Calorie Restriction with or without Time-Restricted Eating in Weight Loss.

BACKGROUND: The long-term efficacy and safety of time-restricted eating for weight loss are not clear. METHODS: We randomly assigned 139 patients with obesity to time-restricted eating (eating only between 8:00 a.m. and 4:00 p.m.) with calorie restriction or daily calorie restriction alone. For 12 months, all the participants were instructed to follow a calorie-restricted diet that consisted of 1500 to 1800 kcal per day for men and 1200 to 1500 kcal per day for women. The primary outcome was the difference between the two groups in the change from baseline in body weight; secondary outcomes included changes in waist circumference, body-mass index (BMI), amount of body fat, and measures of metabolic risk factors. RESULTS: Of the total 139 participants who underwent randomization, 118 (84.9%) completed the 12-month follow-up visit. The mean weight loss from baseline at 12 months was -8.0 kg (95% confidence interval [CI], -9.6 to -6.4) in the time-restriction group and -6.3 kg (95% CI, -7.8 to -4.7) in the daily-calorie-restriction group. Changes in weight were not significantly different in the two groups at the 12-month assessment (net difference, -1.8 kg; 95% CI, -4.0 to 0.4; P = 0.11). Results of analyses of waist circumferences, BMI, body fat, body lean mass, blood pressure, and metabolic risk factors were consistent with the results of the primary outcome. In addition, there were no substantial differences between the groups in the numbers of adverse events. CONCLUSIONS: Among patients with obesity, a regimen of time-restricted eating was not more beneficial with regard to reduction in body weight, body fat, or metabolic risk factors than daily calorie restriction. (Funded by the National Key Research and Development Project [No. 2018YFA0800404] and others; ClinicalTrials.gov number, NCT03745612.).

Akkermansia muciniphila improves heat stress-impaired intestinal barrier function by modulating HSP27 in Caco-2 cells.

OBJECTIVE: Heat stress causes an elevation of intestinal epithelial barrier permeability and leads to multiple organ dysfunction in heatstroke. Akkermansia muciniphila (A. muciniphila) plays a role in maintaining intestinal integrity and improving the inflammatory state. This study aimed to investigate whether A. muciniphila could alleviate heat stress-induced dysfunction of intestinal permeability in Caco-2 monolayers and have the preventive effects on heatstroke. METHODS: Human intestinal epithelial Caco-2 cells were preincubated with live or pasteurized A. muciniphila then exposed to heat stress at 43 °C. Transepithelial electrical resistance (TEER) and the flux of horseradish peroxidase (HRP) across cell monolayers were measured to determine intestinal permeability. The levels of the tight junction proteins Occludin, ZO-1 and HSP27 were analyzed by Western blotting. These proteins were immunostained and localized by fluorescence microscopy. TJ morphology was observed using transmission electron microscopy (TEM). RESULTS: Both live and pasteurized A. muciniphila effectively attenuated the decrease in TEER and impairment of intestinal permeability in HRP flux induced by heat exposure. A. muciniphila significantly elevated the expression of Occludin and ZO-1 by promoting HSP27 phosphorylation. The distortion and redistribution of tight junction proteins and disruption of morphology were also effectively prevented by pretreatment with A. muciniphila. CONCLUSION: This study indicates for the first time that both live and pasteurized A. muciniphila play an important protective role against heat-induced permeability dysfunction and epithelial barrier damage.

Association of the gut microbiota and fecal short-chain fatty acids with skeletal muscle mass and strength in children.

