Evaluation of the evolutionary genetics and population structure of Culex pipiens pallens in Shandong province, China based on knockdown resistance (kdr) mutations and the mtDNA-COI gene.

BACKGROUND: Mosquitoes are important vectors for a range of diseases, contributing to high rates of morbidity and mortality in the human population. Culex pipiens pallens is dominant species of Culex mosquito in northern China and a major vector for both West Nile virus and Bancroftian filariasis. Insecticide application were largely applied to control the mosquito-mediated spread of these diseases, contributing to increasing rates of resistance in the mosquito population. The voltage-gated sodium channel (Vgsc) gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). While these kdr mutations are known to be critical to pyrethroid resistance, their evolutionary origins remain poorly understood. Clarifying the origins of these mutations is potential to guide further vector control and disease prevention efforts. Accordingly, the present study was designed to study the evolutionary genetics of kdr mutations and their association with the population structure of Cx. p. pallens in Shandong province, China. METHODS: Adult Culex females were collected from Shandong province and subjected to morphological identification under a dissection microscope. Genomic DNA were extracted from the collected mosquitoes, the Vgsc gene were amplified via PCR and sequenced to assess kdr allele frequencies, intron polymorphisms, and kdr codon evolution. In addition, population genetic diversity and related population characteristics were assessed by amplifying and sequencing the mitochondrial cytochrome C oxidase I (COI) gene. RESULTS: Totally, 263 Cx. p. pallens specimens were used for DNA barcoding and sequencing analyses to assess kdr allele frequencies in nine Culex populations. The kdr codon L1014 in the Vgsc gene identified two non-synonymous mutations (L1014F and L1014S) in the analyzed population. These mutations were present in the eastern hilly area and west plain region of Shandong Province. However, only L1014F mutation was detected in the southern mountainous area and Dongying city of Shandong Province, where the mutation frequency was low. Compared to other cities, population in Qingdao revealed significant genetic differentiation. Spatial kdr mutation patterns are likely attributable to some combination of prolonged insecticide-mediated selection coupled with the genetic isolation of these mosquito populations. CONCLUSIONS: These data suggest that multiple kdr alleles associated with insecticide resistance are present within the Cx. p. pallens populations of Shandong Province, China. The geographical distributions of kdr mutations in this province are likely that the result of prolonged and extensive insecticide application in agricultural contexts together with frequent mosquito population migrations. In contrast, the low-frequency kdr mutation detected in central Shandong Province populations may originate from the limited selection pressure in this area and the relative genetic isolation. Overall, the 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.

A reassessment of the artificial infection of three predominant mosquito species with Plasmodium vivax in Shandong Province, China.

BACKGROUND & OBJECTIVES: Under certain ecological circumstances, pathogens are able to rapidly adapt to new vectors. The great capacity of Plasmodium spp. to adapt to new anopheline mosquito vectors on different continents and the continuous ecological changes attributed to humans might promote their adaptation to culicine vectors, which are known to infect humans. Based on our current knowledge, it is difficult to predict whether such adaptations will occur. This study was aimed to determine the infection susceptibility of Anopheles sinensis, Culex tritaeniorhynchus and Cx. pipiens pallens to Plasmodium vivax in Shandong Province of China. METHODS: The susceptibility of the three predominant species of mosquitoes -An. sinensis, Cx. tritaeniorhynchus and Cx. pipiens pallens in Shandong Province was compared with a direct membrane feeding assay with 15 batches of Shandong strain mono-infected gametocyte-containing blood collected from Plasmodium vivax-infected patients. Infectivity was measured by dissecting the midguts and salivary glands of the mosquitoes. The presence of oocysts and sporozoites was determined by microscopy at 6 and 22 days post-blood feeding. RESULTS: From the 15 batches of mosquitoes that were fed infected blood, oocysts and sporozoites were detected only in 7th, 13th and 15th batches of infection for An. sinensis, and no oocysts or sporozoites were detected in Cx. tritaeniorhynchus or Cx. pipiens pallens. The positive rate of An. sinensis infection was 21.2, 13 and 36.3% in the three batches of mosquitoes, with an average infection rate of 23.5%. INTERPRETATION & CONCLUSION: The susceptibility of An. sinensis to P. vivax was very high in Shandong Province. Cx. tritaeniorhynchus and Cx. pipiens pallens failed to exhibit susceptibility to P. vivax.

