Objective:To test the effect of voltage-gated sodium channels (VGSCs) blockers on the motility and viability of human spermin-vitro and to evaluate the tested compounds as potential contact spermicidal.Methods: Sperm ...Objective:To test the effect of voltage-gated sodium channels (VGSCs) blockers on the motility and viability of human spermin-vitro and to evaluate the tested compounds as potential contact spermicidal.Methods: Sperm samples were obtained from healthy non-smoking volunteers of age 25-30 years who had not taken any drug 3 months before and during the course of the study. The effect of VGSCs blockers evaluated from two pharmacological classes including antiarrhythmic (amiodarone, procainamide and disopyramide) and antiepileptic (carbamazepine, oxcarbazepine, phenytoin, and lamotrigine) drugs. They were tested on thein-vitro motility and viability of human sperm using Computer Assisted Semen Analyzer.Results:All tested drugs except oxcarbazepine showed dose dependent inhibition of total motility with significant reduction (P<0.05) at the maximum concentration of 200 μM when compared with the control. The concentrations of drugs that reduced total sperm motility to 50% of control (half maximal inhibitory concentration) were 2.76, 14.16 and 20.29 μM for phenytoin, lamotrigine and carbamazepine, respectively;and 2.53, 5.32 and 0.37 μM for amiodarone, procainamide and disopyramide, respectively. The anti-motility effects were reversible to various degrees. There was statistically insignificant difference in the inhibition of sperm viability among amiodarone, procainamide and disopyramide. Phenytoin demonstrated the most potent spermicidal action.Conclusions:VGSCs blockers have significant adverse effects onin-vitro motility of human spermatozoa. Soin-vivo studies are required to determine their potential toxicological effects on human semen quality, which is an important factor regarding fertility. Moreover, these drugs have the potential to be developed into contact spermicidal.展开更多
Objective In this study, the pharmacological kinetics of Buthus martensi Karsch （BmK） AS, a specific modulator of voltage-gated sodium channel site 4, was investigated on Nav1.3 expressed in Xenopus oocytes. Methods...Objective In this study, the pharmacological kinetics of Buthus martensi Karsch （BmK） AS, a specific modulator of voltage-gated sodium channel site 4, was investigated on Nav1.3 expressed in Xenopus oocytes. Methods Two-electrode voltage clamp was used to record the whole-cell sodium current. Results The peak currents of Nav1.3 were depressed by BmK AS over a wide range of concentrations （10, 100, and 500 nmol/L）. Most remarkably, BmK AS at 100 nmol/L hyperpolarized the voltage-dependence and increased the voltage-sensitivity of steady-state activation/inactivation. In addition, BmK AS was capable of hyperpolarizing not only the fast inactivation but also the slow inactivation, with a greater preference for the latter. Moreover, BmK AS accelerated the time constant and increased the ratio of recovery in Nav1.3 at all concentrations. Conclusion This study provides direct evidence that BmK AS facilitates steady-state activation and inhibits slow inactivation by stabilizing both the closed and open states of the Nav1.3 channel, which might result from an integrative binding to two receptor sites on the voltage-gated sodium channels. These results may shed light on therapeutics against Nav1.3-targeted pathology.展开更多
Background The cytochrome P450s-mediated metabolic resistance and the target site insensitivity caused by the knockdown resistance(kdr)mutation in the voltage-gated sodium channel(vgsc)gene were the main mechanisms co...Background The cytochrome P450s-mediated metabolic resistance and the target site insensitivity caused by the knockdown resistance(kdr)mutation in the voltage-gated sodium channel(vgsc)gene were the main mechanisms conferring resistance to deltamethrin in Culex quinquefasciatus from Thailand.This study aimed to investigate the expression levels of cytochrome P450 genes and detect mutations of the vgsc gene in deltamethrin-resistant Cx.quinquefasciatus populations in Thailand.Methods Two field-collected strains of Cx.quinquefasciatus,Cq_SP and Cq_NiH,were selected with deltamethrin to generate the resistant strains Cq_SP-R and Cq_NiH-R,respectively.Bioassays were tested on larvae and adults of each strain according to WHO methods.Eight cytochrome P450 genes were analyzed for the expression level using quantitative real time-PCR.The cDNA of mosquitoes was amplified and sequenced for four fragments of vgsc gene.The kdr L1014F mutation and the haplotype of the CYP9M10 gene were detected in survivor and dead mosquitoes after exposure to the deltamethrin WHO test paper.Statistical analyses were performed using Fisher’s exaction test.Results Bioassay tests revealed a significantly higher resistance level in Cq_SP-R than in Cq_NiH-R strains in both larvae and adults.All eight cytochrome P450 genes were significantly overexpressed in larvae of Cq_NiH-R strain compared to the parent and susceptible Cq_Sus strains.The CYP6AA7 and CYP9J34 genes had the highest expression ratios,exceeding 24-fold in Cq_NiH-R larvae.In Cq_SP-R strain,the CYP4H34 and CYP9J34 genes were overexpressed in both stages.The kdr L1014F mutation was found in Cq_SP-R and its parent Cq_SP strains with a significantly higher mutant allele frequency in the survivor mosquitoes than in dead mosquitoes(P<0.0001).The V240M and novel L925F mutations were found only in Cq_SP-R strain.Heterozygous genotype for the D-Cu(+)/Cu(-)of CYP9M10 gene was detected in Cq_NiH and Cq_NiH-R strains but other strains were mostly homozygous for the Cu(-)/Cu(-).Conclusions Overexpression of multiple cytochrome P450 genes alone has a relatively minor impact on resistance.The combined mechanisms of cytochrome P450-and kdr-mediated resistance result in significantly higher resistance to deltamethrin in Cx.quinquefasciatus.This study supports sustainable public health initiatives in Thailand to address the evolving challenges of insecticide resistance.展开更多
