Identification of multiple odorant receptors essential for pyrethrum repellency in Drosophila melanogaster.

Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. In this study, we found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated our identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-ß-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. Our study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.

Chronology of sodium channel mutations associated with pyrethroid resistance in Aedes aegypti.

Aedes aegypti is the primary mosquito vector of dengue, yellow fever, Zika and chikungunya. Current strategies to control Ae. aegypti rely heavily on insecticide interventions. Pyrethroids are a major class of insecticides used for mosquito control because of their fast acting, highly insecticidal activities and low mammalian toxicity. However, Ae. aegypti populations around the world have begun to develop resistance to pyrethroids. So far, more than a dozen mutations in the sodium channel gene have been reported to be associated with pyrethroid resistance in Ae. aegypti. Co-occurrence of resistance-associated mutations is common in pyrethroid-resistant Ae. aegypti populations. As global use of pyrethroids in mosquito control continues, new pyrethroid-resistant mutations keep emerging. In this microreview, we compile pyrethroid resistance-associated mutations in Ae. aegypti in a chronological order, as they were reported, and summarize findings from functional evaluation of these mutations in an in vitro sodium channel expression system. We hope that the information will be useful for tracing possible evolution of pyrethroid resistance in this important human disease vector, in addition to the development of methods for global monitoring and management of pyrethroid resistance in Ae. aegypti.

Ultrastructure of spinal anterior horn cells in human Niemann-Pick type C (NPC) patient and mouse model of NPC with retroposon insertion in NPC1 genes.

Niemann-Pick disease type C (NPC) is a neurovisceral lipid-storage disease. Although NPC patients show lipid storage in anterior horn cells of the spinal cord, little information is available regarding the electron microscopic analyses of the morphologies of intra-endosomal lipid like-materials in the anterior horn cells of NPC patients. In this study, we elucidated the intra-endosomal ultrastructures in spinal anterior horn cells in an NPC patient, as well as in mutant BALB/c NPC1-/- mice with a retroposon insertion in the NPC1 gene. These morphologies were classified into four types: vesicle, multiple concentric sphere (MCS), membrane, and rose flower. The percentages of the composition in the NPC patient and NPC1-/- mice were: vesicle (55.5% and 14.9%), MCS (15.7% and 3.4%), membrane (23.6% and 57.1%), and rose flower (5.2% and 24.6%), respectively. Formation of the intra-endosomal structures could proceed as follows: (i) a vesicle or MCS buds off the endosome into the lumen; (ii) when a vesicle breaks down, a membrane is formed; and (iii) after an MCS breaks down, a rose flower structure is formed. Our new finding in this study is that ultrastructural morphology is the same between the NPC patient and NPC1-/- mice, although there are differences in the composition.

Genetic landscape of Rett syndrome-like phenotypes revealed by whole exome sequencing.

BACKGROUND: Rett syndrome (RTT) is a characteristic neurological disease presenting with regressive loss of neurodevelopmental milestones. Typical RTT is generally caused by abnormality of methyl-CpG binding protein 2 (MECP2). Our objective to investigate the genetic landscape of MECP2-negative typical/atypical RTT and RTT-like phenotypes using whole exome sequencing (WES). METHODS: We performed WES on 77 MECP2-negative patients either with typical RTT (n=11), atypical RTT (n=22) or RTT-like phenotypes (n=44) incompatible with the RTT criteria. RESULTS: Pathogenic or likely pathogenic single-nucleotide variants in 28 known genes were found in 39 of 77 (50.6%) patients. WES-based CNV analysis revealed pathogenic deletions involving six known genes (including MECP2) in 8 of 77 (10.4%) patients. Overall, diagnostic yield was 47 of 77 (61.0 %). Furthermore, strong candidate variants were found in four novel genes: a de novo variant in each of ATPase H+ transporting V0 subunit A1 (ATP6V0A1), ubiquitin-specific peptidase 8 (USP8) and microtubule-associated serine/threonine kinase 3 (MAST3), as well as biallelic variants in nuclear receptor corepressor 2 (NCOR2). CONCLUSIONS: Our study provides a new landscape including additional genetic variants contributing to RTT-like phenotypes, highlighting the importance of comprehensive genetic analysis.

Molecular basis of selective resistance of the bumblebee BiNav1 sodium channel to tau-fluvalinate.

