A nemaline myopathy-linked mutation inhibits the actin-regulatory functions of tropomodulin and leiomodin.

Actin is a highly expressed protein in eukaryotic cells and is essential for numerous cellular processes. In particular, efficient striated muscle contraction is dependent upon the precise regulation of actin-based thin filament structure and function. Alterations in the lengths of actin-thin filaments can lead to the development of myopathies. Leiomodins and tropomodulins are members of an actin-binding protein family that fine-tune thin filament lengths, and their dysfunction is implicated in muscle diseases. An Lmod3 mutation [G326R] was previously identified in patients with nemaline myopathy (NM), a severe skeletal muscle disorder; this residue is conserved among Lmod and Tmod isoforms and resides within their homologous leucine-rich repeat (LRR) domain. We mutated this glycine to arginine in Lmod and Tmod to determine the physiological function of this residue and domain. This G-to-R substitution disrupts Lmod and Tmod's LRR domain structure, altering their binding interface with actin and destroying their abilities to regulate thin filament lengths. Additionally, this mutation renders Lmod3 nonfunctional in vivo. We found that one single amino acid is essential for folding of Lmod and Tmod LRR domains, and thus is essential for the opposing actin-regulatory functions of Lmod (filament elongation) and Tmod (filament shortening), revealing a mechanism underlying the development of NM.

Nuclear Magnetic Resonance-Guided Structural Analysis of Moderate-Affinity Protein Complexes with Intrinsically Disordered Polypeptides.

Binding affinity of an individual binding site of an intrinsically disordered protein for its folded partner may be moderate. In such cases, a straightforward determination of the structure of the binding interface is difficult. We offer a hybrid protocol combining NMR chemical shift information, NMR spectral data on amino acid residue sequence substitution effects, residual dipolar coupling, and molecular dynamics simulation that allowed us to determine the structure of a complex between the intrinsically disordered tropomyosin-binding site of leiomodin and a coiled-coil peptide modeling the N-terminal fragment of tropomyosin. The protocol can be used for other moderate-affinity complexes composed of an intrinsically disordered peptide bound to a structured protein partner.

Problem gambling severity, gambling behavior, substance use, and mental health in gamblers who do and do not use cannabis: Evidence from a Canadian national sample.

INTRODUCTION: Cannabis use frequently co-occurs with gambling, and evidence indicates that both acute and chronic cannabis use may influence gambling behavior. The primary aim of the present study was to further contribute to the literature on this relationship by examining data collected from a Canadian national study of gambling. METHODS: Respondents consisted of 10,054 Canadian gamblers recruited from Leger Opinion's (LEO) online panel. In this study, gamblers who used cannabis were compared with non-users across a number of gambling as well as demographic and mental health variables. RESULTS: Of the total sample, 25.4 % reported past 12-month cannabis use. Among the 2,553 cannabis-users, 21.3 % reported daily use, and 69.9 % reported using once a month or more. A total of 56.2 % indicated they had used cannabis while gambling in the past 12 months. Bivariate analysis found significant differences between cannabis use and non-use on numerous demographic, mental health, and gambling-related variables. Individuals with greater problem gambling severity scores, more hours gambling, and a larger range of gambling activities were more likely to endorse using cannabis. Hierarchical logistic regression revealed that tobacco use, and having experienced significant child abuse were predictors of cannabis use. Non-use of cannabis was associated with older age, less engagement in online gambling, and being less likely to consume alcohol. CONCLUSION: The present findings both corroborate previous studies and expand upon the relationship between cannabis and gambling.

Harm Minimization Training, Knowledge, and Behaviour of Canadian Casino Employees.

Casino employees regularly interact with problem and at-risk gamblers and thus have considerable potential to both prevent and reduce gambling-related harm. While harm minimization (HM) and responsible gambling (RG) are routinely espoused by the casino industry, the actual level of employee HM/RG training, knowledge, and behaviour is unknown. The present study investigated this issue in the Canadian context by examining employee surveys collected by the RG Check accreditation program (8,262 surveys from 78 Canadian casinos/racinos collected between 2011 and 2020). These surveys revealed that almost all casino employees receive HM/RG training, but the amount of training tends to be quite limited (one hour) except for supervisors, managers, and security personnel. Basic HM/RG knowledge among all employees appears adequate, although their understanding of probability is incomplete. The most important consideration is whether this training and knowledge translates into meaningful HM/RG behaviour towards patrons. The large majority of employees (83.1%) report engaging in at least one HM/RG interaction with a patron at some point during the course of their employment (median length of 4 to 9 years), with security personnel reporting the highest rates. However, the frequency, nature, and impact of these interactions is unknown.