[Research Progress on the Molecular Mechanisms of Mosquito Innate Immunity].

As the important vector of the mosquito-borne diseases, mosquitos rely on their innate immune system against pathogens infection, which includes cellular immunity and humoral immunity. Both of them are interacted and coordinated, via the pattern recognition receptors, immune signaling, antimicrobial peptides, phenoloxidase-based melanization, phagocytosis, and so on. This paper reviews the research progress on the molecular mechanisms of mosquito innate immunity.

Measure post-bloodmeal dispersal of mosquitoes and duration of radioactivity by using the isotope ³²P.

The radioactive isotope (32)P-labeled disodium phosphate (Na2H(32)PO4) was injected via the jugular vein into a cow kept in a shed in Maozhuang Village, Cao Township of Shanxian County, China. Over the following 5 d, mosquitoes feeding on the cow were captured at distances up to 400 m to determine dispersal distance. The duration of radioactivity in the cow and marked mosquitoes was 10 d. The results showed that after blood feeding, Anopheles sinensis and Culex tritaeniorhynchus temporarily rested in the cattle shed and then flew outdoors. In contrast, Culex pipiens pallens remained in the cattle shed after feeding. These findings confirmed that local An. sinensis and Cx. tritaeniorhynchus were partially endophilic and tended to rest out of doors, whereas Cx. pipiens pallens was endophilic. For marked An. sinensis and Cx. tritaeniorhynchus, there was a significant tendency for dispersal to be in a northeast and east direction, probably because of the presence of heavy shading by an agricultural field, a small river for mosquito oviposition sites, and locations downwind from the blood source. The furthest flight distances for An. sinensis and Cx. tritaeniorhynchus were 210 and 240 m; therefore, control of these mosquitoes should include resting places indoors and outdoors within a radius of 250 m from confirmed cases.

Effects of recombinant human growth hormone therapy on carbohydrate, lipid and protein metabolisms of children with Turner syndrome.

OBJECTIVE: To study the effect of recombinant human growth hormone (rhGH) therapy on carbohydrate, lipid and protein metabolisms of Turner syndrome (TS). Metho d s: Total 45 patients with TS admitted between Jul. 2008 and Jun. 2011 were involved in this study. All patients received the clinical evaluation of body fat, plasma lipids, proteins and oral glucose tolerance test (OGTT) before and after rhGH therapy. Results : Our results indicated a significant decrease of body fat (FAT%) from 23.56±4.21 to 18.71±2.23 but no obvious change on the level of fat mass (FM) (p>0.05) was observed after rhGH therapy. We also detected significant changes on plasma high-density lipoprotein cholesterol (HDL-C) from (1.65±0.58 mmol/L) to (2.20±0.65 mmol/L) and low-density lipoprotein cholesterol (LDH-C) from (2.55±0.55 mmol/L) to (2.10±0.54 mmol/L) after rhGH exposure. However, no statistical significance was detected on the level of plasma triglyceride (TG), cholesterol (CHO). Interestingly, the levels of plasma retinol binding protein (RbP) (32.55±4.28mg/L), transferrin (TRF) (2.95±0.40 mg/L), serum albumin (PRE) (250.00±45.50 mg/L) and albumin (propagated) (33.58±4.25 mg/L) were significantly increased. When it goes to the oral glucose tolerance test (OGTT) test, there were 10 impaired glucose tolerance (IGT) cases among all patients before and after rhGH therapy. No significant change was observed on homeostasis model assessment- insulin resistance (HOMA-IR) level during rhGH intervention. Conclusion : Abnormal lipid and protein metabolisms of the children with TS can be improved with rhGH therapy for 6 months.

[Experimental observation on host preference of Anopheles sinensis].