Insecticides are widely used to control pests in agriculture and insect vectors that transmit human diseases. However, these chemicals can have a negative effect on nontarget, beneficial organisms including bees. Discovery and deployment of selective insecticides is a major mission of modern toxicology and pest management. Pyrethroids exert their toxic action by acting on insect voltage-gated sodium channels. Honeybees and bumblebees are highly sensitive to most pyrethroids, but are resistant to a particular pyrethroid, tau-fluvalinate (τ-FVL). Because of its unique selectivity, τ-FVL is widely used to control not only agricultural pests but also varroa mites, the principal ectoparasite of honeybees. However, the mechanism of bee resistance to τ-FVL largely remains elusive. In this study, we functionally characterized the sodium channel BiNav1-1 from the common eastern bumblebee (Bombus impatiens) in Xenopus oocytes and found that the BiNav1-1 channel is highly sensitive to six commonly used pyrethroids, but resistant to τ-FVL. Phylogenetic and mutational analyses revealed that three residues, which are conserved in sodium channels from 12 bee species, underlie resistance to τ-FVL or sensitivity to the other pyrethroids. Further computer modeling and mutagenesis uncovered four additional residues in the pyrethroid receptor sites that contribute to the unique selectivity of the bumblebee sodium channel to τ-FVL versus other pyrethroids. Our data contribute to understanding a long-standing enigma of selective pyrethroid toxicity in bees and may be used to guide future modification of pyrethroids to achieve highly selective control of pests with minimal effects on nontarget organisms.

Mutational analysis of state-dependent contacts in the pore module of eukaryotic sodium channels.

Voltage-gated sodium channels have residues that change or may change contacts upon gating. Contributions of individual contacts in stability of different states are incompletely understood. Pore-lining inner helices contain exceptionally conserved asparagines in positions i20. Here we explored how mutations in positions i20 and i29 affect electrophysiological properties of insect sodium channels. In repeat interfaces I/IV, III/II and IV/III, alanine substitutions caused positive activation shifts in positions i20 and i29, negative shifts of slow inactivation in positions i20 and positive shifts of slow inactivation in positions i29. The results support the hypothesis on open state inter-repeat H-bonding of residues i20 and i29. The shift magnitudes vary between interfaces, reflecting structural asymmetry of the channels. Mutations in positions i20 of repeats III and IV caused much longer recovery delay from the slow and fast inactivation than other mutations. In repeat IV, alanine substitution of tyrosine i30 rescued positive activation shift of mutation in position i29. Our data suggest that polar residues in positions i29 are involved in stabilization of both the open and slow-inactivated states. Transition between the states may involve switching of H-bonding partners of residues i29 from the conserved asparagines to currently unknown residues.

Evidence for Dual Binding Sites for 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) in Insect Sodium Channels.

1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), the first organochlorine insecticide, and pyrethroid insecticides are sodium channel agonists. Although the use of DDT is banned in most of the world due to its detrimental impact on the ecosystem, indoor residual spraying of DDT is still recommended for malaria control in Africa. Development of resistance to DDT and pyrethroids is a serious global obstacle for managing disease vectors. Mapping DDT binding sites is necessary for understanding mechanisms of resistance and modulation of sodium channels by structurally different ligands. The pioneering model of the housefly sodium channel visualized the first receptor for pyrethroids, PyR1, in the II/III domain interface and suggested that DDT binds within PyR1. Previously, we proposed the second pyrethroid receptor, PyR2, at the I/II domain interface. However, whether DDT binds to both pyrethroid receptor sites remains unknown. Here, using computational docking of DDT into the Kv1.2-based mosquito sodium channel model, we predict that two DDT molecules can bind simultaneously within PyR1 and PyR2. The bulky trichloromethyl group of each DDT molecule fits snugly between four helices in the bent domain interface, whereas two p-chlorophenyl rings extend into two wings of the interface. Model-driven mutagenesis and electrophysiological analysis confirmed these propositions and revealed 10 previously unknown DDT-sensing residues within PyR1 and PyR2. Our study proposes a dual DDT-receptor model and provides a structural background for rational development of new insecticides.

The Receptor Site and Mechanism of Action of Sodium Channel Blocker Insecticides.