Background Culex pipiens pallens and Culex pipiens quinquefasciatus are the dominant species of Culex mosquitoes in China and important disease vectors.Long-term use of insecticides can cause mutations in the voltage-...Background Culex pipiens pallens and Culex pipiens quinquefasciatus are the dominant species of Culex mosquitoes in China and important disease vectors.Long-term use of insecticides can cause mutations in the voltage-gated sodium channel(vgsc)gene of mosquitoes,but little is known about the current status and evolutionary origins of vgsc gene in different geographic populations.Therefore,this study aimed to determine the current status of vgsc genes in Cx.p.pallens and Cx.p.quinquefasciatus in China and to investigate the evolutionary inheritance of neighboring downstream introns of the vgsc gene to determine the impact of insecticides on long-term evolution.Methods Sampling was conducted from July to September 2021 in representative habitats of 22 provincial-level administrative divisions in China.Genomic DNA was extracted from 1308 mosquitoes,the IIS6 fragment of the vgsc gene on the nerve cell membrane was amplified using polymerase chain reaction,and the sequence was used to evaluate allele frequency and knockdown resistance(kdr)frequency.MEGA 11 was used to construct neighborjoining(NJ)tree.PopART was used to build a TCS network.Results There were 6 alleles and 6 genotypes at the L1014 locus,which included the wild-type alleles TTA/L and CTA/L and the mutant alleles TTT/F,TTC/F,TCT/S and TCA/S.The geographic populations with a kdr frequency less than 20.00%were mainly concentrated in the regions north of 38°N,and the geographic populations with a kdr frequency greater than 80.00%were concentrated in the regions south of 30°N.kdr frequency increased with decreasing latitude.And within the same latitude,the frequency of kdr in large cities is relatively high.Mutations were correlated with the number of introns.The mutant allele TCA/S has only one intron,the mutant allele TTT/F has three introns,and the wild-type allele TTA/L has 17 introns.Conclusions Cx.p.pallens and Cx.p.quinquefasciatus have developed resistance to insecticides in most regions of China.The neighboring downstream introns of the vgsc gene gradually decreased to one intron with the mutation of the vgsc gene.Mutations may originate from multiple mutation events rather than from a single origin,and populations lacking mutations may be genetically isolated.展开更多
Background The current prevention and control strategy forAedes albopictus heavily relies on comprehensive management, such as environmental management and chemical control. However, the wide application of pyrethroid...Background The current prevention and control strategy forAedes albopictus heavily relies on comprehensive management, such as environmental management and chemical control. However, the wide application of pyrethroids has facilitated the development of insecticide resistance, primarily via mutations in the voltage-gated sodium channel (VGSC) gene. This study aims to develop a novel strategy for detecting mutations in the VGSC gene inAe. albopictus using multiplex PCR-mass spectrometry (MPCR-MS) minisequencing technology.Methods We established a new strategy for detecting mutations in the VGSC gene inAe. albopictus using MPCR-MS minisequencing technology. MPCR amplification and mass probe extension (MPE) were first used, followed by single nucleotide polymorphism (SNP) typing mass spectrometry, which allows the simultaneous detection of multiple mutation sites of the VGSC gene in 96 samples ofAe. albopictus. A total of 70 wild-collectedAe. albopictus were used to evaluate the performance of the method by comparing it with other methods.Results Three target sites (1016, 1532, 1534) in the VGSC gene can be detected simultaneously by double PCR amplification combined with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, achieving a detection limit of 20 fg/μl. We applied this method to 70 wild-collectedAe. albopictus, and the obtained genotypes were consistent with the routine sequencing results, suggesting the accuracy of our method.Conclusions MPCR-MS minisequencing technology provides a sensitive and high-throughput approach toAe. albopictus VGSC gene mutation screening. Compared with conventional sequencing, this method is economical and time-saving. It is of great value for insecticide resistance surveillance in areas with a high risk of vector-borne disease.展开更多
