A subfraction obtained from the venom of the tarantula Poecilotheria regalis contains inhibitor cystine knot peptides and induces relaxation of rat aorta by inhibiting L-type voltage-gated calcium channels.

Venoms from tarantulas contain low molecular weight vasodilatory compounds whose biological action is conceived as part of the envenomation strategy due to its propagative effects. However, some properties of venom-induced vasodilation do not match those described by such compounds, suggesting that other toxins may cooperate with these ones to produce the observed biological effect. Owing to the distribution and function of voltage-gated ion channels in blood vessels, disulfide-rich peptides isolated from venoms of tarantulas could be conceived into potential vasodilatory compounds. However, only two peptides isolated from spider venoms have been investigated so far. This study describes for the first time a subfraction containing inhibitor cystine knot peptides, PrFr-I, obtained from the venom of the tarantula Poecilotheria regalis. This subfraction induced sustained vasodilation in rat aortic rings independent of vascular endothelium and endothelial ion channels. Furthermore, PrFr-I decreased calcium-induced contraction of rat aortic segments and reduced extracellular calcium influx to chromaffin cells by the blockade of L-type voltage-gated calcium channels. This mechanism was unrelated to the activation of potassium channels from vascular smooth muscle, since vasodilation was not affected in the presence of TEA, and PrFr-I did not modify the conductance of the voltage-gated potassium channel Kv10.1. This work proposes a new envenomating function of peptides from venoms of tarantulas, and establishes a new mechanism for venom-induced vasodilation.

Global Pattern of kdr-Type Alleles in Musca domestica (L.).

Purpose of Review: Houseflies, Musca domestica L., are an important sanitary pest that affects human and domesticated animals. They are mechanical carriers of more than 100 human and animal diseases including protozoan, bacterial, helminthic, and viral infections. Recently, it was demonstrated that houseflies acquired, harbored, and transmitted SARS-CoV-2 (COVID-19) for up to 1 day post-exposure. The most widely used control strategy relays on the application of pyrethroid insecticides due to their effectiveness, low mammalian toxicity, low cost, and environmental safety. The main mechanism of action of pyrethroids is to exert their toxic effects through affecting the voltage-sensitive sodium channel (VSSC) modifying the transmission of the nerve impulse and leading to the death of the insects. Target site insensitivity of the VSSC is due to the presence of single nuclear polymorphisms (SNPs) named knockdown mutations (kdr). In this review, we synthetize recent data on the type and distribution of these mutations globally. Recent Findings: Housefly resistance is reported in several countries. Increased applications of pyrethroids to control housefly populations led to the emergence of multiple evolutionary origins of resistance determined by five amino acid substitutions or specific mutations in the VSSC: kdr (L1014F), kdr-his (L1014H), super-kdr (M918T + L1014F), type N (D600N + M918T + L1014F), and 1B (T929I + L1014F). According to the global map obtained, high levels of resistance to pyrethroids are associated with the L1014F mutation found mostly in North America, Europe, and Asia, while the super-kdr mutation was mostly found in the American continent. The level of protection conferred by these alleles against pyrethroids was generally kdr-his < kdr < Type N ≤ super-kdr ≤ 1B. The relative fitness of the alleles under laboratory conditions was susceptible ≅ kdr-his > kdr > super-kdr suggesting that the fitness cost of an allele was relative to the presence of other alleles in a population and that the reversion of resistance in a free insecticide environment might be quite variable from one region to another. Summary: An adequate integrated pest management program should consider monitoring susceptibility to pyrethroids to detect early levels of resistance and predict the spread and evolution of resistant phenotypes and genotypes. From this review, the pyrethroid resistance status of housefly population was determined in very few countries and has evolved independently in different areas of the world affecting chemical control programs.

Modulation of hNav by Tst1, a ß-toxin purified from the scorpion Tityus stigmurus.

Scorpion venoms are known as a rich mixture of components, including peptides that can interact with different ion channels, particularly voltage-gated potassium channels (Kv), calcium channels (Cav) and sodium channels (Nav), essential membrane proteins for various physiological functions in organisms. The present work aimed to characterize the modulation of hNa+-channels by Tst1, a peptide purified from the venom of Tityus stigmurus, using whole-cell patch clamp. Tst1 at 100 nM provoked current inhibition in Nav 1.3 (85.23%), Nav 1.2 (67.26%) and Nav 1.4 (63.43%), while Nav 1.1, 1.5, 1.6, and 1.7 were not significantly affected. Tst1 also shifted the voltage of activation and steady-state inactivation to more hyperpolarized states and altered the recovery from inactivation of the channels, reducing repetitive firing of cells, which was more effective in Nav 1.3. Tst1 also demonstrated that the effect on Nav 1.3 is dose-dependent, with an IC50 of 8.79 nM. Taken together, these results confirmed that Tst1, the first Tityus stigmurus NaScTx assayed in relation to Nav channels, is a ß-toxin, as was previously suggested due to its amino acid sequence. KEY CONTRIBUTION: First ß-toxin purified from the venom of Tityus stigmurus scorpion broadly characterized in hNa+-channels.

