Early-life antibiotic exposure increases the risk of childhood overweight and obesity in relation to dysbiosis of gut microbiota: a birth cohort study.

BACKGROUND: Early-life antibiotic exposure is associated with the development of later obesity through the disruption of gut microbiota in the animal models. However, the related epidemiological evidence is still conflicting. METHODS: A birth cohort was consisted of 2140 mother-infant pairs in Chaoyang District Maternal and Child Health Care Hospital in this study. Here, their available antibiotic exposure during the first one year of life was ascertained using a open-ended questionnaire and related anthropometric parameters from the health screening program. The compositions of gut microbiota were comprehensively analyzed by16S rRNA high throughput sequencing. Then the spearman correlations were performed by the multiple covariance-adjusted regressions between the antibiotic exposure with anthropometric parameters and compositions of gut microbiota. RESULTS: Among the 2140 subjects, the antibiotic exposure during the first one year of life was 53.04%, mainly by Cephalosporins (53.39%) and Erythromycins(27.67%) for the treatment of respiratory tract infection (79.56%), which were not significantly different among the subgroups. Compared to the control group, both childhood overweight and obesity at two and a half years were higher in the antibiotic exposed group, with higher percents of Faecalibacterium, Agathobacter and Klebsiella, and lower percentage of Bifidobacterium. Moreover, there were positively potential associations between early-life antibiotic exposure with the accelerated anthropometric parameters and disruption of Faecalibacterium, Agathobacter, Klebsiella and Bifidobacterium at two and a half years. CONCLUSION: These above results proved that early-life antibiotic exposure was positively associated with the accelerated childhood overweight and obesity from one year to two and a half years by impacting the disorders of Faecalibacterium, Agathobacter, Klebsiella and Bifidobacterium, which would propose the theoretical basis for rationalizing the personalized antibiotic exposure among the infants to truly reflect the fairness of public health.

Requirement of a putative mitochondrial GTPase, GemA, for azole susceptibility, virulence, and cell wall integrity in Aspergillus fumigatus.

Drug resistance in fungal pathogens is a new challenge in clinical aspergillosis treatment. Mitochondria as dynamic organelles are involved in numerous biological processes in fungi, including drug resistance. However, little is known about the mechanism underlying mitochondrial regulation of the response of fungal pathogens to antifungal drugs. Here, we showed that a putative mitochondrial GTPase, GemA, a yeast Gem1 homolog, is crucial for the azole response and cell wall integrity in the opportunistic pathogen Aspergillus fumigatus. The fluorescence observation showed that GFP-labeled GemA is located in mitochondria, and loss of gemA results in aberrant giant mitochondrial morphology and abnormal mitochondrial membrane potential. Moreover, a ΔgemA mutant attenuates fungal virulence in the Galleria mellonella model of aspergillosis. Furthermore, gemA loss increases resistance to azoles and terbinafine but not to amphotericin B. Of note, RNA-seq combined with RT-qPCR showed that a series of drug efflux pumps were upregulated in the gemA deletion mutant. Deleting mdr1 or inhibiting the expression of drug efflux pumps can partially decrease the resistance to azoles resulting from the gemA mutant, implying that GemA influences azole response by affecting the expression of drug efflux pumps. Importantly, the ΔgemA mutant is susceptible to the cell wall-perturbing reagent CR, but not to CFW, and this defect can be partly rescued by hyperosmotic stress. TEM revealed that the cell wall of ΔgemA was thicker than that of the WT strain, demonstrating that GemA plays a role in cell wall composition and integrity. Collectively, we identified a putative mitochondrial GTPase, GemA, which is critical for hyphal growth, virulence, azole susceptibility, and cell wall integrity and acts by affecting mitochondrial function.

Dynamic colonization of gut microbiota and its influencing factors among the breast-feeding infants during the first two years of life.