Sodium channels are excellent targets of both natural and synthetic insecticides with high insect selectivity. Indoxacarb, its active metabolite DCJW, and metaflumizone (MFZ) belong to a relatively new class of sodium channel blocker insecticides (SCBIs) with a mode of action distinct from all other sodium channel-targeting insecticides, including pyrethroids. Electroneutral SCBIs preferably bind to and trap sodium channels in the inactivated state, a mechanism similar to that of cationic local anesthetics. Previous studies identified several SCBI-sensing residues that face the inner pore of sodium channels. However, the receptor site of SCBIs, their atomic mechanisms, and the cause of selective toxicity of MFZ remain elusive. Here, we have built a homology model of the open-state cockroach sodium channel BgNav1-1a. Our computations predicted that SCBIs bind in the inner pore, interact with a sodium ion at the focus of P1 helices, and extend their aromatic moiety into the III/IV domain interface (fenestration). Using model-driven mutagenesis and electrophysiology, we identified five new SCBI-sensing residues, including insect-specific residues. Our study proposes the first three-dimensional models of channel-bound SCBIs, sheds light on the molecular basis of MFZ selective toxicity, and suggests that a sodium ion located in the inner pore contributes to the receptor site for electroneutral SCBIs.

Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel.

Pyrethroid insecticides are widely used as one of the most effective control measures in the global fight against agricultural arthropod pests and mosquito-borne diseases, including malaria and dengue. They exert toxic effects by altering the function of voltage-gated sodium channels, which are essential for proper electrical signaling in the nervous system. A major threat to the sustained use of pyrethroids for vector control is the emergence of mosquito resistance to pyrethroids worldwide. Here, we report the successful expression of a sodium channel, AaNav1-1, from Aedes aegypti in Xenopus oocytes, and the functional examination of nine sodium channel mutations that are associated with pyrethroid resistance in various Ae. aegypti and Anopheles gambiae populations around the world. Our analysis shows that five of the nine mutations reduce AaNav1-1 sensitivity to pyrethroids. Computer modeling and further mutational analysis revealed a surprising finding: Although two of the five confirmed mutations map to a previously proposed pyrethroid-receptor site in the house fly sodium channel, the other three mutations are mapped to a second receptor site. Discovery of this second putative receptor site provides a dual-receptor paradigm that could explain much of the molecular mechanisms of pyrethroid action and resistance as well as the high selectivity of pyrethroids on insect vs. mammalian sodium channels. Results from this study could impact future prediction and monitoring of pyrethroid resistance in mosquitoes and other arthropod pests and disease vectors.

The developmental characteristics of locomotion and sleep in cyclic vomiting syndrome and the effects of drug therapy.

Objective: To evaluate the characteristics of patients with cyclic vomiting syndrome (CVS) and the efficacy of prophylaxis therapy. Methods: We defined the patients as "CV (+) " if they had multiple neuromuscular diseases and as "CV (−) " if they did not. We compared the two groups according to their background and the type of medications and their effects. We also evaluated their locomotion and sleep pattern. Results: There was no significant difference between CV (+) and CV (−) with regard to the association with migraine. Pharmacotherapy with cyproheptadine and valproate had high success rate for both CV (−) and CV (+). The rate of delayed locomotion development and atonia during non-REM sleep was high in both CV (+) and CV (−). Conclusions: We postulate that the association of serotonergic neurons is one of the important factors in both CV (+) and CV (−).

Rotational Symmetry of Two Pyrethroid Receptor Sites in the Mosquito Sodium Channel.

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Although it is well known that specific mutations in insect sodium channels confer knockdown resistance (kdr) to pyrethroids, the atomic mechanisms of pyrethroid-sodium channel interactions are not clearly understood. Previously, computer modeling and mutational analysis predicted two pyrethroid receptors, pyrethroid receptor site 1 (PyR1) (initial) and pyrethroid receptor site 2 (PyR2), located in the domain interfaces II/III and I/II, respectively. The models differ in ligand orientation and the number of transmembrane helices involved. In this study, we elaborated a revised PyR1 model of the mosquito sodium channel. Computational docking in the Kv1.2-based open channel model yielded a complex in which a pyrethroid (deltamethrin) binds between the linker helix IIL45 and transmembrane helices IIS5, IIS6, and IIIS6 with its dibromoethenyl and diphenylether moieties oriented in the intra- and extracellular directions, respectively. The PyR2 and revised PyR1 models explained recently discovered kdr mutations and predicted new deltamethrin-channel contacts. Further model-driven mutagenesis identified seven new pyrethroid-sensing residues, three in the revised PyR1 and four in PyR2. Our data support the following conclusions: 1) each pyrethroid receptor is formed by a linker-helix L45 and three transmembrane helices (S5 and two S6s); 2) IIS6 contains four residues that contribute to PyR1 and another four to PyR2; 3) seven pairs of pyrethroid-sensing residues are located in symmetric positions within PyR1 and PyR2; and 4) pyrethroids bind to PyR1 and PyR2 in similar orientations, penetrating deeply into the respective domain interfaces. Our study elaborates the dual pyrethroid-receptor sites concept and provides a structural background for rational development of new insecticides.