Cribado virtual de metabolitos secundarios de Origanum vulgare y mejorana frente a NaV1. 7 como anestésicos promisorios asociados a la vía neurosensitiva auditiva / Virtual screening of secondary metabolites of Origanum vulgare and marjoram against NaV1. 7 as promising anesthetics associated with the auditory neurosensory pathway

Introducción: en general, el orégano es una planta medicinal usada en los pueblos de la costa Caribe colombiana para tratar afecciones del aparato respiratorio y el oído externo debido a su potencial efecto antiinflamatorio, analgésico y antiséptico; sin embargo, esto no se ha validado mediante ensayos clínicos. Objetivo: realizar un cribado virtual basado en el acoplamiento molecular de metabolitos secundarios identificados en el Origanum vulgare y mejorana frente al receptor Nav1.7 para evaluar el potencial efecto anestésico a nivel del oído externo. Método: el presente es un estudio in silico con un enfoque de cribado virtual por acoplamiento molecular, para lo cual se usó el software AutoDock Vina y para las predicciones farmacocinéticas se usó la herramienta en línea SwissADME del Swiss Institute of Bioinformatics (http://www.sib.swiss). Adicionalmente, se evaluó la toxicidad in silico de las moléculas utilizando el servidor GUSAR-Online. Resultados: de las 99 moléculas que fueron evaluadas por acoplamiento molecular se evidenció que las mayores afinidades con respecto al canal Nav1.7 fueron el ácido clorogénico, la rutina, la luteolina, el luteosido y la apigenina, donde se presentaron energías de afinidad con el sitio de unión en el poro central del canal a valores entre -5,40 ± 0,00 a -5,57 ± 0,06 kcal/mol; de estos, de acuerdo con el análisis ADMET y GUSAR, solo el ácido clorogénico, la luteolina y la apigenina son buenos candidatos potenciales para fármacos anestésicos ya que cumplen con las cinco reglas de Lipinski. Conclusión: con base en los estudios fitoquímicos de O. vulgare y mejorana que han reportado los metabolitos secundarios presentes en los extractos de estas plantas, se evidenció en el presente estudio in silico su acoplamiento al canal Nav1.7 expresado en la vía neurosensitiva del oído. Se demostró, además, que el ácido clorogénico, la luteolina y la apigenina podrían ser potenciales fármacos anestésicos locales para las afecciones del oído.

Introduction: In general, oregano is a medicinal plant used in rural areas of the colombian Caribbean coast to treat conditions of the respiratory system and external ear due to its potential anti-inflammatory, analgesic and antiseptic effect, however, it has not been validated through clinical trials. Objective: To carry out a virtual screening based on molecular coupling of secondary metabolites identified in Origanum vulgare and marjoram against the Nav1.7 receptor to evaluate the potential anesthetic effect at the level of the external ear. Method: This is an in-silico study with a virtual molecular docking screening approach, for which the AutoDock Vina software was used and the Swiss Institute of Bioinformatics (http://www.sib.swiss) online tool SwissADME was used for pharmacokinetic predictions. Additionally, the in-silico toxicity of the molecules was evaluated using the GUSAR-Online server. Results: Of the 99 molecules that were evaluated by molecular coupling, it was shown that the highest affinities with respect to the Nav1.7 channel were chlorogenic acid, rutin, luteolin, luteoside and apigenin, where affinity energies were presented with the binding site in the central pore of the channel at values between -5.40 ± 0.00 to -5.57 ± 0.06 kcal/mol, which according to the ADMET and GUSAR analysis, only chlorogenic acid, luteolin and apigenin are good potential candidates for anesthetic drugs complying with the 5 rules of Lipinsky. Conclusion: Based on the phytochemical studies of O. vulgare and marjoram that have reported the secondary metabolites present in the extracts of these plants, their coupling to the Nav1.7 channel expressed in the neurosensory pathway of the ear was evidenced in this in-silico study. It was also shown that chlorogenic acid, luteolin and apigenin could be potential local anesthetic drugs for ear conditions.

Naview: A d3.js Based JavaScript Library for Drawing and Annotating Voltage-Gated Sodium Channels Membrane Diagrams.