Ca2+ attenuates nucleation activity of leiomodin.

A transient increase in Ca2+ concentration in sarcomeres is essential for their proper function. Ca2+ drives striated muscle contraction via binding to the troponin complex of the thin filament to activate its interaction with the myosin thick filament. In addition to the troponin complex, the myosin essential light chain and myosin-binding protein C were also found to be Ca2+ sensitive. However, the effects of Ca2+ on the function of the tropomodulin family proteins involved in regulating thin filament formation have not yet been studied. Leiomodin, a member of the tropomodulin family, is an actin nucleator and thin filament elongator. Using pyrene-actin polymerization assay and transmission electron microscopy, we show that the actin nucleation activity of leiomodin is attenuated by Ca2+ . Using circular dichroism and nuclear magnetic resonance spectroscopy, we demonstrate that the mostly disordered, negatively charged region of leiomodin located between its first two actin-binding sites binds Ca2+ . We propose that Ca2+ binding to leiomodin results in the attenuation of its nucleation activity. Our data provide further evidence regarding the role of Ca2+ as an ultimate regulator of the ensemble of sarcomeric proteins essential for muscle function. SUMMARY STATEMENT: Ca2+ fluctuations in striated muscle sarcomeres modulate contractile activity via binding to several distinct families of sarcomeric proteins. The effects of Ca2+ on the activity of leiomodin-an actin nucleator and thin filament length regulator-have remained unknown. In this study, we demonstrate that Ca2+ binds directly to leiomodin and attenuates its actin nucleating activity. Our data emphasizes the ultimate role of Ca2+ in the regulation of the sarcomeric protein interactions.

Gambling in Canada During the COVID Lockdown: Prospective National Survey.

The current study investigated the impact of the COVID pandemic lockdown on gambling and problem gambling in Canada. The AGRI National Project's online panel participants (N = 3449) provided baseline gambling data 6 months prior to the pandemic. Re-surveying this sample during the lockdown provided an opportunity to make quantitative comparisons of the changes. Nearly one-third of gamblers reported ceasing gambling altogether during the lockdown. For the continuing gamblers, quantitative data indicated significant decreases in gambling frequency, time spent in gambling sessions, money spent, and the number of game types played. Qualitative perceptions of changes in gambling were examined and the accuracy of these reports were not closely aligned with actual changes in gambling. Gambling platform was the only gambling engagement metric where increases were found with ~ 17% of the gambling sample migrating to online gambling during the lockdown. Although problem gambling within the sample generally declined, consistent with previous literature, it was also found that gambling online-among other biopsychosocial factors-was a significant predictor for classification as a problem gambler during the lockdown. COVID-specific influences on health, employment, leisure time and social isolation were moderately associated with problem gambling scores but were not independent predictors of changes in gambling engagement during lockdown. Future studies are required to assess if the pandemic related changes in gambling evidenced in this study remain stable, or if engagement reverts to pre-pandemic levels when the pandemic response allows for the re-opening of land-based gambling venues.

Responsible Gambling in Canada: An Analysis of the RG Check Patron Surveys.

This study analyzed the Responsible Gambling Check patron survey data from Canadian casinos and racinos collected from 2011-2019 (18,580 patrons and 75 venues). The results indicated increasing awareness and use over time of harm minimization tools among more frequent patrons. Despite these promising trends, it is concerning that a substantial percentage of gamblers are still unaware of the harm minimization tools available. Further, the actual impact of this awareness on responsible gambling behaviour is largely unknown. We suggest greater efforts are needed nation-wide to promote the awareness, utilization, and evaluation of these harm minimization tools.

Predictors of gambling and problem gambling in Canada.

