Kdr Gene Research Articles

Cumulative Effect of Risk Alleles in CFH, ARMS2, and VEGFA on the Response to Ranibizumab Treatment in Age-related Macular Degeneration

Intravitreal ranibizumab injections currently are the standard treatment for neovascular age-related macular degeneration (AMD). However, a broad range of response rates have been observed, the reasons for which are poorly understood. This pharmacogenetic study evaluated the impact of high-risk alleles in CFH, ARMS2, VEGFA, vascular endothelial growth factor (VEGF) receptor KDR, and genes involved in angiogenesis (LRP5, FZD4) on the response to ranibizumab treatment and on the age of treatment onset. In contrast to previous studies, the data were stratified according to the number of high-risk alleles to enable the study of the combined effects of these genotypes on the treatment response. Case series study. A cohort of 420 eyes of 397 neovascular AMD patients. The change in visual acuity (VA) between baseline and after 3 ranibizumab injections was calculated. Genotyping of single nucleotide polymorphisms in the CFH, ARMS2, VEGFA, KDR, LPR5, and FZD4 genes was performed. Associations were assessed using linear mixed models. The VA change after 3 ranibizumab injections and the age of neovascular disease onset. After ranibizumab treatment, AMD patients without risk alleles in the CFH and ARMS2 genes (4.8%) demonstrated a mean VA improvement of 10 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, whereas no VA improvement was observed in AMD patients with 4 CFH and ARMS2 risk alleles (6.9%; P = 0.014). Patients with 4 high-risk alleles in CFH and ARMS2 were 5.2 years younger than patients with 1 or 2 risk alleles, respectively (63.5%; P<0.0001). The mean age at which the first ranibizumab treatment was carried out among AMD patients with all 6 risk alleles in CFH, ARMS2, and VEGFA was 65.9 years (2%) versus 75.3 years in patients with 0 or 1 high-risk allele (8.8%; P = 0.001). After ranibizumab treatment, patients with 6 high-risk alleles demonstrated a mean VA loss of 10 ETDRS letters (P<0.0001). This study evaluated the largest pharmacogenetic AMD cohort reported to date. A cumulative effect of high-risk alleles in CFH, ARMS2, and VEGFA seems to be associated with a younger age of onset in combination with poor response rates to ranibizumab treatment.

Development of an allele-specific, loop-mediated, isothermal amplification method (AS-LAMP) to detect the L1014F kdr-w mutation in Anopheles gambiae s. l.

BackgroundMalaria control relies heavily on treated bed nets and indoor residual spraying with pyrethroid insecticides. Unfortunately, the resistance to pyrethroid insecticides, mainly due to the kdr mutation, is spreading in the main malaria vector Anopheles gambiae s.l., decreasing the insecticides’ efficacy. To manage the insecticide resistance rapidly and flexibly, simple and effective tools for the early detection of resistant mosquitoes are needed. This study aimed to develop an allele-specific, loop-mediated, isothermal amplification (AS-LAMP) method to detect the West African-type kdr mutation (kdr-w; L1014F) in field-collected mosquitoes.MethodsDNA fragments of the wild-type and the mutated kdr gene were used to select the primers and develop the method. The primers were designed with the mutation at the 5’ end of the backward inner primer (BIP). The AS-LAMP method was compared to the AS-PCR method using the genomic DNA of 120 field-collected mosquitoes.ResultsThe AS-LAMP method could discriminate between the wild-type homozygote, the heterozygote, and the kdr-w homozygote within 75 min. The AS-LAMP method has the advantage of being faster and at least as sensitive and specific as the AS-PCR method.ConclusionsThe AS-LAMP method can be used to detect the kdr mutation for quick decision-making, even in less well-equipped laboratories.

Association and interaction between dietary pattern and VEGF receptor-2 (VEGFR2) gene polymorphisms on blood lipids in Chinese Malaysian and Japanese adults.