The maturation of infant gut microbiota has lifelong implications on health, which has been proposed as the major events during the first year of life. However, little is known about their dynamic colonization and influencing elements among the first two-year infancy as well as into the adulthood. So based on the 16S rRNA sequencing data among 30 healthy breast-feeding mother-infant pairs with normal ranges of growth and development indicators from birth to two years old, the dynamic colonization of gut microbiota and its influencing factors were discussed using this birth cohort. Among these, we identified that the diversity of gut microbiota was significantly increased from six-month to two-year subgroups. The significantly dynamic trends of gut microbiota at the phylum (genus) level were that the percents of Firmicutes (Faecalibacterium, Blautia, Enterococcus, Subdoligranulum, Agathobacter, unidentified_Erysipelotrichaceae, Staphylococcus, unidentified_Ruminococcaceae, and Fusicatenibacter), Bacteroidetes and Verrucomicrobia were increased, while Actinobacteria (Bifidobacterium) and Proteobacteria (unidentified-Enterobacteriaceae and Klebsiella) were decreased with the increased ages from six months to two years old, which might simultaneously modulate the host pathways, such as the higher percents of chemoheterotrophy and fermentation, and lower percentages of nitrate_reduction, aerobic_chemoheterotrophy and so on. Furthermore, there were significant associations between maternal (milk microbiota, pre-pregnancy BMI, BMI increment during the pregnancy)/infant characteristics (BMI at birth and BMI gain), and the compositions of gut microbiota. However, no differences of gut microbiota were shown between the different sex and productive mode subgroups. Overall, the colonization of gut microbiota is significantly matured into the adulthood with the increased ages to two-years old and regulated by the above maternal/infant characteristics, which will provide a new direction for the host-gut microbiota interplay during the first two years of life.

Sex-specific maternal calcium requirements for the prevention of nonalcoholic fatty liver disease by altering the intestinal microbiota and lipid metabolism in the high-fat-diet-fed offspring mice.

The significance of maternal appropriate calcium intakes for energy metabolism in the offspring has been recognized. Nonalcoholic fatty liver disease (NAFLD) is considered as the hepatic manifestation of metabolic syndrome. So in this study, we proposed that there were long-term effects of maternal calcium status on the progress of NAFLD by altering the intestinal microbiota and lipid metabolism with attention to potential sex differences among the mouse offspring. Thirty-four-week female C57BL/6 J mice were subjected to obtain low, normal and high calcium reproductive diets throughout the gestation and lactation. After weaning, both the male and female mouse offspring were fed with the high-fat diet for 16 weeks, with the normal diet as control. Biochemical indicators in the plasma and hepatic tissue were measured using ELISA or enzymatic methods. The expression of lipid metabolism, inflammatory and fibrosis related genes was determined by RT-PCR. The intestinal microbiota was analyzed by 16S rRNA high-throughput sequencing. Maternal normal and low calcium intake could, respectively, inhibit the progress of high-fat diet induced NAFLD in the male and female mouse offspring, which was characterized by the least lipid droplets, inflammatory infiltration and fibrosis, the lowest concentrations of free fatty acids and triglyceridethe lowest expression of genes involving in de novo lipogenesis and the highest expression of genes related to lipid oxidation and  hydrolysis, inflammatory, and fibrosis. Pyrosequencing of 16S rRNA genes revealed that the male mouse offspring with maternal normal calcium intake and the female mouse offspring with maternal low calcium intake, after the high-fat diet feeding, had distinct intestinal microbiota, which was closer to thosein mice with the normal diet feeding. Analysis of the functional features for the different microbiota was compatible with the expression of genes associated with lipogenesis, lipid oxidation and hydrolysis. Thus, there is a sex-specific manner for maternal calcium requirement to inhibit the progress of offspring NAFLD, that might be less for the female offspring and more for the male offspring.

Genomic Variant Analyses in Pyrethroid Resistant and Susceptible Malaria Vector, Anopheles sinensis.