OBJECTIVES: The purpose of this study is to provide an updated profile of gamblers and problem gamblers in Canada and to identify characteristics most strongly associated with problem gambling. METHODS: An assessment of gambling participation and problem gambling was included in the 2018 Canadian Community Health Survey and administered to 23,952 individuals 18 years and older. Descriptive statistics provided a demographic profile for each type of gambling involvement as well as category of gambler (non-gambler, non-problem gambler, at-risk gambler, problem gambler). A logistic regression identified characteristics that best distinguished problem from non-problem gamblers. RESULTS: Gambling participation and problem gambling both varied as a function of gender, income, educational attainment, and race/ethnicity. However, multivariate analysis identified electronic gambling machine (EGM) participation to be the primary predictor of problem gambling status, with race/ethnicity, presence of a mood disorder, male gender, casino table game participation, older age, a greater level of smoking, participation in speculative financial activity, instant lottery participation, lower household income, and lottery or raffle ticket participation providing additional predictive power. Provincial EGM density and EGM participation rates are also very strong predictors of provincial rates of at-risk and problem gambling. CONCLUSION: Problem gambling has a biopsychosocial etiology, determined by personal vulnerability factors combined with the presence of riskier types of gambling such as EGMs. Effective prevention requires a multifaceted approach, but constraints on the availability and operation of EGMs would likely have the greatest single public health benefit.

RéSUMé: OBJECTIFS: Présenter un profil actualisé des joueurs et des joueurs pathologiques au Canada et cerner les caractéristiques les plus fortement associées au jeu pathologique. MéTHODE: Une évaluation de la participation au jeu de hasard et du jeu pathologique figurant dans l'Enquête sur la santé dans les collectivités canadiennes de 2018 a été administrée à 23 952 personnes de 18 ans et plus. Le profil démographique de chaque type de participation au jeu de hasard et la catégorie de joueur (non-joueur, joueur non pathologique, joueur à risque, joueur pathologique) ont été établis par statistique descriptive. Une régression logistique a permis de cerner les caractéristiques qui distinguaient le mieux les joueurs pathologiques des joueurs non pathologiques. RéSULTATS: La participation au jeu de hasard et le jeu pathologique variaient tous les deux en fonction du sexe, du revenu, du niveau d'instruction et de la race/l'ethnicité. L'analyse multivariée a cependant déterminé que l'utilisation d'appareils électroniques de jeu (AÉJ) était la principale variable prédictive du jeu pathologique, et que la race/l'ethnicité, la présence d'un trouble de l'humeur, le sexe masculin, la participation aux jeux de table dans les casinos, l'âge avancé, le tabagisme important, la participation à des activités financières spéculatives, la participation aux loteries instantanées, le faible revenu du ménage et l'achat de billets de loterie ou de tirage au sort amélioraient le pouvoir de prédiction. La densité provinciale des AÉJ et les taux d'utilisation des AÉJ étaient aussi de très fortes variables prédictives des taux provinciaux de jeu à risque et de jeu pathologique. CONCLUSION: Le jeu pathologique présente une étiologie biopsychosociale déterminée par des facteurs de vulnérabilité personnels combinés à la présence de types de jeu de hasard plus risqués, comme les AÉJ. Une prévention efficace nécessite une démarche pluridimensionnelle, mais l'imposition de limites à la disponibilité et à l'utilisation des AÉJ serait probablement la solution la plus avantageuse sur le plan de la santé publique.

Structural insights into the tropomodulin assembly at the pointed ends of actin filaments.

Tropomodulins are a family of important regulators of actin dynamics at the pointed ends of actin filaments. Four isoforms of tropomodulin, Tmod1-Tmod4, are expressed in vertebrates. Binding of tropomodulin to the pointed end is dependent on tropomyosin, an actin binding protein that itself is represented in mammals by up to 40 isoforms. The understanding of the regulatory role of the tropomodulin/tropomyosin molecular diversity has been limited due to the lack of a three-dimensional structure of the tropomodulin/tropomyosin complex. In this study, we mapped tropomyosin residues interacting with two tropomyosin-binding sites of tropomodulin and generated a three-dimensional model of the tropomodulin/tropomyosin complex for each of these sites. The models were refined by molecular dynamics simulations and validated via building a self-consistent three-dimensional model of tropomodulin assembly at the pointed end. The model of the pointed-end Tmod assembly offers new insights in how Tmod binding ensures tight control over the pointed end dynamics.