OBJECTIVE: To observe the host preference of Anopheles sinensis captured by outdoor human or cattle baits. METHODS: A large number of non-blood-fed An. sinensis females were collected by overnight trapping outdoor with human and cattle in the rice paddy field in Shan County of Shandong Province, and took back to the lab, and individually labeled as human baits group and cattle baits group, fed with mouse blood. The host preference of parent, F1 and F25 generations of the two groups were observed by mark-release-recapture methods in a large greenhouse. RESULTS: The recapture rates of parent, F1 and F25 generations were 39.02% (1332/3414), 37.97% (2583/6803), and 30.55% (1523/4986), respectively. In parent generation, the proportion of mosquitoes from human baits group and cattle baits group collected by human-bait and cattle-bait was 54.07% (339/627) and 58.01% (409/705), respectively (χ2=19.42, P<0.01); in F1 generation, that of the two groups was 51.03% (669/1311) and 55.11% (701/1272), respectively (χ2= 9.75, P<0.01); in F25 generation, that of the two groups was 51.98% (342/658) and 52.37 (453/865), respectively (χ2=2.82, P>0.05). CONCLUSION: After culture for 25 generations in an experimental condition, the host preference for human or cattle of An. sinensis maybe change.

In Vitro and In Vivo Validation of CYP6A14 and CYP6N6 Participation in Deltamethrin Metabolic Resistance in Aedes albopictus.

The extensive use of chemical insecticides for public health and agricultural purposes has increased the occurrence and development of insecticide resistance. This study used transcriptome sequencing to screen 10 upregulated metabolic detoxification enzyme genes from Aedes albopictus resistant strains. Of these, CYP6A14 and CYP6N6 were found to be substantially overexpressed in the deltamethrin-induced expression test, indicating their role in deltamethrin resistance in Ae. albopictus. Furthermore, the corresponding 60-kDa recombinant proteins, CYP6A14 and CYP6N6, were successfully expressed using the Escherichia coli expression system. Enzyme activity studies revealed that CYP6A14 (5.84 U/L) and CYP6N6 (6.3 U/L) have cytochrome P450 (CYP450) enzyme activity. In vitro, the metabolic analysis revealed that the recombinant proteins degraded deltamethrin into 1-oleoyl-sn-glycero-3-phosphoethanolamine and 2',2'-dibromo-2'-deoxyguanosine. Subsequently, the CYP450 genes in larvae of Ae. albopictus were silenced by RNA interference technology to study deltamethrin resistance in vivo. The silencing of CYP6A14 and CYP6N6 increased the mortality rate of mosquitoes without affecting their survival time, spawning quantity, hatching rate, and other normal life activities. Altogether, CYP6A14 and CYP6N6 belong to the CYP6 family and mutually increase deltamethrin resistance in Ae. albopictus.

Climate change and Aedes albopictus risks in China: current impact and future projection.

BACKGROUND: Future distribution of dengue risk is usually predicted based on predicted climate changes using general circulation models (GCMs). However, it is difficult to validate the GCM results and assess the uncertainty of the predictions. The observed changes in climate may be very different from the GCM results. We aim to utilize trends in observed climate dynamics to predict future risks of Aedes albopictus in China. METHODS: We collected Ae. albopictus surveillance data and observed climate records from 80 meteorological stations from 1970 to 2021. We analyzed the trends in climate change in China and made predictions on future climate for the years 2050 and 2080 based on trend analyses. We analyzed the relationship between climatic variables and the prevalence of Ae. albopictus in different months/seasons. We built a classification tree model (based on the average of 999 runs of classification and regression tree analyses) to predict the monthly/seasonal Ae. albopictus distribution based on the average climate from 1970 to 2000 and assessed the contributions of different climatic variables to the Ae. albopictus distribution. Using these models, we projected the future distributions of Ae. albopictus for 2050 and 2080. RESULTS: The study included Ae. albopictus surveillance from 259 sites in China found that winter to early spring (November-February) temperatures were strongly correlated with Ae. albopictus prevalence (prediction accuracy ranges 93.0-98.8%)-the higher the temperature the higher the prevalence, while precipitation in summer (June-September) was important predictor for Ae. albopictus prevalence. The machine learning tree models predicted the current prevalence of Ae. albopictus with high levels of agreement (accuracy > 90% and Kappa agreement > 80% for all 12 months). Overall, winter temperature contributed the most to Ae. albopictus distribution, followed by summer precipitation. An increase in temperature was observed from 1970 to 2021 in most places in China, and annual change rates varied substantially from -0.22 ºC/year to 0.58 ºC/year among sites, with the largest increase in temperature occurring from February to April (an annual increase of 1.4-4.7 ºC in monthly mean, 0.6-4.0 ºC in monthly minimum, and 1.3-4.3 ºC in monthly maximum temperature) and the smallest in November and December. Temperature increases were lower in the tropics/subtropics (1.5-2.3 ºC from February-April) compared to the high-latitude areas (2.6-4.6 ºC from February-April). The projected temperatures in 2050 and 2080 by this study were approximately 1-1.5 °C higher than those projected by GCMs. The estimated current Ae. albopictus risk distribution had a northern boundary of north-central China and the southern edge of northeastern China, with a risk period of June-September. The projected future Ae. albopictus risks in 2050 and 2080 cover nearly all of China, with an expanded risk period of April-October. The current at-risk population was estimated to be 960 million and the future at-risk population was projected to be 1.2 billion. CONCLUSIONS: The magnitude of climate change in China is likely to surpass GCM predictions. Future dengue risks will expand to cover nearly all of China if current climate trends continue.