We aimed to investigate whether the gut microbiota and fecal short-chain fatty acids (SCFAs) are associated with skeletal muscle mass and strength in healthy Chinese children aged 6-9 years. In this study, 412 children were enrolled. 16S rRNA gene sequencing was used to characterize the gut microbiota compositions. Fecal SCFAs were quantified using high-performance liquid chromatography. Dual X-ray absorptiometry was used to measure the total body lean soft tissue mass (TSM), total body fat mass (TBF), appendicular skeletal muscle mass (ASM), and appendicular fat mass (AFM). TSM/height2 (TSMI), ASM/height2 (ASMI), TSM/weight (TSMR), ASM/weight (ASMR), and the ratio of TSM/TBF and ASM/AFM were calculated. Handgrip strength (HGS) was measured using the Jamar® Plus+ Hand Dynamometer. A multiple regression analysis after adjustment for covariates and multiple test correction showed some operational taxonomic units in partial least squares models identified by Multivariate methods with Unbiased Variable selection analysis such as genera of Faecalibacterium, Lachnospira, Lachnospiraceae_ND3007_group, and Lachnospiraceae_UCG-004 were positively correlated with at least one measure of TSM, TSMI, ASM, ASMI, and ASMI Z-score (ß: 0.103-0.143, pFDR : .008-.032) but negatively correlated with at least one measure of TSMR, TSM/TBF, ASMR, ASM/AFM, and ASMR Z-score (ß: -0.185 to 0.124, pFDR  = .008-.045). Children with higher fecal butyric acid, acetic acid, and total SCFA levels exhibited higher TSM, ASM, TSMI, ASMI, and ASMI Z-score and lower TSM/TBF, ASM/AFM, TSMR, ASMR, and ASMR Z-score. However, after additional adjustment for TBF or body mass index, only the associations for Faecalitalea and Pyramidobacter still existed. Mediation analysis suggested that total body fat significantly mediated 66.3%-95.3% of the estimated association of microbiota and SCFAs with TSM, ASM, and ASMI Z-score. Our results suggest that the associations of gut microbiota and SCFAs with skeletal muscle quality in children may largely depend upon on total body fat content.

Gastric emptying of preoperative drinks is slower in adults with chronic energy deficiency: A 2 hour cross-over study among Chinese.

BACKGROUND AND OBJECTIVES: To investigate the effects of oral preoperative regimens on gastric emptying time in relation to BMI in Chinese adults. METHODS AND STUDY DESIGN: The enrolled 56 adults were divided into three groups (normal-weight, underweight, and overweight) and completed a regimen of two drinks after a 2-week interval. After drinking a carbohydrate regimen (CD, 50 g carbohydrates) or a carbohydrate glutamine regimen (CGD, 44 g carbohydrates and 6 g glutamine) labelled with 99mTc-DTPA (99mTc-diethylenetriaminepentaacetic acid), gastric emptying times T50 and T90 were measured using a curve derived from scintigraphic images. RESULTS: T50 and T90 had no significant difference between the CD and CGD regimens. T50 was significantly delayed in the underweight participants (BMI <18.5 kg/m2, as Chronic Energy Deficiency, CED) compared with the normal-weight participants after drinking CD (p=0.003) or CGD (p=0.002), as well as T90 after CD (p=0.019). There was no difference in glucose concentrations between the three groups. There are negative correlations between body weight and gastric emptying time T50 (r=-0.461, p=0.016) or T90 (r=-0.553, p=0. 003) after drinking CD, as well as T50 (r=-0.553, p=0.003) after drinking CGD. CONCLUSIONS: Underweight adults should be careful to take oral preoperative regimens 2 hours before surgery and consider reducing the volume because of a slower gastric emptying rate.

Genome sequence of Anopheles sinensis provides insight into genetics basis of mosquito competence for malaria parasites.

BACKGROUND: Anopheles sinensis is an important mosquito vector of Plasmodium vivax, which is the most frequent and widely distributed cause of recurring malaria throughout Asia, and particularly in China, Korea, and Japan. RESULTS: We performed 454 next-generation sequencing and obtained a draft sequence of A. sinensis assembled into scaffolds spanning 220.8 million base pairs. Analysis of this genome sequence, we observed expansion and contraction of several immune-related gene families in anopheline relative to culicine mosquito species. These differences suggest that species-specific immune responses to Plasmodium invasion underpin the biological differences in susceptibility to Plasmodium infection that characterize these two mosquito subfamilies. CONCLUSIONS: The A. sinensis genome produced in this study, provides an important resource for analyzing the genetic basis of susceptibility and resistance of mosquitoes to Plasmodium parasites research which will ultimately facilitate the design of urgently needed interventions against this debilitating mosquito-borne disease.