Leiomodin creates a leaky cap at the pointed end of actin-thin filaments.

Improper lengths of actin-thin filaments are associated with altered contractile activity and lethal myopathies. Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance; however, its role is under dispute. Using nuclear magnetic resonance data and molecular dynamics simulations, we generated the first atomic structural model of the binding interface between the tropomyosin-binding site of cardiac leiomodin and the N-terminus of striated muscle tropomyosin. Our structural data indicate that the leiomodin/tropomyosin complex only forms at the pointed end of thin filaments, where the tropomyosin N-terminus is not blocked by an adjacent tropomyosin protomer. This discovery provides evidence supporting the debated mechanism where leiomodin and tropomodulin regulate thin filament lengths by competing for thin filament binding. Data from experiments performed in cardiomyocytes provide additional support for the competition model; specifically, expression of a leiomodin mutant that is unable to interact with tropomyosin fails to displace tropomodulin at thin filament pointed ends and fails to elongate thin filaments. Together with previous structural and biochemical data, we now propose a molecular mechanism of actin polymerization at the pointed end in the presence of bound leiomodin. In the proposed model, the N-terminal actin-binding site of leiomodin can act as a "swinging gate" allowing limited actin polymerization, thus making leiomodin a leaky pointed-end cap. Results presented in this work answer long-standing questions about the role of leiomodin in thin filament length regulation and maintenance.

Iatrogenic Extracorporeal Membrane Oxygenator Rupture: Successful Management of a Unique Crisis.

We are presenting a case of successful resuscitation during accidental rupture of a venoarterial extracorporeal membrane oxygenator after surgical pulmonary embolectomy. This article reports a rare complication related to the mechanical circulatory support and its successful management.

Biophysical Screens Identify Fragments That Bind to the Viral DNA-Binding Proteins EBNA1 and LANA.

The human gamma-herpesviruses Epstein-Barr virus (EBV) (HHV-4) and Kaposi's sarcoma-associated herpesvirus (KSHV) (HHV-8) are responsible for a number of diseases, including various types of cancer. Epstein-Barr nuclear antigen 1 (EBNA1) from EBV and latency-associated nuclear antigen (LANA) from KSHV are viral-encoded DNA-binding proteins that are essential for the replication and maintenance of their respective viral genomes during latent, oncogenic infection. As such, EBNA1 and LANA are attractive targets for the development of small-molecule inhibitors. To this end, we performed a biophysical screen of EBNA1 and LANA using a fragment library by saturation transfer difference (STD)-NMR spectroscopy and surface plasmon resonance (SPR). We identified and validated a number of unique fragment hits that bind to EBNA1 or LANA. We also determined the high-resolution crystal structure of one fragment bound to EBNA1. Results from this screening cascade provide new chemical starting points for the further development of potent inhibitors for this class of viral proteins.

Avoiding gambling harm: An evidence-based set of safe gambling practices for consumers.

Prior studies have identified self-regulatory strategies that are infrequently used by problem-gamblers, but which might be protective if used. However, guidelines with evidence-based safe gambling practices (SGPs) that prevent gambling-related harm are lacking. This study aimed to: 1) identify a parsimonious set of evidence-based SGPs that best predict non-harmful gambling amongst gamblers who are otherwise most susceptible to experiencing gambling harm; 2) examine how widely are they used; and 3) assess whether their use differs by gambler characteristics. A sample of 1,174 regular gamblers in Alberta Canada completed an online survey measuring uptake of 43 potential SGPs, gambling harms and numerous risk factors for harmful gambling. Elastic net regression identified a sub-sample of 577 gamblers most susceptible to gambling harm and therefore most likely to benefit from the uptake of SGPs. A second elastic net predicted gambling harm scores in the sub-sample, using the SGPs as candidate predictors. Nine SGPs best predicted non-harmful gambling amongst this sub-sample. The behaviour most strongly associated with increased harm was using credit to gamble. The behaviour most strongly associated with reduced harm was 'If I'm not having fun gambling, I stop'. These SGPs form the basis of evidence-based safe gambling guidelines which can be: 1) promoted to consumers, 2) form the basis of self-assessment tests, 3) used to measure safe gambling at a population level, and 4) inform supportive changes to policy and practice. The guidelines advise gamblers to: stop if they are not having fun, keep a household budget, keep a dedicated gambling budget, have a fixed amount they can spend, engage in other leisure activities, avoid gambling when upset or depressed, not use credit for gambling, avoid gambling to make money, and not think that strategies can help you win. These guidelines are a promising initiative to help reduce gambling-related harm.