Dietary pattern and genetic predisposition of each population have different impacts on lifestyle-related chronic diseases. This study was conducted to evaluate the association and interaction between dietary patterns and VEGFR2 or KDR gene polymorphisms on physical and biochemical risk factors of cardiovascular disease in two Asian populations (179 Chinese Malaysian and 136 Japanese adults). Dietary patterns were constructed from food frequency questionnaire using factor analysis. Genotyping of rs1870377 and rs2071559 was performed by real-time PCR using TaqMan probes. Physical measurements: body mass index, systolic and diastolic blood pressures and biochemical parameters: glycated hemoglobin A1c and blood lipids (total cholesterol, triglycerides, LDL-cholesterol, HDL-cholesterol and total cholesterol/HDL-C ratio) were measured. Two dietary patterns were extracted for: Japanese ('Japanese diet' and 'Western diet') and Chinese Malaysians ('Balanced diet'; and 'Meat, rice and noodles diet'). In Japanese, 'Western Diet' and rs2071559 were associated with LDL-C and HDL-C, respectively. In Chinese Malaysians, 'Meat, rice and noodles diet' was asso-ciated with triglycerides, HDL-C and total cholesterol/HDL ratio while rs1870377 and rs2071559 were associated with total cholesterol and/or LDL-C. The interaction between 'Western Diet' and rs2071559 in Japanese and 'Meat, rice and noodles diet' and rs1870377 in Malaysians had significant effects on blood lipids after adjusting for confounders. The association and interaction of dietary patterns and VEGFR2 gene polymorphisms on blood lipids differ between Chinese Malaysian and Japanese subjects by either decreasing or increasing the risk of cardiovascular disease.

Paired Tumor and Normal Whole Genome Sequencing of Metastatic Olfactory Neuroblastoma

BackgroundOlfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression.Methodology/Principal FindingsGenomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects.Conclusions/SignificanceThis work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations.

Permethrin and malathion resistance in head lice: Results of ex vivo and molecular assays

Treatment of head lice infestation relies on the application of topical insecticides. Overuse of these products has led to the emergence of resistance to pyrethroids and malathion worldwide. Permethrin resistance in head lice is mostly conferred by the knockdown resistance (kdr) trait. To evaluate the occurrence of permethrin- and malathion-resistant head lice in Paris. A prospective survey was conducted in 74 elementary schools. Live lice collected on schoolchildren were randomly selected and submitted to ex vivo bioassays or underwent individual DNA extraction. A fragment of kdr-like gene was amplified and compared with wild-type sequences. Live head lice were detected in 574 children. Ex vivo assays showed no surviving lice after a 1-hour contact with malathion while most lice died after a 1-hour exposure to permethrin and piperonyl butoxide (85.7%, 95% confidence interval [CI]: 83.9-87.5). Among the 670 lice with workable DNA sequences, 661 lice (98.7%, 95% CI 97.7-99.3) had homozygous kdr mutations. The findings of this large-scale survey of the occurrence of insecticide-resistant head lice indicated a major insecticide pressure in the study population, but it was not sufficient to draw conclusions about other populations. The presence of T917I-L920F mutations in kdr gene may not correlate with treatment failure in prospective studies. The high occurrence of kdr mutant allele suggests that insecticide resistance was already strongly established in the studied population. This finding must be interpreted with caution as it may not be predictive of treatment failure.

The polymorphism and the geographical distribution of the knockdown resistance (kdr) of Anopheles sinensis in the Republic of Korea