Associations of dietary habits, physical activity and cognitive views with gestational diabetes mellitus among Chinese women.

OBJECTIVE: To evaluate the relationship between dietary habits, physical activity and cognitive views and the risk of gestational diabetes mellitus (GDM) in Chinese women. DESIGN: A cross-sectional study to explore the potential risk factors of GMD through the International Physical Activity Questionnaire, an FFQ and a self-designed structured questionnaire, respectively. SETTING: Guangzhou, Guangdong Province, China. SUBJECTS: Chinese pregnant women (n 571) who underwent a 75-g oral glucose tolerance test at their 24th to 28th gestational week. RESULTS: Thirteen per cent of the investigated women were identified as having GDM, and an increased intake of local featured foods and lower physical activity were observed in the GDM-positive group v. the GDM-negative group. Women who regarded early-pregnancy morning sickness as relevant to fetal abnormalities and those with unlimited dietary intake after the ending of morning sickness both had an increased risk for GDM (P = 0·018 and P = 0·038, respectively). After multiple logistic regression analysis, cognitive views for unlimited food intake subsequent to morning sickness, increased consumption of energy-dense snack foods and high-glycaemic-index fruits were strongly associated with the risk of GDM (OR = 1·911, P = 0·032; OR = 1·050, P = 0·001; and OR = 1·002, P = 0·017, respectively). CONCLUSIONS: Local featured foods and incorrect cognitive views on pregnancy-related health were closely related to the risk of GDM in Chinese women. Intensive health education about pregnancy physiology and reasonable dietary and physical exercise behaviours should be strengthened for the control of GDM.

Retraction: Expression of vascular endothelial growth factor C in renal cell carcinoma and its correlation with pathological parameters and prognosis.

[This retracts the article DOI: 10.21037/tau-20-970.].

Ribose-phosphate pyrophosphokinase 1 (PRPS1) associated with deltamethrin resistance in Culex pipiens pallens.

Ribose-phosphate pyrophosphokinase 1 (PRPS1) was identified and isolated as a differentially expressed gene between deltamethrin-susceptible (DS) and deltamethrin-resistant (DR) Culex pipiens pallens and Aedes albopictus C6/36 cell line through microarray and 2D-Gel. An open reading frame of PRPS1 cloned from C. pipiens pallens has 1,011 bp and encodes for a 336 amino acids protein which shares high homology with Culex quinquefasciatus. Real-time polymerase chain reaction was used to determine the transcript expression level of PRPS1 in DS and DR strains. The expression levels of PRPS1 were higher in DR laboratory strains and natural population JXZ-DR, JXZ-LDR. PRPS1 was also detected and expressed at all developmental stages of C. pipiens pallens and increased expression level in DR3 strain than DS strain in the third and fourth instar larvae, female and male stages. In addition, to further investigate the role of PRPS1 in deltamethrin resistance, PRPS1 was transiently expressed in A. albopictus C6/36 cells and detected by western blotting. Cells transfected with PRPS1 had an increased resistance to deltamethrin compared with control cells. These results suggested that the increased expression level of PRPS1 may play roles in the regulation of deltamethrin resistance.

Effects of Time-Restricted Eating on Nonalcoholic Fatty Liver Disease: The TREATY-FLD Randomized Clinical Trial.