Role of intrinsic disorder in muscle sarcomeres.

The role and utility of intrinsically disordered regions (IDRs) is reviewed for two groups of sarcomeric proteins, such as members of tropomodulin/leiomodin (Tmod/Lmod) protein homology group and myosin binding protein C (MyBP-C). These two types of sarcomeric proteins represent very different but strongly interdependent functions, being responsible for maintaining structure and operation of the muscle sarcomere. The role of IDRs in the formation of complexes between thin filaments and Tmods/Lmods is discussed within the framework of current understanding of the thin filament length regulation. For MyBP-C, the function of IDRs is discussed in the context of MYBP-C-dependent sarcomere contraction and actomyosin activation.

A Multivariate Evaluation of 25 Proximal and Distal Risk-Factors for Gambling-Related Harm.

Individual differences in the risk of developing gambling-related harm play an important role in theoretical models and practical interventions. The present study attempted comprehensive measurement and evaluation of 25 known risk factors for gambling-related harm in order to determine which factors provided large and unique explanatory power. We surveyed 1650 regular gamblers from an online panel, screening in 1174 (466 male) who passed all checks of attention and response consistency. We evaluated each risk factor based on bivariate correlations with harms, then made separate multivariate evaluations of proximal (e.g., gambling motivations) and distal (e.g., religiosity) risk factors. Almost all bivariate correlations were significant, but most distal factors were not significant in multivariate models. Trait impulsivity was the most important risk factor by a large margin. Excessive consumption, less use of safe gambling practices, and more fallacies were key proximal risks of harm. Many well-known correlates of gambling harm (e.g., youth, lower educational attainment) do not show a direct role in the development of gambling harm when controlling for other factors. The results support theoretical models that emphasise early conditioning and biological vulnerability (manifested through impulsivity). Since maladaptive cognitive and behavioural schemas appear to be more important than motivations (e.g., escape, excitement, ego), interventions may benefit by targeting these proximal drivers of harm.

Structure-based design of small-molecule inhibitors of EBNA1 DNA binding blocks Epstein-Barr virus latent infection and tumor growth.

Epstein-Barr virus (EBV) is a DNA tumor virus responsible for 1 to 2% of human cancers including subtypes of Burkitt's lymphoma, Hodgkin's lymphoma, gastric carcinoma, and nasopharyngeal carcinoma (NPC). Persistent latent infection drives EBV-associated tumorigenesis. Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all EBV-associated tumors and is therefore an attractive target for therapeutic intervention. It is a multifunctional DNA binding protein critical for viral replication, genome maintenance, viral gene expression, and host cell survival. Using a fragment-based approach and x-ray crystallography, we identify a 2,3-disubstituted benzoic acid series that selectively inhibits the DNA binding activity of EBNA1. We characterize these inhibitors biochemically and in cell-based assays, including chromatin immunoprecipitation and DNA replication assays. In addition, we demonstrate the potency of EBNA1 inhibitors to suppress tumor growth in several EBV-dependent xenograft models, including patient-derived xenografts for NPC. These inhibitors selectively block EBV gene transcription and alter the cellular transforming growth factor-ß (TGF-ß) signaling pathway in NPC tumor xenografts. These EBNA1-specific inhibitors show favorable pharmacological properties and have the potential to be further developed for the treatment of EBV-associated malignancies.

Shifting faunal baselines through the Quaternary revealed by cave fossils of eastern Australia.