BackgroundIn the Republic of Korea (ROK), six sibling species of the Anopheles sinensis complex are considered the vector species of malaria, but data on their susceptibilities to malaria and vector capacities have been controversial. The intensive use of insecticides has contributed to the rapid development and spread of insecticide resistance in the An. sinensis complex. Knockdown resistance (kdr) to pyrethroids and DDT in the An. sinensis complex is associated with a mutation in codon 1014 of the voltage-gated sodium channel (VGSC) gene. Because the degree of insecticide resistance varies among mosquito species and populations, the detection of kdr mutations among the six sibling species of the An. sinensis complex is a prerequisite for establishing effective long-term vector control strategies in the ROKMethodsIn order to investigate species-specific kdr mutations, An. sinensis complex specimens have been collected from 22 sites in the ROK. Because of the difficulties with species identifications that are based only on morphological characteristics, molecular identification methods have been conducted on every specimen. Part of the IIS6 domain of the VGSC was polymerase chain reaction-amplified and directly sequenced.ResultsThe molecular analyses revealed that mutations existed at codon 1014 only in An. sinensis sensu stricto and no mutations were found in the other five Anopheles species. In An. sinensis s.s., one wild type (TTG L1014) and three mutant types (TTT L1014F, TTC L1014F, and TGT L1014C) of kdr alleles were detected. The TTC L1014F mutation was observed for the first time in this species.ConclusionsThe fact that the highly polymorphic kdr gene is only observed in An. sinensis s.s., out of the six Anopheles species and their geographical distribution suggest the need for future studies of insecticide resistance monitoring and investigations of species-specific resistance mechanisms in order to build successful malaria vector control programmes in the ROK.

Association between kinase insert domain-containing receptor gene polymorphisms and silent brain infarction: A Korean study

BackgroundKinase insert domain-containing receptor (KDR), vascular endothelial growth factor receptor-2, play a pivotal role in endothelial dysfunction, which may lead to silent brain infarction (SBI). We evaluated whether single nucleotide polymorphisms (SNPs) of KDR genes are associated with increased risk of SBI in a Korean population. MethodsA total of 383 patients with SBI and 387 controls were genotyped for the KDR −604T>C, 1192G>A, and 1719A>T SNPs. We separately analyzed this association according to the age (age≥65 and age<65) and the gender. We also compared haplotype frequencies between SBI patients and controls. ResultsGenotype frequencies for three SNPs did not differ significantly between SBI patients and controls. In addition, haplotype analysis for three SNPs did not show a difference between patients and controls. However, the frequency of genotype of KDR −604T>C was significantly associated with an increased risk of SBI in the age<65years old group (AOR=1.515, 95% CI, 1.003 to 2.289, p=0.048) and in male group (AOR=1.596, 95% CI, 1.018 to 2.503, p=0.042). ConclusionsKDR −604T>C SNP may serve as genetic markers for the increased risk of SBI among the younger (<65years) or male only Korean subpopulations.

Sorafenib for Patients with Advanced Angiosarcoma: A Phase II Trial from the French Sarcoma Group (GSF/GETO)

Angiosarcomas account for <2% of all soft tissue sarcomas. This subtype is one of the most aggressive forms of soft tissue sarcoma. The prognosis for angiosarcoma patients in the advanced phase remains poor with current cytotoxic agents (progression-free survival [PFS] time of ∼4 months and overall survival [OS] time of ∼8 months). We investigated the antitumor activity of sorafenib in patients with metastatic or advanced angiosarcomas in a phase II trial. We conducted a stratified phase II trial. The primary endpoint was the progression-free rate (PFR) at 9 months according to the Response Evaluation Criteria in Solid Tumors. A two-stage design (optimal Simon design) was used. Patients received sorafenib (400 mg twice daily) for 9 months until unacceptable toxicity or tumor progression. Central pathological and radiological reviews were performed. Data on stratum A (superficial angiosarcoma) and stratum B (visceral angiosarcoma) are currently available. This trial is registered with ClinicalTrials.gov (identifier, NCT00874874). Strata A and B recruited 26 and 15 patients, respectively. The median age was 63 years (range, 31-82 years), with 17 male and 24 female patients. Fourteen cases arose in irradiated fields. Thirty patients (73.0%) had been pretreated with conventional chemotherapy. No unexpected toxicity occurred. The PFR at 9 months was 3.8% in stratum A and 0.0% in stratum B. The median PFS times were 1.8 months and 3.8 months, respectively, whereas the median OS times were 12.0 months and 9.0 months, respectively. No responses were observed in chemotherapy-naïve patients, whereas a 40% tumor control rate and 23% response rate were observed in the pretreated population. In this cohort, no activating mutation of the KDR gene (exons 15, 16, 24) was detected. Sorafenib showed limited antitumor activity in pretreated patients only, for both visceral and superficial angiosarcoma, but tumor control was of short duration.