Anopheles sinensis is a major malaria vector in Southeast Asia. Resistance to pyrethroid insecticides in this species has impeded malaria control in the region. Previous studies found that An. sinensis populations from Yunnan Province, China were highly resistant to deltamethrin and did not carry mutations in the voltage-gated sodium channel gene that cause knockdown resistance. In this study, we tested the hypothesis that other genomic variants are associated with the resistance phenotype. Using paired-end whole genome sequencing (DNA-seq), we generated 108 Gb of DNA sequence from deltamethrin -resistant and -susceptible mosquito pools with an average coverage of 83.3× depth. Using a stringent filtering method, we identified a total of 916,926 single nucleotide variants (SNVs), including 32,240 non-synonymous mutations. A total of 958 SNVs differed significantly in allele frequency between deltamethrin -resistant and -susceptible mosquitoes. Of these, 43 SNVs were present within 37 genes that code for immunity, detoxification, cuticular, and odorant proteins. A subset of 12 SNVs were randomly selected for genotyping of individual mosquitoes by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and showed consistent allele frequencies with the pooled DNA-seq derived allele frequencies. In addition, copy number variations (CNVs) were detected in 56 genes, including 33 that contained amplification alleles and 23 that contained deletion alleles in resistant mosquitoes compared to susceptible mosquitoes. The genomic variants described here provide a useful resource for future studies on the genetic mechanism of insecticide resistance in this important malaria vector species.

A preliminary study on the differential expression of long noncoding RNAs and messenger RNAs in obese and control mice.

Obesity has become one of the public health problems that threatens children's health, but its specific etiology and pathogenesis are still unclear. Recently, many long noncoding RNAs (lncRNAs) have been shown to be involved in the occurrence of obesity. However, their roles are still poorly understood. Thus, the aim of this study was to discover the profiles of the lncRNAs and messenger RNAs (mRNAs) altered in obesity. Epididymal fat samples were collected from mice fed with control and high-fat diets (HFD) for 16 weeks to investigate the differentially expressed lncRNAs and mRNAs by lncRNA microarray, after which seven lncRNAs and nine mRNAs were validated using reverse-transcription polymerase chain reaction (RT-PCR). Bioinformatics analysis and predictions were used to determine the potential biofunctions of these differentially expressed lncRNAs. Then a coexpression network was constructed to determine the transcriptional regulatory relationship of the differentially expressed lncRNAs and mRNAs between the control and HFD groups. The body weight of the HFD group was much higher than that of the control group, as a result of the increased energy intake. In total, 8421 differentially expressed lncRNAs and 6840 mRNAs were profiled using the lncRNAs microarray. Bioinformatics predictions and the coexpression network all indicated that the occurrence of obesity was attributed to those differentially expressed lncRNAs and mRNAs associated with energy metabolism, cell differentiation, and oxidative phosphorylation. The expression levels of Cyp2e1, Atp5b, Hibch, Cnbp, Frmd6, Ptchd3, ENSMUST00000155948, AK140152, ENSMUST00000135194, and ENSMUST00000180861 were significantly different between the control and HFD groups. All these Results suggested that obesity was partially attributed to those lncRNAs associated with energy metabolism, cell differentiation, and oxidative phosphorylation.

Maternal dietary calcium status during pregnancy and lactation affects brain DHA accretion through modifying DNA methylation of fatty acid desaturases in the mouse offspring.

Disturbed calcium homeostasis has detrimental effects on brain development and function, particularly in early life because of epigenetic determination of early nutrition on later health. We hypothesized that the imbalance of calcium status in early life might have long-lasting effects on brain DHA accretion though epigenetic modification on fatty acid desaturases (Fads). Three to four week old C57BL/6J female mice were fed 3 reproductive diets with different calcium concentrations - low (LC, 0.25%), normal (NC, 0.70%) and high-calcium (HC, 1.20%) respectively throughout pregnancy and lactation. Maternal LC diet reduced tissue (brain and hepatic) DHA concentrations in both male and female offsprings at postnatal 21 day, with reductions in male instead of female offsprings in adulthood. Maternal HC diet only reduced hepatic DHA concentration in adult male offsprings. Furthermore, maternal LC diet reduced hepatic but increased brain expressions of Fads1 or Fads2 in 21-days old offsprings, with similar changes in adult male instead of female offsprings. Maternal HC diet reduced hepatic or brain expressions of Fads1 or Fads2 in 21-days old offsprings, and only reduced Fads2 in the liver with adult male offsprings. Determination of DNA methylation (CpG4, CpG5, CpG7,8, CpG14-17 and CpG19) showed that maternal LC diet caused hypermethylation of Fads2 promoter in the liver and hypomethylation in the brain in 21-days old offsprings, as well as in adult male offsprings. These data demonstrate that the imbalance of calcium intake in early life might have long-term gender-specific effects on brain accretion of DHA mediated by altered DNA methylation and associated expressions of Fads.