Importance: The efficacy and safety of time-restricted eating (TRE) on nonalcoholic fatty liver disease (NAFLD) remain uncertain. Objective: To compare the effects of TRE vs daily calorie restriction (DCR) on intrahepatic triglyceride (IHTG) content and metabolic risk factors among patients with obesity and NAFLD. Design, Setting, and Participants: This 12-month randomized clinical trial including participants with obesity and NAFLD was conducted at the Nanfang Hospital in Guangzhou, China, between April 9, 2019, and August 28, 2021. Interventions: Participants with obesity and NAFLD were randomly assigned to TRE (eating only between 8:00 am and 4:00 pm) or DCR (habitual meal timing). All participants were instructed to maintain a diet of 1500 to 1800 kcal/d for men and 1200 to 1500 kcal/d for women for 12 months. Main Outcomes and Measures: The primary outcome was change in IHTG content measured by magnetic resonance imaging; secondary outcomes were changes in body weight, waist circumference, body fat, and metabolic risk factors. Intention-to-treat analysis was used. Results: A total of 88 eligible patients with obesity and NAFLD (mean [SD] age, 32.0 [9.5] years; 49 men [56%]; and mean [SD] body mass index, 32.2 [3.3]) were randomly assigned to the TRE (n = 45) or DCR (n = 43) group. The IHTG content was reduced by 8.3% (95% CI, -10.0% to -6.6%) in the TRE group and 8.1% (95% CI, -9.8% to -6.4%) in the DCR group at the 6-month assessment. The IHTG content was reduced by 6.9% (95% CI, -8.8% to -5.1%) in the TRE group and 7.9% (95% CI, -9.7% to -6.2%) in the DCR group at the 12-month assessment. Changes in IHTG content were comparable between the 2 groups at 6 months (percentage point difference: -0.2; 95% CI, -2.7 to 2.2; P = .86) and 12 months (percentage point difference: 1.0; 95% CI, -1.6 to 3.5; P = .45). In addition, liver stiffness, body weight, and metabolic risk factors were significantly and comparably reduced in both groups. Conclusions and Relevance: Among adults with obesity and NAFLD, TRE did not produce additional benefits for reducing IHTG content, body fat, and metabolic risk factors compared with DCR. These findings support the importance of caloric intake restriction when adhering to a regimen of TRE for the management of NAFLD. Trial Registration: ClinicalTrials.gov Identifiers: NCT03786523 and NCT04988230.

[Cloning, expression and activity analysis of cutinase from Sclerotinia sclerotiorum].

Cutinase can degrade aliphatic and aromatic polyesters, as well as polyethylene terephthalate. Lack of commercially available cutinase calls for development of cost-effective production of efficient cutinase. In this study, eight cutinase genes were cloned from Sclerotinia sclerotiorum. The most active gene SsCut-52 was obtained by PCR combined with RT-PCR, expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography to study its characteristics and pathogenicity. Sscut-52 had a total length of 768 bp and 17 signal peptides at the N terminals. Phylogenetic analysis showed that its amino acid sequence had the highest homology with Botrytis keratinase cutinase and was closely related to Rutstroemia cutinase. Sscut-52 was highly expressed during the process of infecting plants by Sclerotinia sclerotiorum. Moreover, the expression level of Sscut-52 was higher than those of other cutinase genes in the process of sclerotia formation from mycelium. The heterologously expressed cutinase existed in the form of inclusion body. The renatured SsCut-52 was active at pH 4.0-10.0, and mostly active at pH 6.0, with a specific activity of 3.45 U/mg achieved. The optimum temperature of SsCut-52 was 20-30 ℃, and less than 60% of the activity could be retained at temperatures higher than 50 ℃. Plant leaf infection showed that SsCut-52 may promote the infection of Banlangen leaves by Sclerotinia sclerotiorum.

Expression of vascular endothelial growth factor C in renal cell carcinoma and its correlation with pathological parameters and prognosis.