First report on co-occurrence knockdown resistance mutations and susceptibility to beta-cypermethrin in Anopheles sinensis from Jiangsu Province, China.

The increasing prevalence of insecticide resistance in Anopheles sinensis, a major vector of malaria in Jiangsu province in eastern China, threatens to compromise the successful use of insecticides in malaria control strategies. It is therefore vital to understand the insecticide resistance status of An. sinensis in the region. This study examined the nucleotide diversity of the para-sodium channel and knockdown resistance (kdr) in five field populations of adult An. sinensis mosquitoes collected in Jiangsu province, identifying the L1014F and L1014C substitutions for the first time. Competitive polymerase chain reaction (PCR) amplification of specific allele (cPASA) and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) for resistance diagnosis were developed and validated. Comparing the results with direct sequencing revealed that the PCR-RFLP method was more sensitive and specific whereas the cPASA method was more convenient and suitable. The significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014C substitutions in the kdr gene provides a useful molecular marker for monitoring beta-cypermethrin resistance in natural populations of An. sinensis. Our results point to the L1014F substitution as the key mutation associated with beta-cypermethrin resistance. The high resistance and mutation frequency detected in the five populations also suggest cross-resistance with other pyrethroids may occur in An. sinensis, highlighting the need for further surveys to map insecticide resistance in China and the adoption of a rational management of insecticide application for resistance management and mosquito vector control.

Knockdown resistance, Rdl alleles, and the annual entomological Inoculation rate of wild mosquito populations from Lower Moshi, Northern Tanzania

Aim:Understanding vector behavioral response due to ecological factors is important in the control of disease vectors. This study was conducted to determine the knockdown resistance (kdr) alleles, dieldrin resistance alleles, and entomological inoculation rates (EIRs) of malaria vectors in lower Moshi irrigation schemes for the mitigation of disease transmission.Materials and Methods:The study was longitudinal design conducted for 14 months. Mosquitoes were collected fortnightly by using a CDC miniature light trap in 20 houses. Mosquitoes were identified morphologically in the field, of which 10% of this population was identified to species level by using molecular techniques. Samples from this study population were taken for kdr and resistance to dieldrin (rdl) genes detection.Results:A total of 6220 mosquitoes were collected by using a light trap, of which 86.0% (n=5350) were Anopheles gambiae sensu lato and 14.0% (n=870) were Culex quinquefasciatus. Ten percent of the An. gambiae s.l. (n=535) collected were taken for species identification, of which 99.8% (n=534) were identified as An. arabiensis while 0.2% (n=1) were An. gambiae sensu stricto. Of the selected mosquitoes, 3.5% (n=19) were sporozoite positive. None of the mosquitoes tested had the kdr gene. The rdl resistant allele was detected at a frequency of 0.48 throughout the year. EIR was determined to be 0.54 ib/trap/year.Conclusion:The findings of this study suggest that the homozygous and the heterozygous resistance present in rdl genes demonstrated the effect of pesticide residues on resistance selection pressure in mosquitoes. A better insecticide usage protocol needs to be developed for farmers to use in order to avoid excessive use of pesticides.

Using the posterior distribution of deviance to measure evidence of association for rare susceptibility variants

Aitkin recently proposed an integrated Bayesian/likelihood approach that he claims is general and simple. We have applied this method, which does not rely on informative prior probabilities or large-sample results, to investigate the evidence of association between disease and the 16 variants in the KDR gene provided by Genetic Analysis Workshop 17. Based on the likelihood of logistic regression models and considering noninformative uniform prior probabilities on the coefficients of the explanatory variables, we used a random walk Metropolis algorithm to simulate the distributions of deviance and deviance difference. The distribution of probability values and the distribution of the proportions of positive deviance differences showed different locations, but the direction of the shift depended on the genetic factor. For the variant with the highest minor allele frequency and for any rare variant, standard logistic regression showed a higher power than the novel approach. For the two variants with the strongest effects on Q1 under a type I error rate of 1%, the integrated approach showed a higher power than standard logistic regression. The advantages and limitations of the integrated Bayesian/likelihood approach should be investigated using additional regions and considering alternative regression models and collapsing methods.