Abnormality in Maternal Dietary Calcium Intake During Pregnancy and Lactation Promotes Body Weight Gain by Affecting the Gut Microbiota in Mouse Offspring.

SCOPE: To investigate the effects of calcium status in early life on adult body weight and the underlying mechanisms involved in gut microbiota and related lipid metabolism. METHODS AND RESULTS: Three to four-week-old C57BL/6J female mice were fed diets with normal, insufficient, and excessive calcium respectively throughout pregnancy and lactation. The weaning male pups were fed with a high-fat diet for 16 weeks, with a normal-fat diet to the normal calcium group as control. The offspring fecal microbiota was analyzed by 16S rRNA high-throughput sequencing, and mRNA expressions of genes were determined by the real-time RT-PCR. Maternal insufficient or excessive calcium intake exacerbated offspring obesity, with expressional changes in the Fasn, Acc1, LPL, Fiaf, and PPAR-α genes in the liver or fat. The dysbiosis in gut microbiota in obese offspring was exacerbated by maternal imbalanced calcium intake, with increased Firmicutes and decreased Bacteroidetes in calcium insufficiency, and decreased Verrucomicrobia in calcium excess. Several genera, including Bacteroides, were reduced, and Lachnospiraceae and Lactobacillus were increased by maternal insufficient or excessive calcium intake. CONCLUSION: Imbalance in maternal calcium intake promotes body weight gain in offspring, which may be mediated by calcium's modulation on the gut microbiota and lipid metabolism.

Dietary calcium status during maternal pregnancy and lactation affects lipid metabolism in mouse offspring.

Calcium plays important roles in lipid metabolism and adipogenesis, but whether its status in early life affects later lipid profiles needs to be clarified. Three to four-week old C57BL/6J female mice were fed with three different reproductive diets containing normal, low (insufficient) and high (excessive) calcium concentrations respectively throughout pregnancy and lactation. At postnatal 21 days, the weaning male and female pups from each group were sacrificed for experiments and the remaining were fed with the normal chow diet for 16 weeks. Meanwhile, some of the weaning female pups from maternal low calcium diet group were fed with the normal calcium, low calcium and high calcium mature diets respectively for 8 weeks. Maternal insufficient or excessive calcium status during pregnancy and lactation programmed an abnormal expression of hepatic and adipose genes (PPAR-γ, C/EBP-α, FABP4, Fasn, UCP2, PPAR-α, HMG-Red1, Acc1, and SREBP-1c) in the offspring and this may lead to dyslipidemia and accumulation of hepatic triglyceride (TG) and total cholesterol (TC) in later life. The effects of maternal calcium status on lipid metabolism were found only in the female adult offspring, but were similar between offspring males and females at postnatal 21 days. Additionally, the dyslipidemia and hepatic lipid accumulation caused by insufficient calcium status in early life may be reversed to some extent by dietary calcium supplementation in later life.

[Component analysis of excretory/secretory protein from Trichinella spiralis adult worm].

Objective: To analyze the component of adult worm excretory/secretory protein(AWESP) from Trichinella spiralis using the shotgun method, and find out the active component underlying its regulatory effect on colitis in humans. Methods: The T. spiralis AWESP was prepared, separated by SDS-PAGE, lysed with trypsin, and analyzed by shotgun LC-MS/MS. The protein components were determined with the Masco software and classified using the Gene Ontology(GO) method in cellular components, molecular functions, and biological processes. Results: The AWESPs isolated by SDS-PAGE had a Mr of 15 000-116 000. A total of 280 proteins were revealed by LC-MS/MS, of which 96 were identified by Masco software, 98 were putative, and the remaining 86 were unclear. Preliminary results showed that 4 proteins had regulatory potential for colitis, including cysteine protease inhibitor, serine protease, 53 000 excretory/secretory antigen, and glutathione-S-transferase. GO enrichment analysis showed that the identified proteins had 104 different molecular functions, involved in 363 biological processes. Conclusion: As revealed by the Masco software, T. spiralis AWESP has complex components and 96 have been identified in this study. Four of them are preliminarily shown to be associated with the anti-colitis effect of T. spiralis.

