unclear. We investigated whether ablation of the cavotricuspid isthmus (CTI) for atrial flutter (AFL) would be improved using ME signals. METHODS: Sixty-one patients (76% male, 63 ± 10 years) with CTI-dependent AFL underwent ablation using a maximum voltage-guided approach, randomized to either standard 8 mm non-irrigated catheter with bipolar signals or IntellaTip MiFi catheter using ME signals alone. RESULTS: Acute bidirectional block was achieved in 97%. Mean follow-up was 16.7 ± 10 months. The median number of ablation lesions was 13 in both groups (range 3-62 vs. 1-43, p = .85). No significant differences were observed in AFL recurrences (17% vs. 11%, p = .7), median procedure durations (97 min [interquartile range (IQR), 71-121] vs. 87 min [IQR, 72-107], p = .55) or fluoroscopy times (31 min [IQR, 21-52] vs. 38 min [IQR, 25-70], p = .56). Amplitudes of ME signals were on average 160% greater than blinded bipolar signals. In 23.7% of lesions where bipolar signals were difficult to interpret, 13.6% showed a clear ME signal. CONCLUSIONS: There was no difference in the effectiveness of CTI ablation guided by ME signals, compared with using bipolar signals from a standard 8 mm ablation catheter. While ME signal amplitudes were larger and sometimes present when the bipolar signal was unclear, this did not improve procedural characteristics or outcomes. The results suggest future research should focus on lesion integrity rather than signal sensing.

Structural and Functional Characterization of a Novel Scorpion Toxin that Inhibits NaV1.8 via Interactions With the DI Voltage Sensor and DII Pore Module.

Voltage-gated sodium channel NaV1.8 regulates transmission of pain signals to the brain. While NaV1.8 has the potential to serve as a drug target, the molecular mechanisms that shape NaV1.8 gating are not completely understood, particularly mechanisms that couple activation to inactivation. Interactions between toxin producing animals and their predators provide a novel approach for investigating NaV structure-function relationships. Arizona bark scorpions produce Na+ channel toxins that initiate pain signaling. However, in predatory grasshopper mice, toxins inhibit NaV1.8 currents and block pain signals. A screen of synthetic peptide toxins predicted from bark scorpion venom showed that peptide NaTx36 inhibited Na+ current recorded from a recombinant grasshopper mouse NaV1.8 channel (OtNaV1.8). Toxin NaTx36 hyperpolarized OtNaV1.8 activation, steady-state fast inactivation, and slow inactivation. Mutagenesis revealed that the first gating charge in the domain I (DI) S4 voltage sensor and an acidic amino acid (E) in the DII SS2 - S6 pore loop are critical for the inhibitory effects of NaTx36. Computational modeling showed that a DI S1 - S2 asparagine (N) stabilizes the NaTx36 - OtNaV1.8 complex while residues in the DI S3 - S4 linker and S4 voltage sensor form electrostatic interactions that allow a toxin glutamine (Q) to contact the first S4 gating charge. Surprisingly, the models predicted that NaTx36 contacts amino acids in the DII S5 - SS1 pore loop instead of the SS2 - S6 loop; the DII SS2 - S6 loop motif (QVSE) alters the conformation of the DII S5 - SS1 pore loop, enhancing allosteric interactions between toxin and the DII S5 - SS1 pore loop. Few toxins have been identified that modify NaV1.8 gating. Moreover, few toxins have been described that modify sodium channel gating via the DI S4 voltage sensor. Thus, NaTx36 and OtNaV1.8 provide tools for investigating the structure-activity relationship between channel activation and inactivation gating, and the connection to alternative pain phenotypes.