Voltage-gated sodium channels (Nav) are membrane proteins essential to initiating and propagating action potential in neurons and other excitable cells. For a given organism there are often multiple, specialized sodium channels found in different tissues, whose mutations can cause deleterious effects observed in numerous diseases. Consequently, there is high medical and pharmacological interest in these proteins. Scientific literature often uses membrane diagrams to depict important patterns in these channels including the six transmembrane segments (S1-S6) present in four different homologous domains (D1-D4), the S4 voltage sensors, the pore-lining residue segments and the ion selectivity filter residues, glycosylation and phosphorylation residues, toxin binding sites and the inactivation loop, among others. Most of these diagrams are illustrated either digitally or by hand and programs specifically dedicated to the interactive and data-friendly generation of such visualizations are scarce or non-existing. This paper describes Naview, an open-source javascript visualization compatible with modern web browsers for the dynamic drawing and annotation of voltage-gated sodium channels membrane diagrams based on the D3.js library. By using a graphical user interface and combining user-defined annotations with optional UniProt code as inputs, Naview allows the creation and customization of membrane diagrams. In this interface, a user can also map and display important sodium channel properties, residues, regions and their relationships through symbols, colors, and edge connections. Such features can facilitate data exploration and provide fast, high-quality publication-ready graphics for this highly active area of research.

Gastrointestinal Symptoms and Channelopathy-Associated Epilepsy.

OBJECTIVE: To determine the prevalence of and identify factors associated with gastrointestinal (GI) symptoms among children with channelopathy-associated developmental and epileptic encephalopathy (DEE). STUDY DESIGN: Parents of 168 children with DEEs linked to SCN1A (n = 59), KCNB1 (n = 31), or KCNQ2 (n = 78) completed online CLIRINX surveys about their children's GI symptoms. Our analysis examined the prevalence, frequency, and severity of GI symptoms, as well as DEE type, functional mobility, feeding difficulties, ketogenic diet, antiseizure medication, autism spectrum disorder (ASD), and seizures. Statistical analyses included the χ2 test, Wilcoxon rank-sum analysis, and multiple logistic regression. RESULTS: GI symptoms were reported in 92 of 168 patients (55%), among whom 63 of 86 (73%) reported daily or weekly symptoms, 29 of 92 (32%) had frequent or serious discomfort, and 13 of 91 (14%) had frequent or serious appetite disturbances as a result. The prevalence of GI symptoms varied across DEE cohorts with 44% of SCN1A-DEE patients, 35% of KCNB1-DEE patients, and 71% of KCNQ2-DEE patients reporting GI symptoms in the previous month. After adjustment for DEE type, current use of ketogenic diet (6% reported), and gastrostomy tube (13% reported) were both associated with GI symptoms in a statistically, but not clinically, significant manner (P < .05). Patient age, functional mobility, feeding difficulties, ASD, and seizures were not clearly associated with GI symptoms. Overall, no individual antiseizure medication was significantly associated with GI symptoms across all DEE cohorts. CONCLUSIONS: GI symptoms are common and frequently severe in patients with DEE.

Small cyclic sodium channel inhibitors.

Voltage-gated sodium (NaV) channels play crucial roles in a range of (patho)physiological processes. Much interest has arisen within the pharmaceutical industry to pursue these channels as analgesic targets following overwhelming evidence that NaV channel subtypes NaV1.7-NaV1.9 are involved in nociception. More recently, NaV1.1, NaV1.3 and NaV1.6 have also been identified to be involved in pain pathways. Venom-derived disulfide-rich peptide toxins, isolated from spiders and cone snails, have been used extensively as probes to investigate these channels and have attracted much interest as drug leads. However, few peptide-based leads have made it as drugs due to unfavourable physiochemical attributes including poor in vivo pharmacokinetics and limited oral bioavailability. The present work aims to bridge the gap in the development pipeline between drug leads and drug candidates by downsizing these larger venom-derived NaV inhibitors into smaller, more "drug-like" molecules. Here, we use molecular engineering of small cyclic peptides to aid in the determination of what drives subtype selectivity and molecular interactions of these downsized inhibitors across NaV subtypes. We designed a series of small, stable and novel NaV probes displaying NaV subtype selectivity and potency in vitro coupled with potent in vivo analgesic activity, involving yet to be elucidated analgesic pathways in addition to NaV subtype modulation.

Voltage-Dependent Sodium Channel Blocker Anticonvulsants: An Approach to the Structure-Activity Relationship.