Combining indoor residual spraying with chlorfenapyr and long-lasting insecticidal bed nets for improved control of pyrethroid-resistant Anopheles gambiae: an experimental hut trial in Benin

BackgroundNeither indoor residual spraying (IRS) nor long-lasting insecticidal nets (LLINs) are able to fully interrupt transmission in holoendemic Africa as single interventions. The combining of IRS and LLINs presents an opportunity for improved control and management of pyrethroid resistance through the simultaneous presentation of unrelated insecticides.MethodChlorfenapyr IRS and a pyrethroid-impregnated polyester LLIN (WHO approved) were tested separately and together in experimental huts in southern Benin against pyrethroid resistant Anopheles gambiae and Culex quinquefasciatus. The bed nets were deliberately holed with either six or 80 holes to examine the effect of increasing wear and tear on protectiveness. Anopheles gambiae were genotyped for the kdr gene to assess the combination's potential to prevent the selection of pyrethroid resistance.ResultsThe frequency of kdr was 84%. The overall mortality rates of An. gambiae were 37% and 49% with the six-hole and 80-hole LLINs, respectively, and reached 57% with chlorfenapyr IRS. Overall mortality rates were significantly higher with the combination treatments (82-83%) than with the LLIN or IRS individual treatments. Blood feeding (mosquito biting) rates were lowest with the 6-hole LLIN (12%), intermediate with the 80-hole LLIN (32%) and highest with untreated nets (56% with the 6-hole and 54% with the 80-hole nets). Blood feeding (biting) rates and repellency of mosquitoes with the combination of LLIN and chlorfenapyr IRS showed significant improvement compared to the IRS treatment but did not differ from the LLIN treatments indicating that the LLINs were the primary agents of personal protection. The combination killed significantly higher proportions of Cx. quinquefasciatus (51%, 41%) than the LLIN (15%, 13%) or IRS (32%) treatments.ConclusionThe chlorfenapyr IRS component was largely responsible for controlling pyrethroid-resistant mosquitoes and the LLIN component was largely responsible for blood feeding inhibition and personal protection. Together, the combination shows potential to provide additional levels of transmission control and personal protection against pyrethroid-resistant mosquitoes, thereby justifying the additional resources required. Chlorfenapyr has potential to manage pyrethroid resistance in the context of an expanding LLIN/IRS strategy.

The CpG Island Methylation Regulated Expression of Endothelial Proangiogenic Genes in Response to β-Carotene and Arachidonic Acid

β-carotene (BC) and arachidonic acid (AA) were demonstrated to modulate carcinogenesis by influencing angiogenesis. DNA methylation is an epigenetic mechanism regulating gene expression. The aim of this study was to investigate whether BC and AA change DNA methylation and expression of the proangiogenic genes, which might help explain their impact on carcinogenesis. Human umbilical vein endothelial cells (HUVECs) and endothelial progenitors (EPCs) were incubated with BC or AA for 24 h. Based on microarray results, we selected 18 genes for DNA methylation analysis. CpG island methylation was quantified using the combined bisulphite restriction analysis method and methylation sensitive restriction enzymes. Relative gene expression was quantified using a quantitative real-time PCR (qRT-PCR) method. Incubation with AA significantly decreased methylation of the promoters of both KDR (P = 0.048) and Notch4 (P = 0.027) genes in HUVECs. In EPCs, BC increased methylation of the connexin 43 gene (P = 0.036). qRT-PCR showed that AA (P = 0.059) and BC (P = 0.044) upregulated the KDR gene expression in HUVECs. Connexin 43 gene expression was induced in the presence of 1 μM (P = 0.039) and 3 μM (P = 0.043) BC in EPCs. No significant changes in the Notch4 gene expression were found. The impact of BC and AA on carcinogenesis may be due, at least in part, to changes in expression of angiogenic genes and these transcriptional effects may be mediated by changes in methylation of CpG islands in the gene promoters. However more research is necessary to confirm this hypothesis.