A mutation in the intracellular loop III/IV of mosquito sodium channel synergizes the effect of mutations in helix IIS6 on pyrethroid resistance.

Activation and inactivation of voltage-gated sodium channels are critical for proper electrical signaling in excitable cells. Pyrethroid insecticides promote activation and inhibit inactivation of sodium channels, resulting in prolonged opening of sodium channels. They preferably bind to the open state of the sodium channel by interacting with two distinct receptor sites, pyrethroid receptor sites PyR1 and PyR2, formed by the interfaces of domains II/III and I/II, respectively. Specific mutations in PyR1 or PyR2 confer pyrethroid resistance in various arthropod pests and disease vectors. Recently, a unique mutation, N(1575)Y, in the cytoplasmic loop linking domains III and IV (LIII/IV) was found to coexist with a PyR2 mutation, L(1014)F in IIS6, in pyrethroid-resistant populations of Anopheles gambiae. To examine the role of this mutation in pyrethroid resistance, N(1575)Y alone or N(1575)Y + L(1014)F were introduced into an Aedes aegypti sodium channel, AaNav1-1, and the mutants were functionally examined in Xenopus oocytes. N(1575)Y did not alter AaNav1-1 sensitivity to pyrethroids. However, the N(1575)Y + L(1014)F double mutant was more resistant to pyrethroids than the L(1014)F mutant channel. Further mutational analysis showed that N(1575)Y could also synergize the effect of L(1014)S/W, but not L(1014)G or other pyrethroid-resistant mutations in IS6 or IIS6. Computer modeling predicts that N(1575)Y allosterically alters PyR2 via a small shift of IIS6. Our findings provide the molecular basis for the coexistence of N(1575)Y with L(1014)F in pyrethroid resistance, and suggest an allosteric interaction between IIS6 and LIII/IV in the sodium channel.

Genetics and pathophysiology of primary dystonia with special emphasis on DYT1 and DYT5.

DYT1 and DYT5 are early-onset dominant inherited dystonias. DYT1 is caused by mutations of the TOR1A gene, located on 9q34, which causes dysfunction of the D1 direct pathway or the indirect pathway. Dysfunction of the former causes postural-type and segmental dystonia; the latter causes action-type dystonia. In families with action-type dystonia, there are cases with focal and segmental dystonia. Ages of onset of postural-type dystonia are around 6 years, and 8 to 10 years in cases of action-type dystonia. Focal and segmental dystonia develops in the teens. Mental and psychological functions are preserved. DYT5 is caused by heterozygous mutations of the GCH1 gene, located on 14q22.1-q22.2. Again, mental and psychological functions are preserved. Clinically, there are two types: postural and action. Postural-type dystonia occurs around 6 years of age, with postural dystonia of one leg, and all extremities and trunk muscles are involved by the late teens. Action-type dystonia shows dystonic movements from around 8 to 10 years of age. In both types, all symptoms show diurnal fluctuations that diminish with age and are no longer apparent in the late teens. L-dopa produces dramatic effects, which continue throughout the course of the illness. In both postural and action types, each family or sporadic case has a particular mutation. It remains unclear why specific mutations cause certain age- and gender-specific symptoms.

Age- and sex-related oculomotor manifestation of dopamine deficiency in Segawa disease.