BACKGROUND: Anticonvulsants are drugs used in the treatment of seizures; their pharmacology includes promoters of brain inhibition and inhibitors of brain activity. Of the latter, voltagedependent sodium channel blockers (VGSCB) are the most widely used in therapeutics. OBJECTIVE: The study aimed at proposing the structural requirements of VGSC blockers through a quantitative structure-activity relationship analysis of drugs with proven activity. METHODS: IC50 values of anticonvulsant drugs on VGSCs were considered under similar experimental conditions; some physicochemical properties of the molecules that were correlated with their biological activity were determined in silico. RESULTS: Relationships were observed between the dipole moment, pKa, EHOMO, and MR with the biological activity, which infers that between greater polarity and basicity of the drugs, their activity as blockers will increase. Subsequently, the structural subclassification of the drugs was carried out, based on the urea derivation, the groups of which were: Group 1 (direct and bioisostere derivatives) and Group 2 (homologue and vinylogue derivatives of urea). CONCLUSION: The biological activity depends on the polarity, basicity, and electronic density of the drugs. The derivation of urea is essential, which is present in its original substituted form or a bioisosteric form. Urea can be in the form of a homologue or a vinylogue at the ends of the molecule. Aromatic substitution to the urea portion is necessary.

Genetic study in Aedes (Stegomyia) aegypti (Linnaeus, 1762) from Londrina (Paraná State, Brazil): an approach to population structure and pyrethroid resistance

Abstract Londrina is the fourth most populous city in southern Brazil. Its subtropical weather with rain in all seasons, as well as its high population density, make the city perfect for the Aedes aegypti (Linnaeus, 1762) life cycle. Over the last few years, Londrina presented high infestation indexes and was one of the cities with the most reported cases of dengue. Uncontrolled use of synthetic insecticides may influence the mosquito's genetic composition. In this paper, we studied mitochondrial DNA and kdr mutations in Aedes aegypti. The analysis of the ND4 gene in 330 specimens showed the presence of 27 haplotypes. The pyrethroid resistance alleles (kdr) evaluated are present in the collected populations, with a 50% frequency of the Val1016Ile and 48% of the Phe1534Cys mutations. Such analysis of the mutations in the populations collected at the State University of Londrina's campus - a microenvironment that differs from the rest of the city - showed frequencies of 57% and 62%, respectively. The low gene flow observed, Nm = 0.11 and Nm = 0.10, along with the elevated differentiation, Fst = 0.19 and Fst = 0.18, among populations suggest an influence of genetic drift. The strong presence of resistance alleles kdr in the city is evident, which demonstrates that even with the interruption of the use of pyrethroids by the National Dengue Control Program, resistance may be maintained due to domestic use. Thus, the results have shown the need for genetic monitoring, alongside other entomological surveillance monitoring tools, to create strategies of mosquito control.

Entomotoxicity of jaburetox: revisiting the neurotoxic mechanisms in insects.

Ureases are metalloenzymes that hydrolyze urea to ammonia and carbamate. The main urease isoforms present in the seeds of Canavalia ensiformis (jack bean urease - JBU and canatoxin) exert a variety of biological activities. The insecticidal activity of JBU is mediated, at least in part, by jaburetox (Jbtx), a recombinant peptide derived from the JBU amino acid sequence. In this article, we review the neurotoxicity of Jbtx in insects. The insecticidal activity of Jbtx has been investigated in a variety of insect orders and species, including Blattodea (the cockroaches Blatella germânica, Nauphoeta cinerea, Periplaneta americana e Phoetalia pallida), Bruchidae (Callosobruchus maculatus - cowpea weevil), Diptera (Aedes aegypti - mosquito), Hemiptera (Dysdercus peruvianus - cotton stainer bug; Oncopeltus fasciatus - large milkweed bug, and the kissing bugs Rhodnius prolixus and Triatoma infestans), Lepidoptera (Spodoptera frugiperda - fall army worm) and Orthoptera (Locusta migratoria - locust). In N. cinerea, the injection of Jbtx induces marked alteration of locomotor and grooming behavior, whereas in T. infestans Jbtx causes leg paralysis, an extension of the proboscis and abnormal antennal movements. Electromyographical analysis showed that Jbtx causes complete neuromuscular blockade in P. pallida. The same treatment in N. cinerea and L. migratoria causes a decrease in the action potential firing rate. Jbtx forms membrane pore-channels compatible with cations in bilipid membranes. A study using B. germanica voltage-gated sodium (Nav1.1) channels that were heterologously expressed in Xenopus laevis oocytes correlated the entomotoxicity of Jbtx with the activation of these channels. Taken together, these findings demonstrate the potential of this peptide as a natural pesticide.