Short Chain Fatty Acids Prevent High-fat-diet-induced Obesity in Mice by Regulating G Protein-coupled Receptors and Gut Microbiota.

Elucidating the mechanisms by which short chain fatty acids (SCFA) reduce body weight may assist in the development of an effective weight control strategy. Dietary supplementation of acetate, propionate, butyrate or their admixture was shown to significantly inhibit the body weight gain induced by high-fat diet feeding. Supplementation of SCFAs caused significant changes in the expressions of G-protein coupled receptor 43 (GPR43) and GPR41 characterized by increases in the adipose tissue and reductions in the colon. Additionally, they influenced the bacterial community structure in feces, with a reduction in the proportion of Firmicutes and an increase in the proportion of Bacteroidetes. The effects of dietary SCFAs on the GPR expression and gut microbiota composition may further result in body weight reduction by enhancing triglyceride hydrolysis and FFA oxidation in the adipose tissue, promoting beige adipogenesis and mitochondrial biogenesis, and inhibiting chronic inflammation.

A neural network prediction of environmental determinants of Anopheles sinensis knockdown resistance mutation to pyrethroids in China.

Selection pressure caused by long-term intensive use of insecticides is the key driving force in resistance development. Additional parameters such as environmental conditions may affect both the mosquito response to insecticides and the selection of resistance mechanisms. In this context, we analyzed the environmental determinants of kdr prevalence in Anopheles sinensis across China. We collected kdr frequency from 48 sites across central and southern China, together with key environmental factors including long-term climatic data, topographic features, main crops, and land cover types. Trend surface analysis found that the distribution of kdr frequency can be partitioned into three regions, namely central China (kdr frequency >80%), western China (kdr frequency varies from 0% to 60%), and southern China (kdr frequency <10%). Seven predictor variables were selected based on a radial basis function neural network model. A multilayer perceptron (MLP) network model revealed that the number of crops in a year was the most important predictor for the kdr mutation rate. Topography, long-term mean climate and land cover all contributed to the kdr mutation rate. The observed mean kdr frequency was 53.0% and the MLP network model-predicted mean was 52.6%, a 0.1% relative error. Predicted kdr frequencies closely matched the observed values. The model explained 92% of the total variance in kdr frequency. The results indicated that kdr was associated with the intensity of pesticide usage. Crop cultivation information, together with environmental factors, may well predict the spatial heterogeneity of kdr mutations in An. sinensis in China.

Landscape genetic structure and evolutionary genetics of insecticide resistance gene mutations in Anopheles sinensis.

BACKGROUND: Anopheles sinensis is one of the most abundant vectors of malaria and other diseases in Asia. Vector control through the use of insecticides is the front line control method of vector-borne diseases. Pyrethroids are the most commonly used insecticides due to their low toxicity to vertebrates and low repellency. However, the extensive use of insecticides has imposed strong selection pressure on mosquito populations for resistance. High levels of resistance to pyrethroid insecticides and various mutations and haplotypes in the para sodium channel gene that confers knockdown resistance (kdr) have been detected in An. sinensis. Despite the importance of kdr mutations in pyrethroid resistance, the evolutionary origin of the kdr mutations is unknown. This study aims to examine the evolutionary genetics of kdr mutations in relation to spatial population genetic structure of An. sinensis. METHODS: Adults or larvae of Anopheles sinensis were collected from various geographic locations in China. DNA was extracted from individual mosquitoes. PCR amplification and DNA sequencing of the para-type sodium channel gene were conducted to analyse kdr allele frequency distribution, kdr codon upstream and downstream intron polymorphism, population genetic diversity and kdr codon evolution. The mitochondrial cytochrome c oxidase COI and COII genes were amplified and sequenced to examine population variations, genetic differentiation, spatial population structure, population expansion and gene flow patterns. RESULTS: Three non-synonymous mutations (L1014F, L1014C, and L1014S) were detected at the kdr codon L1014 of para-type sodium channel gene. A patchy distribution of kdr mutation allele frequencies from southern to central China was found. Near fixation of kdr mutation was detected in populations from central China, but no kdr mutations were found in populations from southwestern China. More than eight independent mutation events were detected in the three kdr alleles, and at least one of them evolved multiple times subsequent to their first divergence. Based on sequence analysis of the mitochondrial COI and COII genes, significant and large genetic differentiation was detected between populations from southwestern China and central China. The patchy distribution of kdr mutation frequencies is likely a consequence of geographic isolation in the mosquito populations and the long-term insecticide selection. CONCLUSION: Our results indicate multiple origins of the kdr insecticide-resistant alleles in An. sinensis from southern and central China. Local selection related to intense and prolonged use of insecticide for agricultural purposes, as well as frequent migrations among populations are likely the explanations for the patchy distribution of kdr mutations in China. On the contrary, the lack of kdr mutations in Yunnan and Sichuan is likely a consequence of genetic isolation and absence of strong selection pressure. The present study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure.