Interactions between the gut micro-community and transcriptome of Culex pipiens pallens under low-temperature stress.

BACKGROUND: Culex pipiens pallens (Diptera: Culicidae) can survive at low temperature for long periods. Understanding the effects of low-temperature stress on the gut microflora and gene expression levels in Cx. pipiens pallens, as well as their correlation, will contribute to the study of the overwintering mechanism of Cx. pipiens pallens. METHODS: The gut bacteria were removed by antibiotic treatment, and the survival of Cx. pipiens pallens under low-temperature stress was observed and compared with the control group. Then, full-length 16S rRNA sequencing and the Illumina HiSeq X Ten sequencing platform were used to evaluate the gut microflora and gene expression levels in Cx. pipiens pallens under low-temperature stress. RESULTS: Under the low-temperature stress of 7 °C, the median survival time of Cx. pipiens pallens in the antibiotic treatment group was significantly shortened by approximately 70% compared to that in the control group. The species diversity index (Shannon, Simpson, Ace, Chao1) of Cx. pipiens pallens decreased under low-temperature stress (7 °C). Non-metric multidimensional scaling (NMDS) analysis divided all the gut samples into two groups: control group and treatment group. Pseudomonas was the dominant taxon identified in the control group, followed by Elizabethkingia and Dyadobacter; in the treatment group, Pseudomonas was the dominant taxon, followed by Aeromonas and Comamonas. Of the 2417 differentially expressed genes (DEGs), 1316 were upregulated, and 1101 were downregulated. Functional GO terms were enriched in 23 biological processes, 20 cellular components and 21 molecular functions. KEGG annotation results showed that most of these genes were related to energy metabolism-related pathways. The results of Pearson's correlation analysis showed a significant correlation between the gut microcommunity at the genus level and several DEGs. CONCLUSIONS: These results suggest that the mechanism of adaptation of Cx. pipiens pallens to low-temperature stress may be the result of interactions between the gut bacterial community and transcriptome.

New insights into cypermethrin insecticide resistance mechanisms of Culex pipiens pallens by proteome analysis.

BACKGROUND: Due to the development of insecticide resistance in mosquitoes, with worldwide mosquito-borne diseases resurgence in recent years, recent advances in proteome technology have facilitated a proteome-wide analysis of insecticide resistance-associated proteins in mosquitoes. Understanding the complexity of the molecular basis of insecticide resistance mechanisms employed by mosquitoes will help in designing the most effective and sustainable mosquito control methods. RESULTS: After 30 generations, insecticide-selected strains showed elevated resistance levels to the cypermethrin used for selection. Proteome data allowed the detection of 2892 proteins, of which 2885 differentially expressed proteins (DEPs) achieved quantitative significances in four stages (egg, larvae, pupae, adult) of Culex pipiens pallens cypermethrin-resistant strain as compared to the susceptible strain. Among them, a significant enrichment of proteins, including cuticular proteins, enzymes involved in the detoxification (cytochrome P450, glutathione S-transferases, esterase, ATP-binding cassette) and some biological pathways (oxidative phosphorylation, hippo signalling) that are potentially involved in cypermethrin resistance, was observed. Thirty-one representative DEPs (cytochrome P450, glutathione S-transferase, cuticle protein) during Cx. pipiens pallens developmental stages were confirmed by a parallel reaction monitoring strategy. CONCLUSIONS: The present study confirmed the power of isobaric tags for relative and absolute quantification for identifying concomitantly quantitative proteome changes associated with cypermethrin in Cx. pipiens pallens. Proteome analysis suggests that proteome modifications can be selected rapidly by cypermethrin, and multiple resistance mechanisms operate simultaneously in cypermethrin-resistance of Cx. pipiens pallens, Our results interpret that an up-regulated expression of proteins and enzymes like cytochrome P450, glutathione S-transferases, esterase etc. has an impact in insecticide resistance. Previously neglected penetration resistance (cuticular proteins) may play an important role in the adaptive response of Cx. pipiens pallens to insecticides. This information may serve as a basis for future work concerning the possible role of these proteins in cypermethrin resistance in mosquito Cx. pipiens pallens. © 2022 Society of Chemical Industry.