BACKGROUND: Renal cell carcinoma (RCC) is a common malignant tumor of the urinary system. This study aimed to analyze the expression of vascular endothelial growth factor C (VEGF-C) in RCC and its relationship with pathological parameters and prognosis. METHODS: The clinical data of 68 patients who underwent surgical treatment and confirmed to be RCC by pathology from February 2012 to January 2014 were collected. The specimens of carcinoma tissues of the 68 patients were collected, among which 20 patients were collected from normal tissue specimens adjacent to the cancer more than 5 cm away from the tumor as controls. The VEGF-C expression level was detected by immunohistochemistry, and the relationship between VEGF-C expression and clinical pathological parameters and prognosis of RCC was analyzed. RESULTS: The positive expression rate of VEGF-C in cancer tissues of RCC patients was significantly higher than the adjacent tissues (85.29% vs. 15.00%) (P<0.05). The positive expression rate of VEGF-C in cancer tissues of RCC patients with low-to- moderate differentiation, stage III-IV, and lymph node metastasis was higher than that of RCC patients with high differentiation, stage I-II, and no lymph node metastasis (P<0.05). The survival rates of RCC patients at 1-, 3- and 5-year follow-up were 82.35% (56/68), 54.41% (37/68), and 32.35% (22/68), and the survival time of patients with positive VEGF-C expression was shorter than patients with negative expression (P<0.05). There was no significant difference in the 5-year survival rate among RCC patients according to sex or presence of muscular infiltration (P>0.05). Meanwhile, the 5-year survival rate was higher in patients with tumor diameter <5 cm, high differentiation, stage I-II, no lymph node metastasis, VEGF-C-negative expression, and aged <55 years old (P<0.05). The Cox regression model analysis showed that differentiation degree, clinical stage, lymph node metastasis, and VEGF-C expression were all independent risk factors affecting the prognosis of RCC patients (P<0.05). CONCLUSIONS: VEGF-C is highly expressed in cancer tissues of RCC patients, and is related to clinical stage, pathological differentiation, and lymph node metastasis, which maybe an effective factor of prognosis prediction.

[Role of lipophagy in the regulation of lipid metabolism and the molecular mechanism].

Recent studies have discovered a selective autophagy-lipophagy, which can selectively identify and degrade lipids and plays an important role in regulating cellular lipid metabolism and maintaining intracellular lipid homeostasis. The process of lipophagy can be directly or indirectly regulated by genes, enzymes, transcriptional regulators and other factors. This review examines the role of lipophagy in reducing liver lipid content, regulating pancreatic lipid metabolism, and regulating adipose tissue differentiation, and summarizes the findings of the molecules (Rab GTPase, enzymes, ion channels, transcription factors, small molecular substances) involved in the regulation of lipophagy, which points to new directions for the treatment of diseases caused by lipid accumulation.

[Blockade of TLR4/NF-κB pathway promotes the inflammation of Acinetobacter baumannii-infected rats].

Objective To explore the effects of TLR4 on Acinetobacter baumannii (A. baumannii) infection in a rat model. Methods Healthy male SD rats were divided into normal control group, TAK-242 treated group, A. baumannii treated group, TAK-242 and A. baumannii combined treatment group. Rats of TAK-242-treated group were prepared by caudal vein injection of TAK-242 (1 mg/kg). A. baumannii were isolated from intensive care unit (ICU) and the freshly grown bacteria (1×108 CFU/mL) were prepared. Each normal or TAK-242-treated rat was inoculated with 50 µL A. baumannii through trachea. The bronchoalveolar lavage fluid (BALF) and blood were collected at 72 hours after inoculation. The histopathology of lung was evaluated by HE staining. TNF-α and IL-6 were detected by ELISA. The level of phosphorylated NF-κBp65 (p-NF-κBp65) in peripheral blood mononuclear cells (PBMCs) was detected by Western blot analysis. Results A. baumannii were eliminated within 72 hours in normal rats, whereas bacteria continued to replicate rapidly in the lungs of TAK-242 A. baumannii treated group. The pulmonary inflammatory was more severe than the normal rats. The levels of TNF-α and IL-6 increased markedly after the infection. However, the levels of TNF-α and IL-6 in the TAK-242 combined with A. baumannii treated group were lower than those in the A. baumannii treated group. The level of p-NF-κBp65 increased significantly in the PBMCs of the normal rats 72 hours after infected with A. baumannii, but increased slightly in the TAK-242 combined with A. baumannii treated group. Conclusion TLR4/NF-κB pathway plays an important role in the process of A. baumannii infection, and TLR4 can be used as a target molecule in the treatment of A. baumannii infection.