Evaluación de primers reportados para detección de KDR en especies de Anopheles colectadas en los departamentos de Caaguazú y Alto Paraná en Paraguay / Evaluation of primers reported for KDR detection in Anopheles species collected in the departments of Caaguazú and Alto Paraná in Paraguay

Las mutaciones KDR en el gen del canal del sodio (VGSC) han sido ya detectadas en al menos 13 especies de mosquitos Anopheles en su mayoría especies de África, pero aún resta por determinar los cebadores específicos para la detección en especies de Latinoamérica. En nuestro país la especie Anopheles darlingi es el vector principal de la malaria, y el A. albitarsis, el vector secundario. Se emplearon muestras de mosquitos Anoheles de las especies A. strodei, A. albitarsis, A. fluminensis, A. evansae, A. nuneztovari, A. nyssorhynchela lutzi y A. oswaldoi capturadas en los departamentos de Caaguazú y Alto Paraná en Paraguay. Para la amplificación y secuenciación se usaron cebadores reportados para el gen VGSC de A. albimanus en Guatemala, que resultaron ser específicos solo para la especie A. strodei. La secuencia revela el codón TTA que codifica para una Leucina como la secuencia TTG, reportada para la versión susceptible en la posición L1014. El fragmento amplificado es de aproximadamente 225 pares de bases. A nuestro entender, esta es la primera caracterización del gen VGSC en mosquitos Anopheles del Paraguay y para la especie A. strodei

KDR mutations in the sodium channel gene (VGSC) have already been detected in at least 13 species of Anopheles mosquitoes, mostly African species, but the molecular techniques for detection in Latin American species have yet to be determined. In our country, Anopheles darlingi species is the main vector of Malaria, and A. albitarsis, the secondary vector. We used samples of Anoheles from the species A. strodei, A. albitarsis, A. fluminensis, A. evansae, A. nuneztovari, A. nyssorhynchela lutzi and A. oswaldoi collected at the departments of Caaguazú and Alto Paraná in Paraguay. For the amplification and sequentiation, primers reported for the VGSC gen of A. strodei in Guatemala were used and were specific only for A. strode in this case. The sequence revealed the TTA codon that codifies for a leucine as the TTG sequence, reported for the susceptible version at position L1014. The amplified fragment is approximately 225 base pairs. To our knowledge, this is the first characterization of the VGSC gene in Anopheles mosquitoes in Paraguay and for the species A. strodei

Neurotoxicity of Tityus bahiensis (brown scorpion) venom in sympathetic vas deferens preparations and neuronal cells.

Systemic scorpion envenomation is characterized by massive neurotransmitter release from peripheral nerves mediated primarily by scorpion venoms neurotoxins. Tityus bahiensis is one of the medically most important species in Brazil, but its venom pharmacology, especially regarding to peripheral nervous system, is poorly understood. Here, we evaluated the T. bahiensis venom activity on autonomic (sympathetic) neurotransmission by using a variety of approaches, including vas deferens twitch-tension recordings, electrophysiological measurements (resting membrane potentials, spontaneous excitatory junctional potentials and whole-cell patch-clamp), calcium imaging and histomorphological analysis. Low concentrations of venom (≤ 3 µg/mL) facilitated the electrically stimulated vas deferens contractions without affecting postsynaptic receptors or damaging the smooth muscle cells. Transient TTX-sensitive sustained contractions and resting membrane depolarization were mediated mainly by massive spontaneous ATP release. High venom concentrations (≥ 10 µg/mL) blocked the muscle contractions and induced membrane depolarization. In neuronal cells (ND7-23wt), the venom increased the peak sodium current, modified the current-voltage relationship by left-shifting the Nav-channel activation curve, thereby facilitating the opening of these channels. The venom also caused a time-dependent increase in neuronal calcium influx. These results indicate that the sympathetic hyperstimulation observed in systemic envenomation is presynaptically driven, probably through the interaction of α- and ß-toxins with neuronal sodium channels.