Effects of oxygen concentrations in modified atmosphere packaging on pork quality and protein oxidation.

This study aimed to investigate the effects of oxygen concentrations in modified atmosphere packaging (MAP) on pork color and protein oxidation under refrigerated storage. Pork steaks were vacuum packaged or MAP packed with different levels of oxygen (40%, 60% and 80%), and stored for 14 days at 4 °C. After storage, the instrumental color, purge loss, cooking loss and shear fore were measured, and levels of protein oxidation in both the sarcoplasmic and myofibrillar proteins were quantified separately by the determination of carbonyl contents, thiol contents, loss of specific amino acid residues, and formation of α-aminoadipic semialdehyde. Results suggested that 40% of oxygen in MAP was sufficient to obtain highest redness, and higher oxygen levels showed little improvement but led to further protein carbonylation and meat toughening. Meanwhile, the sarcoplasmic proteins seemed to be more vulnerable to protein oxidation than the myofibrillar proteins. In particular, the formation of α-aminoadipic semialdehyde might contribute little to the carbonylation of both protein fractions, and possible reasons were discussed.

A symbiotic gut bacterium enhances Aedes albopictus resistance to insecticide.

BACKGROUND: The increasing insecticide resistance of Aedes albopictus puts many countries in Asia and Africa, including China, at great risk of a mosquito-borne virus epidemic. To date, a growing number of researches have focused on the relationship between intestinal symbiotic bacteria and their hosts' resistance to insecticides. This provides a novel aspect to the study of resistant mechanisms. METHODS/FINDINGS: This study reveals significant composition and dynamic changes in the intestinal symbiotic bacteria of Ae. albopictus between the resistant and susceptible strains based on full-length sequencing technology. The relative abundance of Serratia oryzae was significantly higher in the resistance strain than in the susceptible strains; also, the relative abundance of S. oryzae was significantly higher in deltamethrin-induced Ae. albopictus than in their counterpart. These suggested that S. oryzae may be involved in the development of insecticide resistance in Ae. albopictus. To explore the insecticide resistance mechanism, adult mosquitoes were fed with GFP-tagged S. oryzae, which resulted in stable bacterial enrichment in the mosquito gut without affecting the normal physiology, longevity, oviposition, and hatching rates of the host. The resistance measurements were made based on bioassays as per the WHO guidelines. The results showed that the survival rate of S. oryzae-enriched Ae. albopictus was significantly higher than the untreated mosquitoes, indicating the enhanced resistance of S. oryzae-enriched Ae. albopictus. Also, the activities of three metabolic detoxification enzymes in S. oryzae-enriched mosquitoes were increased to varying degrees. Meanwhile, the activity of extracellular enzymes released by S. oryzae was measured, but only carboxylesterase activity was detected. HPLC and UHPLC were respectively used to measure deltamethrin residue concentration and metabolite qualitative analysis, showing that the deltamethrin degradation efficiency of S. oryzae was positively correlated with time and bacterial amount. Deltamethrin was broken down into 1-Oleoyl-2-hydroxy-sn-glycero-3-PE and 2',2'-Dibromo-2'-deoxyguanosine. Transcriptome analysis revealed that 9 cytochrome P450s, 8 GSTs and 7 CarEs genes were significantly upregulated. CONCLUSIONS: S. oryzae can be accumulated into adult Ae. albopictus by artificial feeding, which enhances deltamethrin resistance by inducing the metabolic detoxification genes and autocrine metabolic enzymes. S. oryzae is vertically transmitted in Ae. albopictus population. Importantly, S. oryzae can degrade deltamethrin in vitro, and use deltamethrin as the sole carbon source for their growths. Therefore, in the future, S. oryzae may also be commercially used to break down the residual insecticides in the farmland and lakes to protect the environment.