[Activation of platelet activating factor receptor by Acinetobacter baumannii results in oxidative stress and apoptosis in human bronchial epithelial cells].

Objective To investigate the effect of platelet activating factor receptor (PAFR) on human bronchial epithelial (HBE) cells infected by Acinetobacter baumannii (A. baumannii). Methods HBE cells were divided into control group, ginkgolide B (GB) group, A. baumannii infected group, A. baumannii infection and inhibitor group. HBE cells were infected with low dose (1×103 CFU/mL), medium dose (1×105 CFU/mL) and high dose (1×107 CFU/mL) A. baumannii separated from clinical samples. The PAFR activity was blocked by the 10 µmol/L GB. The expression of PAFR was detected using Western blotting in HBE cells. The proliferation ability of HBE cells was detected using CCK-8 assay. The oxidative stress level was evaluated by superoxide dismutase (SOD) and malondialdehyde(MDA) kits. Apoptosis of HBE cells was observed by annexin V-FITC-V/PI staining. The phosphorylation level of PAFR and its downstream molecule JAK1/STAT1 in HBE cells were examined by Western blot analysis. Results Compared with the control group, the expression of PAFR increased significantly in A. baumannii infected group. A. baumannii infection could decrease cell vitality, but increase intracellular oxidative stress, apoptosis, and JAK1/STAT1 phosphorylation. Conclusion PAFR is an important mediator molecule for A. baumannii infection in HBE cells, and PAFR/JAK1/STAT1 signaling pathway plays an important role in the pulmonary infection of A. baumannii.

Assessment of marine debris in beaches or seawaters around the China Seas and coastal provinces.

Compared with United States of America (USA), Brazil, Chile, Australia, limited attention has been paid to marine debris research in China and few studies have attempted to quantify the abundance and mass of marine debris. In this study, firstly the general status and sources of marine debris in China were assessed in the time period between 2007 and 2014, and secondly marine debris situation was evaluated in three China Sea Areas (the North China Sea, the East China Sea and the South China Sea) from 2009 to 2013, and finally marine debris conditions and sources were analyzed in beaches or seawaters around some coastal provinces of China during 2007-2013. Based on above analysis, the primary conclusions were as follows: (1) The mean number and weight densities of beached marine debris (BMD) and submerged marine debris (SMD) were 4.30, 0.13items/100m(2) and 133.80, 22.60g/100m(2) in China from 2007 to 2014, respectively. The average number density of the large size FMD (LOSFMD) was 0.0024items/100m(2) and that of the small and medium size FMD (SMSFMD) was 0.30items/100m(2), and the mean weight density of the SMSFMD was 1.40g/100m(2) from 2008 to 2014. The SMD and FMD densities were at the low level and the BMD density was at the high level in China. (2) The marine debris primarily was comprised of plastic, Styrofoam, wood, glass, rubber, fabric/fiber and metal, which included almost all major categories of marine debris. (3) Sources of BMD and FMD were as follows: the first source was coastal/recreational activities, followed by other disposal sources, navigation/fishing activities and the activities related smoking, and the least source being those associated with medical/sanitary activities, while the source of SMD remained unknown. (4) The mean number and weight densities of BMD were the biggest in the North China Sea, while those of FMD and SMD were the highest in the northern South China Sea. The results of this study were beneficial to the establishment of management measures for dealing with ecological and environmental problems that were generated by the high speed socio-economic development in China.

[Association of dietary habits with gestational diabetes mellitus among Cantonese women].