[Construction and analysis of a breast cancer gene-drug network model].

OBJECTIVE: To construct a breast cancer gene-drug network model for extracting and predicting the correlations between breast cancer-related genes and drugs. METHODS: We developed an algorithm based on the ABC principle and the association rules to obtain the correlations between the biological entities. For breast cancer, we constructed 3 different correlations (gene-gene, drug-drug and gene-drug) and used the R language to implement the associated network model. The reliability of the algorithm was verified by ROC curve. RESULTS: We identified 185 breast cancer-associated genes and 98 associations between them, 97 drugs and 170 associations between them. The breast cancer genes-drugs network contained 127 genes and 77 drugs with 384 associations between them. CONCLUSIONS: We identified a large number of different correlations between the breast cancer-related genes and drugs and close correlations between some biological entity pairs that have not yet been reported, which may provide a new strategy for experimental design for testing personalized breast cancer treatment.

[In vitro killing of Trichinella spiralis muscle larvae by exogenous nitric oxide from SNP].

OBJECTIVE: To study the lethal effect of exogenous nitric oxide donor sodium nitroprusside (SNP) on the muscle larvae of Trichinella spiralis in vitro cultivation. METHODS: T. spiralis muscle larvae isolated from the infected BALB/c mice were formulated into a 1,000 larva/ml suspension with RPMI 1640 medium, and 0.1 ml suspension per orifice was cultured with SNP at 37°C in a humidified 5% CO2 atmosphere. The final concentrations of SNP were 0.02, 0.05, 0.10, 0.20, 0.50 and 1.00 mmol/L, respectively, and then the experiments were divided into 5 groups:1.00 mmol/L SNP (control group, Group A), 0.15 mmol/L FeSO4+ 1.00 mmol/L SNP (Group B), 1.00 mmol/L L-cysteine + 1.00 mmol/L SNP (Group C), 0.15 mmol/L FeSO4+ 1.00 mmol/L L-cysteine + 1.00 mmol/L SNP (Group D) and 0.15 mmol/L Hemoglobin + 1.00 mmol/L SNP (Group E). All the groups were incubated with T. spiralis muscle larvae in RPMI 1640 medium. The survivability of the muscle larvae was observed by steromicroscope and the differences of inhibition ratio among these groups were analyzed 4 d after the incubation. Results SNP 0.02 mmol/L was not cytotoxic to the muscle larvae with an inhibition of (5.50 ± 1.80) %. The mortality rates of SNP 0.05, 0.10, 0.20, 0.50, 1.00 mmol/L groups were (20.19±2.71)%, (29.21±2.12)%, (41.81±2.03)%, (47.85±3.79)%, (60.98±5.19)%, respectively, significantly higher than that of the control group[(4.93±0.25) %, all P < 0.051]. There was a positive liner correlation between the mortality of muscle larvae and SNP concentrations in the range of 0.02-1.00 mmol/L. Next, Group A, B, C, D and E led to the mortalities from (60.98±5.19)% to (49.48±1.34)%, (47.29±2.79)%, (26.28±1.37)%, (17.93±3.49)%, respectively, and all the differences between Group A and the other four groups were statistically significant (all P < 0.05). CONCLUSIONS: Exogenous nitric oxide released from SNP can kill the muscle larvae of T. spiralis. However, hemoglobin, L-cysteine, and FeSO4 can reverse the lethal effect on the parasites. The best inhibitor was hemoglobin.