Increased VEGFR-2 Gene Copy Is Associated with Chemoresistance and Shorter Survival in Patients with Non–Small-Cell Lung Carcinoma Who Receive Adjuvant Chemotherapy

VEGF receptor-2 (VEGFR-2 or kinase insert domain receptor; KDR) is a known endothelial target also expressed in NSCLC tumor cells. We investigated the association between alterations in the KDR gene and clinical outcome in patients with resected non-small-cell lung carcinoma (NSCLC; n = 248). KDR copy number gains (CNG), measured by quantitative PCR and fluorescence in situ hybridization, were detected in 32% of tumors and associated with significantly higher KDR protein and higher microvessel density than tumors without CNGs. KDR CNGs were also associated with significantly increased risk of death (HR = 5.16; P = 0.003) in patients receiving adjuvant platinum-based chemotherapy, but no differences were observed in patients not receiving adjuvant therapy. To investigate potential mechanisms for these associations, we assessed NSCLC cell lines and found that KDR CNGs were significantly associated with in vitro resistance to platinum chemotherapy as well as increased levels of nuclear hypoxia inducible factor-1α (HIF-1α) in both NSCLC tumor specimens and cell lines. Furthermore, KDR knockdown experiments using small interfering RNA reduced platinum resistance, cell migration, and HIF-1α levels in cells bearing KDR CNGs, providing evidence for direct involvement of KDR. No KDR mutations were detected in exons 7, 11, and 21 by PCR-based sequencing; however, two variant single nucleotide polymorphism genotypes were associated with favorable overall survival in adenocarcinoma patients. Our findings suggest that tumor cell KDR CNGs may promote a more malignant phenotype including increased chemoresistance, angiogenesis, and HIF-1α levels, and that KDR CNGs may be a useful biomarker for identifying patients at high risk for recurrence after adjuvant therapy, a group that may benefit from VEGFR-2 blockade.

First Detection of a Putative Knockdown Resistance Gene in Major Mosquito Vector, &lt;i&gt;Aedes albopictus&lt;/i&gt;

The Asian tiger mosquito, Aedes albopictus (Skuse), is the major vector of Chikungunya fever and the secondary vector of dengue fever. We collected Ae. albopictus from Singapore and performed genotyping assay to detect mutations of the voltage-gated sodium channel, which is the target site of pyrethroid insecticides. We detected an amino acid substitution, F1534C, which is suspected to confer knockdown resistance (kdr) to pyrethroid insecticides. Of the collected mosquitoes, 53.8% were homozygous for this mutation, and the allele frequency of this mutation was estimated to be 73.1%. No kdr mutation was detected in the 5 other loci of domains II and IV. This is the first evidence for the presence of the kdr gene in Ae. albopictus, and our findings highlight the need for studying the global distribution of this allele in this important vector insect.

Cotton pest management practices and the selection of pyrethroid resistance in Anopheles gambiae population in Northern Benin