Comparative proteomics reveals mechanisms that underlie insecticide resistance in Culex pipiens pallens Coquillett.

Mosquito control based on chemical insecticides is considered as an important element of the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. In contrast to target site resistance, other mechanisms are far from being fully understood. Global protein profiles among cypermethrin-resistant, propoxur-resistant, dimethyl-dichloro-vinyl-phosphate-resistant and susceptible strain of Culex pipiens pallens were obtained and proteomic differences were evaluated by using isobaric tags for relative and absolute quantification labeling coupled with liquid chromatography/tandem mass spectrometric analysis. A susceptible strain of Culex pipiens pallens showed elevated resistance levels after 25 generations of insecticide selection, through iTRAQ data analysis detected 2,502 proteins, of which 1,513 were differentially expressed in insecticide-selected strains compared to the susceptible strain. Finally, midgut differential protein expression profiles were analyzed, and 62 proteins were selected for verification of differential expression using iTRAQ and parallel reaction monitoring strategy, respectively. iTRAQ profiles of adaptation selection to three insecticide strains combined with midgut profiles revealed that multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Culex pipiens pallens. Significant molecular resources were developed for Culex pipiens pallens, potential candidates were involved in metabolic resistance and reducing penetration or sequestering insecticide. Future research that is targeted towards RNA interference of the identified metabolic targets, such as cuticular proteins, cytochrome P450s, glutathione S-transferases and ribosomal proteins proteins and biological pathways (drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, oxidative phosphorylation, ribosome) could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Culex pipiens pallens.

Effects of l-Lysine on the physiochemical properties and sensory characteristics of salt-reduced reconstructed ham.

This paper aimed to investigate the dose-effects of l-Lysine (Lys, 0.2% to 0.8%) on the water holding capacity (WHC), textural properties, water mobility and distribution, microstructures and sensory acceptance of reconstructed ham with 50% reduction of added salt. Results showed that reducing salt from 2.50% to 1.25% caused significant increase in cooking loss and centrifuge loss, and decrease in hardness, springiness and chewiness. 0.8% Lys significantly improved the WHC and textural properties of salt-reduced hams, whereas 0.2% Lys further impaired these attributes. Moreover, 0.8% Lys improved the sensory scores for mouthfeel, appearance, taste and global acceptance. The NMR analysis reveals that water distribution and mobility were affected by salt reduction and Lys addition, and hams with 0.8% Lys contained more entrapped water with decreased mobility. Furthermore, a fine network with more bridge-linkage among myofibrils was formed in hams with 0.8% Lys. Therefore, addition of 0.8% Lys showed great potential in developing salt-reduced reconstructed ham with premium technological and sensory qualities.

Analysis of Epidemiological Changes and Prevention Effects for Malaria in Weifang, Shandong Province, China from 1957 to 2017.

BACKGROUND: We aimed to conduct a retrospective analysis of the epidemiological changes and prevention effects for malaria in Weifang, Shandong Peninsula, China from 1957 to 2017. METHODS: The malaria data from a web-based reporting system were analyzed to explore malaria epidemiological characteristics and prevention effects in Weifang. RESULTS: Overall, 1, 704, 890 malaria cases were reported in Weifang from 1957 to 2017, of which two major malaria epidemics occurred in 1961 (827.28/10, 000) and 1971 (366.14/10, 000). Prior to 1997, all malaria patients (1, 704, 829) were infected with Plasmodium vivax (P. vivax). After 2007, the cases of Plasmodium falciparum (P. falciparum) showed an upward trend (76.8%). The reported cases after the 21st century were mainly imported cases, and the last indigenous case was a patient that infected with P. vivax in 2006. Overall, 36 imported cases were reported from 2010 to 2017, of which 88.9% were acquired in Africa. Except for one 32-year-old woman, the rest were male (97.2%), in which laborers and farmers represented the vast majority (66.6%). From 1987 to 2017, there were 1, 224, 474 cases of fever with blood tests, and the average blood test rate was 4.9%. From 1957 to 2017, a total of 1, 704, 890 malaria patients were treated, 96 cases were treated during resting phase from 1987 to 2017. CONCLUSION: Weifang should continue to strengthen the management of the migrant population, making blood tests for fever patients and patient treatment as important means of malaria control and monitoring.