Epilepsia refractaria con convulsiones febriles plus en niños con mutaciones de la subunidad alfa de canales de sodio (SCN1A): un reporte de caso / Refractory epilepsy with febrile seizures in a child with sodium channel alpha subunit mutation (SCN1A): A case report

RESUMEN Se presenta caso de una niña de 10 años con cuadro de epilepsia mioclónica juvenil severa en la infancia, que a los 6 meses de edad debutó con crisis tónico-clónicas generalizadas (TCG) luego de la administración de una dosis de vacuna DPT, con posteriores crisis TCG, mioclónicas y múltiples episodios de estado epiléptico refractarios a fármacos antiepilépticos (FAE) de primera y segunda línea durante los primeros 5 años. Las crisis se asociaron a retraso global en el desarrollo luego del primer episodio. Durante la evolución se realizaron estudios que incluyen resonancia magnética cerebral que fue normal y tomografía por emisión de positrones (PET-CT) que evidenció alteraciones en el metabolismo en región temporal izquierda, además de estudios para inmunodeficiencias y trombofilias sin alteraciones. Los electroencefalogramas iniciales fueron normales, pero video electroencefalograma de 12 horas mostró actividad irritativa en la región central con diseminación bilateral. Los estudios genéticos identificaron una mutación en el marco de lectura de tipo "frameshift" del gen SCN1A mediante secuenciación de la región codificante. Luego de los primeros años de vida, la paciente presenta, atípicamente, remisión progresiva de las crisis con posterior desmonte de FAE y mejoría del neuro-desarrollo en el proceso interdisciplinario de rehabilitación.

SUMMARY We report the case of a 10-year-old female with a history of severe myoclonic epilepsy of infancy who presents with generalized tonic-clonic (GTC) seizures at 6 months of age after administration of a DPT vaccine, who then begins to present frequent and severe GTC seizures, myoclonic seizures and multiple refractory status epilepticus poorly controlled with first and second line anti-epileptic drugs (AEDs). This was accompanied by development delay. Studies performed on the patient included brain MRI which was normal, immunodeficiency and trombophilic studies which were normal and electroencephalographs: studies (EEG) that were at first mostly normal. The most significant findings were seen during a 12-hour video-EEG which reported epileptogenic activity in central region with bilateral dissemination and a PET-CT that showed metabolism alterations in the left temporal region. Due to this presentation a channelopathy was suspected and a coding region sequentiation study was performed which identified a frameshift mutation of the SCN1A gene confirming the diagnosis. Atipically, after 5 years the patient begins to present a favorable evolution with significant seizure remission even allowing the progressive weaning of AEDs and a remarkable stalemate of developmental delay after interdisciplinary rehabilitation process was started.

Co-occurrence of kdr Mutations V1016I and F1534C and Its Association With Phenotypic Resistance to Pyrethroids in Aedes aegypti (Diptera: Culicidae) Populations From Costa Rica.

Aedes aegypti (Linnaeus, 1762) is considered the most important mosquito vector species for several arboviruses (e.g., dengue, chikungunya, Zika) in Costa Rica. The primary strategy for the control and prevention of Aedes-borne diseases relies on insecticide-based vector control. However, the emergence of insecticide resistance in the mosquito populations presents a significant threat to these prevention actions. The characterization of the mechanisms driving the insecticide resistance in Ae. aegypti is vital for decision making in vector control programs. Therefore, we analyzed the voltage-gated sodium channel (VGSC) gene for the presence of the V1016I and F1534C kdr mutations in Ae. aegypti populations from Puntarenas and Limon provinces, Costa Rica. The CDC bottle bioassays showed that both Costa Rican Ae. aegypti populations were resistant to permethrin and deltamethrin. In the case of kdr genotyping, results revealed the co-occurrence of V1016I and F1534C mutations in permethrin and deltamethrin-resistant populations, as well as the fixation of the 1534C allele. A strong association between these mutations and permethrin and deltamethrin resistance was found in Puntarenas. Limon did not show this association; however, our results indicate that the Limon population analyzed is not under the same selective pressure as Puntarenas for the VGSC gene. Therefore, our findings make an urgent call to expand the knowledge about the insecticide resistance status and mechanisms in the Costa Rican populations of Ae. aegypti, which must be a priority to develop an effective resistance management plan.

Neurotoxicity of Tityus bahiensis (brown scorpion) venom in sympathetic vas deferens preparations and neuronal cells

Systemic scorpion envenomation is characterized by massive neurotransmitter release from peripheral nerves mediated primarily by scorpion venoms neurotoxins. Tityus bahiensis is one of the medically most important species in Brazil, but its venom pharmacology, especially regarding to peripheral nervous system, is poorly understood. Here, we evaluated the T. bahiensis venom activity on autonomic (sympathetic) neurotransmission by using a variety of approaches, including vas deferens twitch-tension recordings, electrophysiological measurements (resting membrane potentials, spontaneous excitatory junctional potentials and whole-cell patch-clamp), calcium imaging and histomorphological analysis. Low concentrations of venom (= 3 µg/mL) facilitated the electrically stimulated vas deferens contractions without affecting postsynaptic receptors or damaging the smooth muscle cells. Transient TTX-sensitive sustained contractions and resting membrane depolarization were mediated mainly by massive spontaneous ATP release. High venom concentrations (= 10 µg/mL) blocked the muscle contractions and induced membrane depolarization. In neuronal cells (ND7-23wt), the venom increased the peak sodium current, modified the current-voltage relationship by left-shifting the Nav-channel activation curve, thereby facilitating the opening of these channels. The venom also caused a time-dependent increase in neuronal calcium influx. These results indicate that the sympathetic hyperstimulation observed in systemic envenomation is presynaptically driven, probably through the interaction of a- and ß-toxins with neuronal sodium channels.