OBJECTIVE: To investigate the oculomotor manifestations of Segawa disease (SD), considered to represent mild dopamine deficiency and discuss their pathophysiological basis. METHODS: We recorded visually- (VGS) and memory-guided saccade (MGS) tasks in 31 SD patients and 153 age-matched control subjects to study how basal ganglia (BG) dysfunction in SD evolves with age for male and female subjects. RESULTS: SD patients were impaired in initiating MGS, showing longer latencies with occasional failure. Patients showed impaired ability to suppress reflexive saccades; saccades to cues presented in MGS were more frequent and showed a shorter latency than in control subjects. These findings were more prominent in male patients, particularly between 13 and 25 years. Additionally, male patients showed larger delay in MGS latency in trials preceded by saccades to cue than those unpreceded. CONCLUSIONS: The findings can be explained by a dysfunction of the BG-direct pathway impinging on superior colliculus (SC) due to dopamine deficiency. The disturbed inhibitory control of saccades may be explained by increased SC responsivity to visual stimuli. SIGNIFICANCE: Oculomotor abnormalities in SD can be explained by dysfunction of the BG inhibitory pathways reaching SC, with a delayed maturation in male SD patients, consistent with previous pathological/physiological studies.

Dopa-responsive dystonia is caused by particular impairment of nigrostriatal dopamine neurons different from those involved in Parkinson disease: evidence observed in studies on Segawa disease.

From the characteristics of its clinical features, Segawa disease is considered to be caused by deficiency of the tyrosine hydroxylase (TH) of the nigrostriatal dopamine neurons, which have high TH activities in the terminal but not in the perikaryon. This hypothesis was confirmed by two autopsied cases. However, these cases were younger than 40 years and left a question as to whether these abnormalities turned to those of Parkinson disease in older ages. An autopsy of a 90-year-old woman with Segawa disease confirmed the hypothesis that Segawa disease has a completely different pathophysiology and pathology than Parkinson disease.

The Pathophysiology of Gilles de la Tourette Syndrome: Changes in Saccade Performance by Low-Dose L-Dopa and Dopamine Receptor Blockers.

AIM: To elucidate the pathophysiology of Gilles de la Tourette syndrome (GTS), which is associated with prior use of dopamine receptor antagonists (blockers) and treatment by L-Dopa, through saccade performance. METHOD: In 226 male GTS patients (5-14 years), we followed vocal and motor tics and obsessive-compulsive disorder (OCD) after discontinuing blockers at the first visit starting with low-dose L-Dopa. We recorded visual- (VGS) and memory-guided saccades (MGS) in 110 patients and 26 normal participants. RESULTS: At the first visit, prior blocker users exhibited more severe vocal tics and OCD, but not motor tics, which persisted during follow-up. Patients treated with L-Dopa showed greater improvement of motor tics, but not vocal tics and OCD. Patients with and without blocker use showed similarly impaired MGS performance, while patients with blocker use showed more prominently impaired inhibitory control of saccades, associated with vocal tics and OCD. DISCUSSION: Impaired MGS performance suggested a mild hypodopaminergic state causing reduced direct pathway activity in the (oculo-)motor loops of the basal ganglia-thalamocortical circuit. Blocker use may aggravate vocal tics and OCD due to disinhibition within the associative and limbic loops. The findings provide a rationale for discouraging blocker use and using low-dose L-Dopa in GTS.

Nine Hereditary Movement Disorders First Described in Asia: Their History and Evolution.

Clinical case studies and reporting are important to the discovery of new disorders and the advancement of medical sciences. Both clinicians and basic scientists play equally important roles leading to treatment discoveries for both cures and symptoms. In the field of movement disorders, exceptional observation of patients from clinicians is imperative, not just for phenomenology but also for the variable occurrences of these disorders, along with other signs and symptoms, throughout the day and the disease course. The Movement Disorders in Asia Task Force (TF) was formed to help enhance and promote collaboration and research on movement disorders within the region. As a start, the TF has reviewed the original studies of the movement disorders that were preliminarily described in the region. These include nine disorders that were first described in Asia: Segawa disease, PARK-Parkin, X-linked dystonia-parkinsonism, dentatorubral-pallidoluysian atrophy, Woodhouse-Sakati syndrome, benign adult familial myoclonic epilepsy, Kufor-Rakeb disease, tremulous dystonia associated with mutation of the calmodulin-binding transcription activator 2 gene, and paroxysmal kinesigenic dyskinesia. We hope that the information provided will honor the original researchers and help us learn and understand how earlier neurologists and basic scientists together discovered new disorders and made advances in the field, which impact us all to this day.

Pharmacological therapy for Tourette syndrome: What medicine can do and cannot do.