Toward the Elimination of Malaria in China: A Retrospective Analysis of Malaria-Endemic Characteristics and Prevention Effects in Yantai, Shandong Province, 1951 to 2017.

Objectives: To study the epidemiological characteristics of malaria and the effects of comprehensive malaria control implementation in Yantai, as well as to provide a scientific basis for future malaria elimination. Materials and Methods: A retrospective analysis was conducted to explore the epidemiological changes and prevention effects in Yantai from 1951 to 2017. The malaria data before 2004 were collected from paper-based annual reports, and data after 2004 came from the Infectious Diseases Information Reporting Management System. Results: A total of 81,286 malaria cases were identified in Yantai from 1957 to 2017, with two peaks occurring in 1966 (120.93/100,000) and 1970-1972 (366.12/100,000). From 1951 to 2003, the case classification was mainly Plasmodium vivax (99.80%); the first case attributed to Plasmodium falciparum occurred in 2004, and P. falciparum became the main cause of malaria after 2011. All cases reported since 2010 have been imported cases, and the last indigenous case was caused by P. vivax in 2008. A total of 129 imported cases were reported from 2010 to 2017, most of which originated in Africa (93.80%), mainly in men aged 30-49 years (68.28%). From 1951 to 1973, 73,868 cases of malaria were diagnosed with clinical symptoms, and from 1974 to 2017, 818,943 cases in fever patients were diagnosed with blood tests. Conclusions: To achieve the goal of eliminating malaria by 2020, Yantai should continue to strengthen the management of migrant population, including improving malaria surveillance for returnees and immigrants from overseas endemic areas, and continue to increase the training of medical personnel to improve their diagnostic ability.

Understanding the regulation of overwintering diapause molecular mechanisms in Culex pipiens pallens through comparative proteomics.

To reveal overwintering dormancy (diapause) mechanisms of Culex pipiens pallens (L.), global protein expression differences at three separate time points represent nondiapause, diapause preparation and overwintering diapause phases of Cx. pipiens pallens were compared using iTRAQ. Cx. pipiens pallens females accumulate more lipid droplets during diapause preparation and overwintering diapause maintenance than during the nondiapause phase. A total of 1030 proteins were identified, among which 1020 were quantified and compared. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), Domain and Clusters of Orthologous Groups (COG) analyses revealed key groups of proteins, pathways and domains differentially regulated during diapause preparation and overwintering diapause maintenance phases in this mosquito, including major shifts in energy production and conversion, fatty acid metabolism, the citrate (TCA) cycle, and the cytoskeletal reorganization pathway. Our results provide novel insight into the molecular bases of diapause in mosquitoes and corroborate previously reported diapause-associated features in invertebrates. More interestingly, the phototransduction pathway exists in Cx. pipiens pallens, in particular, actin, rather than other proteins, appears to have substantial role in diapause regulation. In addition, the differential changes in calmodulin protein expression in each stage implicate its important regulatory role of the Cx. pipiens pallens biological clock. Finally, 24 proteins were selected for verification of differential expression using a parallel reaction monitoring strategy. The findings of this study provide a unique opportunity to explore the molecular modifications underlying diapause in mosquitoes and might therefore enable the future design and development of novel genetic tools for improving management strategies in mosquitoes.

miR-202 Inhibits Cell Proliferation, Migration, and Invasion by Targeting Epidermal Growth Factor Receptor in Human Bladder Cancer.