BackgroundPyrethroid insecticides, carbamate and organophosphate are the classes of insecticides commonly used in agriculture for crop protection in Benin. Pyrethroids remain the only class of insecticides recommended by the WHO for impregnation of bed nets. Unfortunately, the high level of pyrethroid resistance in Anopheles gambiae s.l., threatens to undermine the success of pyrethroid treated nets. This study focuses on the investigation of agricultural practices in cotton growing areas, and their direct impact on larval populations of An. gambiae in surrounding breeding sites.MethodsThe protocol was based on the collection of agro-sociological data where farmers were subjected to semi-structured questionnaires based on the strategies used for crop protection. This was complemented by bioassay tests to assess the susceptibility of malaria vectors to various insecticides. Molecular analysis was performed to characterize the resistance genes and the molecular forms of An. gambiae. Insecticide residues in soil samples from breeding sites were investigated to determine major factors that can inhibit the normal growth of mosquito larvae by exposing susceptible and resistant laboratory strains.ResultsThere is a common use by local farmers of mineral fertilizer NPK at 200 kg/ha and urea at 50 kg/hectare following insecticide treatments in both the Calendar Control Program (CCP) and the Targeted Intermittent Control Program (TICP). By contrast, no chemicals are involved in Biological Program (BP) where farmers use organic and natural fertilizers which include animal excreta.Susceptibility test results confirmed a high resistance to DDT. Mean mortality of An. gambiae collected from the farms practicing CCP, TICP and BP methods were 33%, 42% and 65% respectively. An. gambiae populations from areas using the CCP and TICP programs showed resistance to permethrin with mortality of 50% and 58% respectively. By contrast, bioassay test results of An. gambiae from BP areas gave a high level of susceptibility to permethrin with an average mortality of 94%.Molecular analysis identified An. gambiae s.s, and An. arabiensis with a high predominance of An. gambiae s.s (90%). The two molecular forms, M and S, were also determined with a high frequency of the S form (96%).The Kdr gene seemed the main target- site resistance mechanism detected in CCP, TICP, and BP areas at the rates ranging from 32 to 78%. The frequency of ace-1R gene was very low (< 0.1).The presence of inhibiting factors in soil samples under insecticide treatments were found and affected negatively in delaying the development of An. gambiae larval populations.ConclusionsThis research shows that Kdr has spread widely in An. gambiae, mainly in CCP and TICP areas where pyrethroids are extensively used. To reduce the negative impact of pesticides use in cotton crop protection, the application of BP-like programs, which do not appear to select for vector resistance would be useful. These results could serve as scientific evidence of the spread of resistance due to a massive agricultural use of insecticides and contribute to the management of pesticides usage on cotton crops hence reducing the selection pressure of insecticides on An. gambiae populations.

Amplification of the PDGFRA, KIT and KDR genes in glioblastoma: a population-based study

The aim of this study was to establish the frequency of amplification of tyrosine kinase receptor genes PDGFRA, KIT and KDR (VEGFR2) at 4q12 in glioblastomas at a population level, and to assess whether such alterations have any clinical impact. Screening of 390 glioblastomas from a population-based study by differential PCR revealed amplification of the PDGFRA, KIT and KDR genes in 33 (8.5%), 17 (4.4%) and 13 (3.3%) glioblastomas, respectively. None of these alterations was prognostic for overall survival. Patients with glioblastoma showing KIT amplification were significantly younger than those with glioblastoma showing no amplification (51.7 ± 21.7 years vs. 59.3 ± 13.1 years; P=0.0231). Twelve glioblastomas showed concurrent amplification of the PDGFRA, KIT and KDR genes, whereas 18 glioblastomas showed PDGFRA amplification only. A significant inverse association was observed between KIT amplification and EGFR amplification (P=0.0260), whereas a borderline positive association was found between KIT amplification and TP53 mutation (P=0.0579). No significant difference was observed in the frequency of amplification of these genes in primary and secondary glioblastomas or in glioblastomas with and without IDH1 mutations, suggesting that amplification of PDGFRA, KIT and KDR may be implicated in the pathogenesis of a small fraction of both subtypes of glioblastoma.

Enzymes-based resistant mechanism in pyrethroid resistant and susceptible Aedes aegypti strains from northern Thailand