Development of Resistance to Pyrethroid in Culex pipiens pallens Population under Different Insecticide Selection Pressures.

Current vector control programs are largely dependent on pyrethroids, which are the most commonly used and only insecticides recommended by the World Health Organization for insecticide-treated nets (ITNs). However, the rapid spread of pyrethroid resistance worldwide compromises the effectiveness of control programs and threatens public health. Since few new insecticide classes for vector control are anticipated, limiting the development of resistance is crucial for prolonging efficacy of pyrethroids. In this study, we exposed a field-collected population of Culex pipiens pallens to different insecticide selection intensities to dynamically monitor the development of resistance. Moreover, we detected kdr mutations and three detoxification enzyme activities in order to explore the evolutionary mechanism of pyrethroid resistance. Our results revealed that the level of pyrethroid resistance was proportional to the insecticide selection pressure. The kdr and metabolic resistance both contributed to pyrethroid resistance in the Cx. pipiens pallens populations, but they had different roles under different selection pressures. We have provided important evidence for better understanding of the development and mechanisms of pyrethroid resistance which may guide future insecticide use and vector management in order to avoid or delay resistance.

[Analysis of population genetic structure of Anopheles sinensis based on mitochondrial DNA cytochrome oxidase subunit I gene fragment].

OBJECTIVE: To study the population genetic variation, genetic diversity and phylogenesis of Anopheles sinensis in China. METHODS: Anopheles sinensis samples collected from Shandong, Anhui, Jiangsu, Guizhou, and Yunnan Provinces and Guangxi Zhuang Autonomous Region with different geographical conditions between 2010 and 2012 were analyzed by mitochondrial DNA cytochrome oxidase subunit I (mtDNA-COI) gene amplification and sequencing. Bioedit 7.0 and DnaSP 5.0 software was used to compare the gene sequences and analyze the population genetic structure, respectively. Arlequin 3.1 was used to calculate the genetic distance and parameters of population differentiation. The relationship between the geographic and genetic distances was analyzed using IBD Web Service. PHYLIP 3.6 was used to construct the phylogenetic tree. RESULTS: PCR amplification and sequencing was performed successfully for 6 Anopheles sinensis populations containing 123 female mosquitoes. The length of mtDNA-COI gene fragment was 841 bp with an average A+T content of 71.2% and G+C content of 28.8%. High nucleotide diversity and genetic differentiation were observed among the Anopheles sinensis populations based on mtDNA-COI gene. Analysis of the molecular variance revealed a greater variation between populations than that within populations with isolation by distance between the populations. The Anopheles sinensis populations appeared to have undergone expansion, but the Yunnan population constituted an isolated branch in the phylogenetic tree. CONCLUSION: mtDNA-COI can serve as the molecular marker to analyze population genetic variation and phylogenesis of Anopheles sinensis. The Yunnan population shows a phylogenetic difference from the other populations analyzed in this study.

Optimized codon usage enhances the expression and immunogenicity of DNA vaccine encoding Taenia solium oncosphere TSOL18 gene.

Cysticercosis due to larval cysts of Taenia solium, is a serious public health problem affecting humans in numerous regions worldwide. The oncospheral stage-specific TSOL18 antigen is a promising candidate for an anti-cysticercosis vaccine. It has been reported that the immunogenicity of the DNA vaccine may be enhanced through codon optimization of candidate genes. The aim of the present study was to further increase the efficacy of the cysticercosis DNA vaccine; therefore, a codon optimized recombinant expression plasmid pVAX1/TSOL18 was developed in order to enhance expression and immunogenicity of TSOL18. The gene encoding TSOL18 of Taenia solium was optimized, and the resulting opt-TSOL18 gene was amplified and expressed. The results of the present study showed that the codon-optimized TSOL18 gene was successfully expressed in CHO-K1 cells, and immunized mice vaccinated with opt-TSOL18 recombinant expression plasmids demonstrated opt­TSOL18 expression in muscle fibers, as determined by immunohistochemistry. In addition, the codon-optimized TSOL18 gene produced a significantly greater effect compared with that of TSOL18 and active spleen cells were markedly stimulated in vaccinated mice. 3H-thymidine incorporation was significantly greater in the opt-TSOL18 group compared with that of the TSOL18, pVAX and blank control groups (P<0.01). In conclusion, the eukaryotic expression vector containing the codon-optimized TSOL18 gene was successfully constructed and was confirmed to be expressed in vivo and in vitro. The expression and immunogenicity of the codon-optimized TSOL18 gene were markedly greater compared with that of the un-optimized gene. Therefore, these results may provide the basis for an optimized TSOL18 gene vaccine against cysticercosis.