文摘Objective:To test the effect of voltage-gated sodium channels (VGSCs) blockers on the motility and viability of human spermin-vitro and to evaluate the tested compounds as potential contact spermicidal.Methods: Sperm samples were obtained from healthy non-smoking volunteers of age 25-30 years who had not taken any drug 3 months before and during the course of the study. The effect of VGSCs blockers evaluated from two pharmacological classes including antiarrhythmic (amiodarone, procainamide and disopyramide) and antiepileptic (carbamazepine, oxcarbazepine, phenytoin, and lamotrigine) drugs. They were tested on thein-vitro motility and viability of human sperm using Computer Assisted Semen Analyzer.Results:All tested drugs except oxcarbazepine showed dose dependent inhibition of total motility with significant reduction (P<0.05) at the maximum concentration of 200 μM when compared with the control. The concentrations of drugs that reduced total sperm motility to 50% of control (half maximal inhibitory concentration) were 2.76, 14.16 and 20.29 μM for phenytoin, lamotrigine and carbamazepine, respectively;and 2.53, 5.32 and 0.37 μM for amiodarone, procainamide and disopyramide, respectively. The anti-motility effects were reversible to various degrees. There was statistically insignificant difference in the inhibition of sperm viability among amiodarone, procainamide and disopyramide. Phenytoin demonstrated the most potent spermicidal action.Conclusions:VGSCs blockers have significant adverse effects onin-vitro motility of human spermatozoa. Soin-vivo studies are required to determine their potential toxicological effects on human semen quality, which is an important factor regarding fertility. Moreover, these drugs have the potential to be developed into contact spermicidal.
基金supported by grants from the National Basic Research Development Program of China (2010CB529806)the National Natural Science Foundation of China (31171064)+1 种基金the Key Research Program of Science and Technology Commissions of Shanghai Municipality (11JC1404300)the Leading Academic Discipline Project of Shanghai Municipal Education Commission (J50108)
文摘Objective In this study, the pharmacological kinetics of Buthus martensi Karsch （BmK） AS, a specific modulator of voltage-gated sodium channel site 4, was investigated on Nav1.3 expressed in Xenopus oocytes. Methods Two-electrode voltage clamp was used to record the whole-cell sodium current. Results The peak currents of Nav1.3 were depressed by BmK AS over a wide range of concentrations （10, 100, and 500 nmol/L）. Most remarkably, BmK AS at 100 nmol/L hyperpolarized the voltage-dependence and increased the voltage-sensitivity of steady-state activation/inactivation. In addition, BmK AS was capable of hyperpolarizing not only the fast inactivation but also the slow inactivation, with a greater preference for the latter. Moreover, BmK AS accelerated the time constant and increased the ratio of recovery in Nav1.3 at all concentrations. Conclusion This study provides direct evidence that BmK AS facilitates steady-state activation and inhibits slow inactivation by stabilizing both the closed and open states of the Nav1.3 channel, which might result from an integrative binding to two receptor sites on the voltage-gated sodium channels. These results may shed light on therapeutics against Nav1.3-targeted pathology.
基金supported by CMU Mid-Career Research Fellowship program to J.Y.
文摘Background The cytochrome P450s-mediated metabolic resistance and the target site insensitivity caused by the knockdown resistance(kdr)mutation in the voltage-gated sodium channel(vgsc)gene were the main mechanisms conferring resistance to deltamethrin in Culex quinquefasciatus from Thailand.This study aimed to investigate the expression levels of cytochrome P450 genes and detect mutations of the vgsc gene in deltamethrin-resistant Cx.quinquefasciatus populations in Thailand.Methods Two field-collected strains of Cx.quinquefasciatus,Cq_SP and Cq_NiH,were selected with deltamethrin to generate the resistant strains Cq_SP-R and Cq_NiH-R,respectively.Bioassays were tested on larvae and adults of each strain according to WHO methods.Eight cytochrome P450 genes were analyzed for the expression level using quantitative real time-PCR.The cDNA of mosquitoes was amplified and sequenced for four fragments of vgsc gene.The kdr L1014F mutation and the haplotype of the CYP9M10 gene were detected in survivor and dead mosquitoes after exposure to the deltamethrin WHO test paper.Statistical analyses were performed using Fisher’s exaction test.Results Bioassay tests revealed a significantly higher resistance level in Cq_SP-R than in Cq_NiH-R strains in both larvae and adults.All eight cytochrome P450 genes were significantly overexpressed in larvae of Cq_NiH-R strain compared to the parent and susceptible Cq_Sus strains.The CYP6AA7 and CYP9J34 genes had the highest expression ratios,exceeding 24-fold in Cq_NiH-R larvae.In Cq_SP-R strain,the CYP4H34 and CYP9J34 genes were overexpressed in both stages.The kdr L1014F mutation was found in Cq_SP-R and its parent Cq_SP strains with a significantly higher mutant allele frequency in the survivor mosquitoes than in dead mosquitoes(P<0.0001).The V240M and novel L925F mutations were found only in Cq_SP-R strain.Heterozygous genotype for the D-Cu(+)/Cu(-)of CYP9M10 gene was detected in Cq_NiH and Cq_NiH-R strains but other strains were mostly homozygous for the Cu(-)/Cu(-).Conclusions Overexpression of multiple cytochrome P450 genes alone has a relatively minor impact on resistance.The combined mechanisms of cytochrome P450-and kdr-mediated resistance result in significantly higher resistance to deltamethrin in Cx.quinquefasciatus.This study supports sustainable public health initiatives in Thailand to address the evolving challenges of insecticide resistance.