Development of molecular assays to detect target-site mechanisms associated with insecticide resistance in malaria vectors from Latin America.

BACKGROUND: Malaria remains an important public health problem in Latin America, and the development of insecticide resistance in malaria vectors poses a major threat to malaria elimination efforts. Monitoring of insecticide susceptibility and the determination of the mechanisms involved in insecticide resistance are needed to effectively guide the deployment of appropriate vector control measures. Here, molecular assays have been developed to screen for mutations associated with insecticide resistance on the voltage-gated sodium channel (VGSC) and acetylcholinesterase-1 (Ace-1) genes in four malaria vectors from Latin America. METHODS: Degenerate primers were designed to amplify a partial fragment on the VGSC and Ace-1 genes. Wild-caught individuals for Anopheles albimanus (also historical samples and individuals from a laboratory strain), Anopheles darlingi, Anopheles vestitipennis and Anopheles pseudopunctipennis were used to optimize the PCR assays. All samples were sequenced to validate the PCR results and DNA alignments were constructed for each gene using the unique haplotypes observed. RESULTS: Primers designed successfully amplified the VGSC gene in An. albimanus, An. darlingi, An. vestitipennis and An. pseudopunctipennis, and the Ace-1 gene in both An. albimanus and An. darlingi. DNA sequencing revealed that compared with Anopheles gambiae, there were a total of 29, 28, 21 and 24 single nucleotide polymorphisms (SNPs) on the VGSC gene for An. albimanus (308 bp), An. darlingi (311 bp), An. pseudopunctipennis (263 bp) and An. vestitipennis (254 bp), respectively. On the 459 bp fragment of the Ace-1 gene, a total of 70 SNPs were detected in An. darlingi and 59 SNPs were detected in An. albimanus compared with An. gambiae. The SNPs detected on the VGSC gene were all synonymous. On the Ace-1 gene, non-synonymous substitutions were identified on three different codons. All species showed the homozygous wild-type kdr allele (coding for leucine) at codon 995 (formerly reported as codon 1014) on the VGSC gene, but one sample was heterozygous at codon 280 (formerly reported as codon 119) on the Ace-1 gene, coding for both the resistant (serine) and susceptible (glycine) amino acids. CONCLUSIONS: New molecular assays to amplify and screen the regions of the VGSC and Ace-1 genes associated with insecticide resistance are reported for An. albimanus, An. darlingi, An. vestitipennis, and An. pseudopunctipennis. The development of these PCR assays presents an important advance in the analysis of target-site resistance in malaria vectors in the Americas, and will further facilitate the characterization of insecticide resistance mechanisms in these species.

Contribution of voltage-gated sodium channel ß-subunits to cervical cancer cells metastatic behavior.

BACKGROUND: Voltage-gated sodium (NaV) channels are heteromeric proteins consisting of a single pore forming α-subunit associated with one or two auxiliary ß-subunits. These channels are classically known for being responsible of action potential generation and propagation in excitable cells; but lately they have been reported as widely expressed and regulated in several human cancer types. We have previously demonstrated the overexpression of NaV1.6 channel in cervical cancer (CeCa) biopsies and primary cultures, and its contribution to cell migration and invasiveness. Here, we investigated the expression of NaV channels ß-subunits (NaVßs) in the CeCa cell lines HeLa, SiHa and CaSki, and determined their contribution to cell proliferation, migration and invasiveness. METHODS: We assessed the expression of NaVßs in CeCa cell lines by performing RT-PCR and western blotting experiments. We also evaluated CeCa cell lines proliferation, migration, and invasion by in vitro assays, both in basal conditions and after inducing changes in NaVßs levels by transfecting specific cDNAs or siRNAs. The potential role of NaVßs in modulating the expression of NaV α-subunits in the plasma membrane of CeCa cells was examined by the patch-clamp whole-cell technique. Furthermore, we investigated the role of NaVß1 on cell cycle in SiHa cells by flow cytometry. RESULTS: We found that the four NaVßs are expressed in the three CeCa cell lines, even in the absence of functional NaV α-subunit expression in the plasma membrane. Functional in vitro assays showed differential roles for NaVß1 and NaVß4, the latter as a cell invasiveness repressor and the former as a migration abolisher in CeCa cells. In silico analysis of NaVß4 expression in cervical tissues corroborated the downregulation of this protein expression in CeCa vs normal cervix, supporting the evidence of NaVß4's role as a cell invasiveness repressor. CONCLUSIONS: Our results contribute to the recent conception about NaVßs as multifunctional proteins involved in cell processes like ion channel regulation, cell adhesion and motility, and even in metastatic cell behaviors. These non-canonical functions of NaVßs are independent of the presence of functional NaV α-subunits in the plasma membrane and might represent a new therapeutic target for the treatment of cervical cancer.