Fossils from caves in the Manning Karst Region, New South Wales, Australia have long been known, but until now have never been assessed for their palaeontological significance. Here, we report on late Quaternary faunal records from eight caves in the region. Extinct Pleistocene megafaunal taxa are recognised in two systems and include giant echidnas (Tachyglossidae gen. et sp. indet.), devils (Sarcophilus laniarius), koalas (Phascolarctos stirtoni), marsupial 'lions' (Thylacoleo carnifex), and kangaroos (Macropus giganteus titan). Some caves contain skeletal remains of introduced exotics such as sheep and dogs, but also provide a rich record of small-bodied native species including Eastern Bettongs (Bettongia gaimardi), Eastern Chestnut Mice (Pseudomys gracilicaudatus), and White-footed Rabbit Rats (Conilurus albipes). These endemics are either locally extirpated or have suffered total extinction in the historic period. Their skeletal and dental remains were recorded as unmineralised surface specimens in the caves, indicating that they are recent in age. Extant populations have never been recorded locally, thus, their probable loss from the region in historic times had gone unnoticed in the absence of palaeo-evidence. Our findings suggest that the supposed habitat tolerances of such species have been substantially underestimated. It is highly likely that modern populations have suffered niche contraction since the time of European colonisation of the continent. The local extirpations of several species of digging mammal has likely led to decreased functionality of the current ecosystem.

Dipeptide Prodrugs of the Glutamate Modulator Riluzole.

We have previously reported a prodrug strategy based on the marketed drug riluzole (2-amino-6-trifluoromethoxybenzothiazole), associated with the benefits of lower patient to patient variability of exposure and potentially once daily oral dosing, as opposed to the large variance and twice daily dosing, which is currently observed with the parent drug. Riluzole is a glutamate modulator that is currently approved by the US FDA to treat amyotrophic lateral sclerosis (ALS). Riluzole also strongly suppresses the growth of melanoma cells that express the type 1 metabotropic glutamate receptor (GRM1, mGluR1). Riluzole is a substrate for the variably expressed liver isozyme CYP1A2, which has been shown to contribute to the variance in exposure of riluzole in humans upon oral administration. In addition, an elevated Cmax following oral administration is a probable cause of increased liver enzyme levels in some patients. In order to mitigate these issues, a series of natural and unnatural dipeptide prodrugs of riluzole were prepared as products that bear lower first-pass hepatic clearance. The prodrugs were evaluated for their ability to produce riluzole in serum while remaining intact prior to absorption from the GI tract, characteristic of a type IIB prodrug. Here, we describe dipeptide conjugates of riluzole and report that the t-Bu-Gly-Sar-riluzole analog FC-3423 (6) is absorbed well and converts to riluzole in rats and mice in a regular and well-defined manner. FC-3423 strongly suppress tumor cell growth in mouse xenograft models of melanoma at a molar dose 10-fold less than that of riluzole itself.

Chemokines and 'bath salts': CXCR4 receptor antagonist reduces rewarding and locomotor-stimulant effects of the designer cathinone MDPV in rats.

BACKGROUND AND PURPOSE: Little is known about how chemokine systems influence the behavioral effects of designer cathinones and psychostimulants. The chemokine CXCL12 and its principal receptor target, CXCR4, are of particular interest because CXCR4 activation enhances mesolimbic dopamine output that facilitates psychostimulant reward, reinforcement, and locomotor activation. Repeated cocaine enhances CXCL12 gene expression in the midbrain and produces conditioned place preference (CPP) that is inhibited by a CXCR4 antagonist. Yet, interactions between chemokines and synthetic cathinones remain elusive. METHODS: We tested the hypothesis that an FDA-approved CXCR4 antagonist (AMD3100) inhibits MDPV-induced reward, locomotor activation and positive affective state in rats using a triad of behavioral assays (CPP, open field, and 50-kHz ultrasonic vocalizations [USVs]). KEY RESULTS: AMD3100 (1, 2.5, 5, 10 mg/kg, ip) significantly reduced MDPV (2 mg/kg, ip)-evoked hyper-locomotion in a dose-related manner. AMD3100 (1, 5, 10 mg/kg) administered during CPP conditioning caused a significant, dose-dependent reduction of MDPV (2 mg/kg x 4 days) place preference. MDPV injection elicited significantly greater 50 kHz USVs in vehicle-pretreated rats but not in AMD3100-pretreated rats. CONCLUSION AND IMPLICATION: A CXCR4 antagonist reduced the rewarding and locomotor-activating effects of MDPV. Our results identify the existence of chemokine/cathinone interactions and suggest the rewarding and stimulant effects of MDPV, similar to cocaine, require an active CXCL12/CXCR4 system.