Tourette syndrome (TS) is a frequently observed developmental neuropsychological disorder occurring in children. The pathophysiology involves both genetic and environmental factors. In this review, clinical characteristics, pathophysiology, and treatment approaches based on the pathophysiology of TS are presented. The pathophysiology is the acceleration of developmental decrement of dopamine (DA) activity at the terminal of nigro-striatal (NS)-DA system causing DA D2 receptor up-ward regulation. Serotonergic neurons involving in development of the biphasic sleep-wake-rhythm, and locomotion may be involved. Pharmacological treatments constitute an important part in managing TS. Small dose of levodopa and aripiprazole showed the good effect controlling the tics, without side effects. Intervention with enhancing the day time activity and keeping the regular sleep-wake-rhythm, and encouraging locomotion are important. The data from Yoshiko Nomura Neurological Clinic for Children regarding the clinical features and outcomes, medication effects, and OCD and outcomes are shown. To discuss about the environmental factor, how the COVID-19 pandemic affected the TS patients is also presented.

Characterization of two kdr mutations at predicted pyrethroid receptor site 2 in the sodium channels of Aedes aegypti and Nilaparvata lugens.

Pyrethroid insecticides prolong the opening of insect sodium channels by binding to two predicted pyrethroid receptor sites (PyR), PyR1 and PyR2. Many naturally-occurring sodium channel mutations that confer pyrethroid resistance (known as knockdown resistance, kdr) are located at PyR1. Recent studies identified two new mutations, V253F and T267A, at PyR2, which co-exist with two well-known mutations F1534C or M918T, at PyR1, in pyrethroid-resistant populations of Aedes aegypti and Nilaparvata lugens, respectively. However, the role of the V253F and T267A mutations in pyrethroid resistance has not been functionally examined. Here we report functional characterization of the V253F and T267A mutations in the Ae. aegypti sodium channel AaNav2-1 and the N. lugens sodium channel NlNav1 expressed in Xenopus oocytes. Both mutations alone reduced channel sensitivity to pyrethroids, including etofenprox. We docked etofenprox in a homology model of the pore module of the NlNav1 channel based on the crystal structure of an open prokaryotic sodium channel NavMs. In the low-energy binding pose etofenprox formed contacts with V253, T267 and a previously identified L1014 within PyR2. Combining of V253F or T267A with F1534C or M918T results in a higher level of pyrethroid insensitivity. Furthermore, both V253F and T267A mutations altered channel gating properties. However, V253F- and T267A-induced gating modifications was not observed in the double mutant channels. Our findings highlight the first example in which naturally-found combinational mutations in PyR1 and PyR2 not only confer higher level pyrethroid insensitivity, but also reduce potential fitness tradeoff in pyrethroid-resistant mosquitoes caused by kdr mutation-induced sodium channel gating modifications.

Nationwide survey of Lambert-Eaton myasthenic syndrome in Japan.

Background: There was no nationwide epidemiological study of Lambert-Eaton myasthenic syndrome (LEMS) in Japan; therefore, we conducted a nationwide survey. Methods: For the first survey, we sent survey sheets to randomly selected medical departments (n=7545) to obtain the number of LEMS who visited medical departments between 1 January 2017 and 31 December 2017. For the second survey, we sent survey sheets to the corresponding medical departments to obtain clinical information on LEMS. Results: We received 2708 responses (recovery rate: 35.9%) to the first survey. We estimated the number of LEMS as 348 (95% CI 247 to 449). The prevalence was 2.7 (95% CI 1.9 to 3.5) in 1 000 000 population. As a result of the second survey, we obtained 30 case records of 16 men and 14 women. Fourteen patients (46.7%) had a tumour, and 10 out of 14 tumours were small-cell lung carcinoma (71.4%). There was a predominance of men in the LEMS with tumour (paraneoplastic LEMS, P-LEMS) (n=11, 78.6%) and women in the LEMS without tumour (a primary autoimmune form of LEMS, AI-LEMS) (n=11, 68.8%) (p=0.0136). The onset age (mean (SD)) for the P-LEMS was 67.1 (9.0), and that for AI-LEMS was 57.8 (11.2) years old (p=0.0103). The disease duration (median) for P-LEMS was 2 years, and for AI-LEMS was 7.5 years (p=0.0134). Conclusions: The prevalence of LEMS in Japan is similar to that in other countries. There are predominances of men in P-LEMS and women in AI-LEMS.