Presence of the point mutations Val1016Gly in the voltage-gated sodium channel detected in a single mosquito from Panama.

BACKGROUND: In Panama, arboviroses such as dengue fever, and more recently chikungunya fever and Zika disease, are transmitted by Aedes aegypti and Aedes albopictus. Their control is based on the elimination of breeding sites and fogging with pyrethroid insecticides. However, one of the significant issues derived from the prolonged use of pyrethroid insecticide is the development of resistance mechanisms, such as knockdown resistance or kdr. The objective of this study was to evaluate the presence of kdr mutations in a partial region of the VGSC gene in samples of wild-caught Aedes mosquitoes from different locations of the Metropolitan Region of Panama. RESULTS: Based on the analysis of 194 sequences of the VGSC gene, two kdr mutations (Ile1011Met and Val1016Gly) were detected in a specimen of Ae. aegypti. The frequency of kdr mutations in the evaluated samples of Ae. aegypti was 0.01. CONCLUSIONS: This study provides evidence for a low frequency of kdr mutations in Ae. aegypti populations in Panama. It is possible that these changes have no impact on vector control interventions. To our knowledge, we report, for the first time in America the Val1016Gly mutation documented in Asia. In general terms, this result is highly relevant to the Aedes Control Programme in Panama since it constitutes a feasible approach for the timely detection of resistance as well as for the development of strategies.

First report of the Phe1534Cys kdr mutation in natural populations of Aedes albopictus from Brazil.

BACKGROUND: Knockdown resistance (kdr), caused by alterations in the voltage-gated sodium channel (NaV), is one of the mechanisms responsible for pyrethroid (PY) resistance. In the Asian tiger mosquito, Aedes albopictus, at least four different mutations were described in the IIIS6 NaV segment in populations from Asia, North America and Europe. In contrast, in Aedes aegypti at least 12 non-synonymous mutations have been reported at nine different codons, mostly in the IIS6 and IIIS6 NaV segments. The Phe1534Cys kdr mutation in the IIIS6 NaV segment is the most prevalent in populations of Ae. aegypti worldwide, also found in Ae. albopictus from Singapore. Herein, we investigated the DNA diversity corresponding to the IIS6 and IIIS6 NaV segments in natural populations of Ae. albopictus from Brazil. METHODS: DNA from eight Brazilian Ae. albopictus natural populations were individually extracted and pooled by states of origin, amplified, cloned and sequenced for the corresponding IIS6 and IIIS6 NaV segments. Additionally, samples from each location were individually genotyped by an allelic specific PCR (AS-PCR) approach to obtain the genotypic and allelic frequencies for the 1534 NaV site. RESULTS: No non-synonymous substitutions were observed in the IIS6 sequences. However, the Phe1534Cys kdr mutation was evidenced in the Ae. albopictus NaV IIIS6 segment sequences from Paraná (PR) and Rondônia (RO) states, but not from Mato Grosso (MT) state. The 1534Cys kdr allele varied from 3% (Marilena/PR and Porto Velho/RO) to 10% (Foz do Iguaçu/PR). To our knowledge, this paper reports the first occurrence and provides distribution data of a possible kdr mutation in Ae. albopictus in South America. CONCLUSION: The emergence of a likely kdr mutation in Ae. albopitus natural populations is a signal of alert for vector control measures since PY are the most popular insecticides adopted by residents. Additionally, once the kdr allele is present, its frequency tends to increase faster under exposition to those compounds. Although the Asian tiger mosquito is not incriminated as an important vector of dengue, chikungunya and Zika viruses in South America, its importance in this regard has been extensively discussed since Ae. albopictus is rapidly spreading and can also migrate between sylvatic and urban environments. Therefore, insecticide resistance monitoring initiatives should also be extended to Ae. albopictus in Brazil in order to maintain chemical compounds as an efficient vector control tool when needed.