Previous studies have shown that permethrin resistance in our selected PMD-R strain of Aedes aegypti from Chiang Mai, Thailand, was associated with a homozygous mutation in the knockdown resistance (kdr) gene and other mechanisms. In this study, we investigated the metabolic mechanism of resistance of this strain compared to the PMD strain which is susceptible to permethrin. The permethrin susceptibility of larvae was determined by a dose-response bioassay. Two synergists, namely piperonyl butoxide (PBO) and bis(4-nitrophenyl)-phosphate (BNPP), were also added to determine if the resistance is conferred by oxidase or esterase enzymes, respectively. The LC(50) value for PMD-R (25.42 ppb) was ∼25-fold higher than for PMD (1.02 ppb). The LC(50) was reduced 3.03-fold in PMD-R and 2.27-fold in PMD when the oxidase inhibitor (PBO) was added, but little or no reduction was observed in the presence of BNPP, indicating that oxidative enzymes play an important role in resistance. However, the LC(50) previously observed in the heterozygous mutation form was reduced ∼eightfold, indicating that metabolic resistance is inferior to kdr. The levels of cytochrome P450 (P450) extracted from fourth instar larvae were similar in both strains and were about 2.3-fold greater in microsomal fractions than in crude supernatant and cytosol fractions. Microsome oxidase activities were determined by incubation with each of three substrates, i.e., permethrin, phenoxybenzyl alcohol (PBOH), and phenoxybenzaldehyde (PBCHO), in the presence or absence of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NAD(+)), PBO, and BNPP. It is known that hydrolysis of permethrin produces PBOH which is further oxidized to PBCHO by alcohol dehydrogenase (ADH) and then to phenoxybenzoic acid (PBCOOH) by aldehyde dehydrogenase (ALDH). When incubated with permethrin, a small amount of PBCOOH was detected in both strains (about 1.1-1.2 nmol/min/mg protein), regardless of the addition of NADPH. The addition of PBO resulted in about 70% and 50% reduction of PBCOOH in PMD and PMD-R, respectively. The addition of BNPP reduced PBCOOH about 50% and 35% in PMD and PMD-R, respectively. Using PBOH as substrate increased PBCOOH ∼16-fold and ∼40-fold in PMD and PMD-R, respectively. Using PBCHO as substrate increased PBCOOH ∼26-fold and ∼50-fold in PMD and PMD-R, respectively. The addition of NADPH, and particularly NAD(+), increased the level of PBCOOH. Together, the results have indicated the presence of a metabolic metabolism involving P450, ADHs, and ALDHs in both PMD and PMD-R strains, with greater enzyme activity in the latter.

Controlling malaria in Niger with bednets: how to take the Big Picture

Results and discussion During the first year of follow-up, vector dynamics showed an overall decrease in malaria transmission, but this was highly variable from village to village. Indeed, the second year of survey showed a return to transmission levels close to the pre-intervention period. A study of microsatellite markers showed no modification of the genetic structure in the two malaria vectors An. gambiae and An. arabiensis. However, a clear increase of the resistant allele of the kdr gene resistence to the pyrethroid insecticide used in the bed nets was observed. This clearly demonstrated that the main target vectors were reached. As measured by ovarian tracheoles observation, the parity rate of vectors was not significantly decreased. However, the index sporozoite decreased in the first year and remained low in the second year. Indeed, the parasite prevalence and the gametocyte carriage were shown decreasing in the children under five. This continued in 2007 as the National Malaria Control Program provided free artemisinin-based combination therapies as first line treatment for malaria in children under five. The national survey network showed a decrease in malaria cases, as confirmed in our sentinelle sites. However the biological confirmation of cases by HRP2 plasmodial antigen research has increased during the two years of follow-up. This could be explained by the more accurate diagnosis by peripheral health personal with the help of rapid diagnostic tests.

Molecular Ecology of Pyrethroid Knockdown Resistance in Culex pipiens pallens Mosquitoes

Pyrethroid insecticides have been extensively used in China and worldwide for public health pest control. Accurate resistance monitoring is essential to guide the rational use of insecticides and resistance management. Here we examined the nucleotide diversity of the para-sodium channel gene, which confers knockdown resistance (kdr) in Culex pipiens pallens mosquitoes in China. The sequence analysis of the para-sodium channel gene identified L1014F and L1014S mutations. We developed and validated allele-specific PCR and the real-time TaqMan methods for resistance diagnosis. The real-time TaqMan method is more superior to the allele-specific PCR method as evidenced by higher amplification rate and better sensitivity and specificity. Significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014S mutations in the kdr gene can be used as a molecular marker for deltamethrin resistance monitoring in natural Cx. pipiens pallens populations in the East China region. The laboratory selection experiment found that L1014F mutation frequency, but not L1014S mutation, responded to deltamethrin selection, suggesting that the L1014F mutation is the key mutation conferring resistance to deltamethrin. High L1014F mutation frequency detected in six populations of Cx. pipens pallens suggests high prevalence of pyrethroid resistance in Eastern China, calling for further surveys to map the resistance in China and for investigating alternative mosquito control strategies.