基金funded by the National Science and Technology Major Project of China(No.2018ZX10101002-002)
文摘Background Culex pipiens pallens and Culex pipiens quinquefasciatus are the dominant species of Culex mosquitoes in China and important disease vectors.Long-term use of insecticides can cause mutations in the voltage-gated sodium channel(vgsc)gene of mosquitoes,but little is known about the current status and evolutionary origins of vgsc gene in different geographic populations.Therefore,this study aimed to determine the current status of vgsc genes in Cx.p.pallens and Cx.p.quinquefasciatus in China and to investigate the evolutionary inheritance of neighboring downstream introns of the vgsc gene to determine the impact of insecticides on long-term evolution.Methods Sampling was conducted from July to September 2021 in representative habitats of 22 provincial-level administrative divisions in China.Genomic DNA was extracted from 1308 mosquitoes,the IIS6 fragment of the vgsc gene on the nerve cell membrane was amplified using polymerase chain reaction,and the sequence was used to evaluate allele frequency and knockdown resistance(kdr)frequency.MEGA 11 was used to construct neighborjoining(NJ)tree.PopART was used to build a TCS network.Results There were 6 alleles and 6 genotypes at the L1014 locus,which included the wild-type alleles TTA/L and CTA/L and the mutant alleles TTT/F,TTC/F,TCT/S and TCA/S.The geographic populations with a kdr frequency less than 20.00%were mainly concentrated in the regions north of 38°N,and the geographic populations with a kdr frequency greater than 80.00%were concentrated in the regions south of 30°N.kdr frequency increased with decreasing latitude.And within the same latitude,the frequency of kdr in large cities is relatively high.Mutations were correlated with the number of introns.The mutant allele TCA/S has only one intron,the mutant allele TTT/F has three introns,and the wild-type allele TTA/L has 17 introns.Conclusions Cx.p.pallens and Cx.p.quinquefasciatus have developed resistance to insecticides in most regions of China.The neighboring downstream introns of the vgsc gene gradually decreased to one intron with the mutation of the vgsc gene.Mutations may originate from multiple mutation events rather than from a single origin,and populations lacking mutations may be genetically isolated.
基金National Science and Technology Major Project of China(No. 2018ZX10101002-002)。
文摘Background The current prevention and control strategy forAedes albopictus heavily relies on comprehensive management, such as environmental management and chemical control. However, the wide application of pyrethroids has facilitated the development of insecticide resistance, primarily via mutations in the voltage-gated sodium channel (VGSC) gene. This study aims to develop a novel strategy for detecting mutations in the VGSC gene inAe. albopictus using multiplex PCR-mass spectrometry (MPCR-MS) minisequencing technology.Methods We established a new strategy for detecting mutations in the VGSC gene inAe. albopictus using MPCR-MS minisequencing technology. MPCR amplification and mass probe extension (MPE) were first used, followed by single nucleotide polymorphism (SNP) typing mass spectrometry, which allows the simultaneous detection of multiple mutation sites of the VGSC gene in 96 samples ofAe. albopictus. A total of 70 wild-collectedAe. albopictus were used to evaluate the performance of the method by comparing it with other methods.Results Three target sites (1016, 1532, 1534) in the VGSC gene can be detected simultaneously by double PCR amplification combined with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, achieving a detection limit of 20 fg/μl. We applied this method to 70 wild-collectedAe. albopictus, and the obtained genotypes were consistent with the routine sequencing results, suggesting the accuracy of our method.Conclusions MPCR-MS minisequencing technology provides a sensitive and high-throughput approach toAe. albopictus VGSC gene mutation screening. Compared with conventional sequencing, this method is economical and time-saving. It is of great value for insecticide resistance surveillance in areas with a high risk of vector-borne disease.