Transcriptome profiling of pyrethroid resistant and susceptible mosquitoes in the malaria vector, Anopheles sinensis.

BACKGROUND: Anopheles sinensis is a major malaria vector in China and other Southeast Asian countries, and it is becoming increasingly resistant to the insecticides used for agriculture, net impregnation, and indoor residual spray. Very limited genomic information on this species is available, which has hindered the development of new tools for resistance surveillance and vector control. We used the 454 GS FLX system and generated expressed sequence tag (EST) databases of various life stages of An. sinensis, and we determined the transcriptional differences between deltamethrin resistant and susceptible mosquitoes. RESULTS: The 454 GS FLX transcriptome sequencing yielded a total of 624,559 reads (average length of 290 bp) with the pooled An. sinensis mosquitoes across various development stages. The de novo assembly generated 33,411 contigs with average length of 493 bp. A total of 8,057 ESTs were generated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. A total of 2,131 ESTs were differentially expressed between deltamethrin resistant and susceptible mosquitoes collected from the same field site in Jiangsu, China. Among these differentially expressed ESTs, a total of 294 pathways were mapped to the KEGG database, with the predominant ESTs belonging to metabolic pathways. Furthermore, a total of 2,408 microsatellites and 15,496 single nucleotide polymorphisms (SNPs) were identified. CONCLUSIONS: The annotated EST and transcriptome databases provide a valuable genomic resource for further genetic studies of this important malaria vector species. The differentially expressed ESTs associated with insecticide resistance identified in this study lay an important foundation for further functional analysis. The identified microsatellite and SNP markers will provide useful tools for future population genetic and comparative genomic analyses of malaria vectors.

Multiple resistances and complex mechanisms of Anopheles sinensis mosquito: a major obstacle to mosquito-borne diseases control and elimination in China.

Malaria, dengue fever, and filariasis are three of the most common mosquito-borne diseases worldwide. Malaria and lymphatic filariasis can occur as concomitant human infections while also sharing common mosquito vectors. The overall prevalence and health significance of malaria and filariasis have made them top priorities for global elimination and control programmes. Pyrethroid resistance in anopheline mosquito vectors represents a highly significant problem to malaria control worldwide. Several methods have been proposed to mitigate insecticide resistance, including rotational use of insecticides with different modes of action. Anopheles sinensis, an important malaria and filariasis vector in Southeast Asia, represents an interesting mosquito species for examining the consequences of long-term insecticide rotation use on resistance. We examined insecticide resistance in two An. Sinensis populations from central and southern China against pyrethroids, organochlorines, organophosphates, and carbamates, which are the major classes of insecticides recommended for indoor residual spray. We found that the mosquito populations were highly resistant to the four classes of insecticides. High frequency of kdr mutation was revealed in the central population, whereas no kdr mutation was detected in the southern population. The frequency of G119S mutation in the ace-1 gene was moderate in both populations. The classification and regression trees (CART) statistical analysis found that metabolic detoxification was the most important resistance mechanism, whereas target site insensitivity of L1014 kdr mutation played a less important role. Our results indicate that metabolic detoxification was the dominant mechanism of resistance compared to target site insensitivity, and suggests that long-term rotational use of various insecticides has led An. sinensis to evolve a high insecticide resistance. This study highlights the complex network of mechanisms conferring multiple resistances to chemical insecticides in mosquito vectors and it has important implication for designing and implementing vector resistance management strategies.