OBJECTIVE: To evaluate the relationship between dietary habits and the risk of gestational diabetes mellitus (GDM) in Cantonese women. METHDS: This a cross-sectional study included 571 pregnant women who underwent a 75-g oral glucose challenge test at the 24th to 28th gestational week. Food frequency questionnaire (FFQ) was used by three dieticians to evaluate all the subjects' dietary habits. RESULTS: 13% of the investigated subjects were identified to have GDM (GDM⁺). No significant differences were found between the GDM⁺ and GDM⁻ groups in the intake of energy, macronutrients, fibers, or cholesterol. The amount of low- and middle-GI fruits consumed daily in the two groups was not statistically different, but the GDM⁺ subjects reported a significantly greater amount of high-GI fruit intake as well as energy-dense foods than the GDM⁻ subjects (P<0.05). Multiple logistic regression analysis revealed that increased consumption of energy-dense snack foods and high glycemic-index fruits were strongly associated with the risk of GDM. CONCLUSION: Local dietary composition (high-GI fruit and energy-dense foods) is closely related to the risk of GDM in Cantonese women, for whom intensive health education of dietary behavior is needed to control GDM.

Identification of proteins associated with pyrethroid resistance by iTRAQ-based quantitative proteomic analysis in Culex pipiens pallens.

BACKGROUND: Mosquito control based on chemical insecticides is considered as an important element in the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of pyrethroid resistance in important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. To date, the mechanisms of pyrethroid resistance are still unclear. Recent advances in proteomic techniques can facilitate to identify pyrethroid resistance-associated proteins at a large-scale for improving our understanding of resistance mechanisms, and more importantly, for seeking some genetic markers used for monitoring and predicting the development of resistance. METHODS: We performed a quantitative proteomic analysis between a deltamethrin-susceptible strain and a deltamethrin-resistant strain of laboratory population of Culex pipiens pallens using isobaric tags for relative and absolute quantitation (iTRAQ) analysis. Gene Ontology (GO) analysis was used to find the relative processes that these differentially expressed proteins were involved in. One differentially expressed protein was chosen to confirm by Western blot in the laboratory and field populations of Cx. pipiens pallens. RESULTS: We identified 30 differentially expressed proteins assigned into 10 different categories, including oxidoreductase activity, transporter activity, catalytic activity, structural constituent of cuticle and hypothetical proteins. GO analysis revealed that 25 proteins were sub-categorized into 35 hierarchically-structured GO classifications. Western blot results showed that CYP6AA9 as one of the up-regulated proteins was confirmed to be overexpressed in the deltamethrin-resistant strains compared with the deltamethrin-susceptible strains both in the laboratory and field populations. CONCLUSIONS: This is the first study to use modern proteomic tools for identifying pyrethroid resistance-related proteins in Cx. pipiens. The present study brought to light many proteins that were not previously thought to be associated with pyrethroid resistance, which further expands our understanding of pyrethroid resistance mechanisms. CYP6AA9 was overexpressed in the deltamethrin-resistant strains, indicating that CYP6AA9 may be involved in pyrethroid resistance and may be used as a potential genetic marker to monitor and predict the pyrethroid resistance level of field populations.

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.

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.

Cloning and characterization of prophenoloxidase A3 (proPOA3) from Culex pipiens pallens.

The prophenoloxidase subunit A3 (proPOA3) gene was cloned from Culex pipiens pallens, which had an open reading frame of 2061 bp encoding a putative 686 amino acid protein. The deduced amino acid sequence shares 98% with proPOA3 from Culex quinquefasciatus. ProPOA3 is expressed at all developmental stages of C. pipiens pallens. Significant negative correlation was observed between proPOA3 expression and deltamethrin resistance in resistant C. pipiens pallens. Furthermore, proPOA3 expression levels were significantly lower in deltamethrin-resistant mosquitoes than in susceptible mosquitoes collected at four locations in Eastern China. However, we did not find any substantial change in proPOA3 expression in field-collected resistant Anopheles mosquitoes. Moreover, overexpressing proPOA3 in C6/36 cells led to more sensitivity to deltamethrin treatment. In laboratory and field-collected resistant C. pipiens pallens, a valine to isoleucine mutation (769G>A) and two synonymous mutations (1116G>C and 1116G>A) were identified in proPOA3. In addition, the mutation frequency of 769G>A and 1116G>C increased gradually, which corresponded with raised deltamethrin resistance levels. Taken together, our study provides the first evidence that proPOA3 may play a role in the regulation of deltamethrin-resistance in C. pipiens pallens.