Recent studies have demonstrated that miR-202 is associated with several types of cancer; however, the expression and function of miR-202 have not been investigated in bladder cancer. We analyzed the expression of miR-202 in bladder cancer tissues and adjacent noncancerous tissues. The effect of miR-202 on the proliferation, migration, and invasion was evaluated by in vitro assays. The target gene of miR-202 was assessed by luciferase reporter assay. In this study, miR-202 was found to be significantly downregulated in bladder cancer cell lines and tissues and was highly correlated with the T classification, N classification, grade, and recurrence. Ectopic expression of miR-202 suppressed cell viability, colony formation, cell migration, and invasion in vitro and inhibited xenograft tumor growth in vivo. Inversely, downregulation of miR-202 had contradictory effects. The 3'-untranslated region (3'-UTR) of epidermal growth factor receptor (EGFR) was identified as a direct target of miR-202 using luciferase reporter assays, and knockdown of EGFR enhanced miR-202-inhibited cell proliferation, migration, and invasion. In conclusion, miR-202 suppresses bladder cancer carcinogenesis and progression by targeting EGFR, thereby representing a potential target for miRNA-based therapy for bladder cancer in the future.

Synergistic Inhibitory Effect of Traditional Chinese Medicine Astragaloside IV and Curcumin on Tumor Growth and Angiogenesis in an Orthotopic Nude-Mouse Model of Human Hepatocellular Carcinoma.

AIM: The aim of the present study was to investigate the efficacy of the traditional Chinese medicine (TCM), astragaloside IV (AS-IV) and curcumin on tumor growth and angiogenesis in an orthotopic nude-mouse model of human hepatocellular carcinoma (HCC). We have previously shown the usefulness of orthotopic models of human cancer for evaluation of the efficacy of TCM. MATERIALS AND METHODS: Nude mice with orthotopic HepG2 HCC were treated with vehicle control (0.01 ml/g normal saline), cisplatinum (2 mg/kg), AS-IV (20 mg/kg), curcumin (100 mg/kg) or AS-IV plus curcumin (20 mg/kg + 100 mg/kg). Tumor inhibition in each group was evaluated by tumor weight at autopsy. The effect of AS-IV and curcumin on tumor angiogenesis was assessed by CD34 staining and expression of fibroblast growth factor-2 (FGF2), matrix metalloproteinase 2 (MMP2), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), thrombosis-related factor tissue factor (TF) and coagulation factor VII (FVII), as well as microRNAs miR-122 and miR-221. RESULTS: AS-IV and curcumin alone and in combination significantly reduced mean tumor weight compared to vehicle control (p<0.05). Tumor microvessel count was reduced by AS-IV and curcumin alone. Expression of FGF2, MMP2, VEGF, HGF, TF and FVII was reduced by AS-IV and curcumin alone. AS-IV and curcumin alone up-regulated expression of miR-122 and down-regulated that of miR-221. The combination of AS-IV and curcumin demonstrated significant synergistic effects on microvessel count as well as on expression of angiogenic and thrombosis-related factors and microRNAs. CONCLUSION: The present study indicates future clinical potential of combination therapy with AS-IV and curcumin for HCC.

Epidemiological analysis of 133 malaria cases in Shanxian county, Shandong Province, China.

OBJECTIVE: To conduct an analysis of the epidemiological changes in malaria that have occurred in Shanxian county from 2002 to 2016. METHODS: A retrospective study was conducted and data were collected from web-based reporting system to explore the epidemiological characteristics in Shanxian county from 2002 to 2016. All individual case information was obtained from village malaria servicers organized by the local Shandong Institute of Parasitic Diseases. RESULTS: A total of 133 cases were identified as malaria in Shanxian county during this period, including 124 indigenous cases (93.2%) and 9 imported cases (6.8%). The 124 indigenous malaria cases were infected with Plasmodium vivax (P. vivax), whereas 7 of the 9 confirmed imported cases were infected with Plasmodium falciparum (P. falciparum), 1 was infected with Plasmodium ovale (P. ovale) and 1 patient was infected with P. falciparum mixed with P. vivax. The total number of malaria cases included 86 males (64.7%) and 47 females (35.3%). Age of the patients ranged from 1 to 83 years, although most (64.7%) infections occurred in the 21-to 60-year-old age group. Remarkably, 117 of the total malaria cases (98.0%) were reported from 2006 to 2011. The epidemic season was from June to October, with the peak occurring yearly from July to September. The most common occupation of the infected patients was farmer. In total, 58.1% of the cases occurred in 3 townships, namely, Fugang, Huanggang and Caozhuang. CONCLUSIONS: In Shanxian county, the local malaria incidence experienced an emerge-peak-control-eliminate status. However, due to the numbers of migrant labourers returning from Africa, imported cases were continuous and presented an increasing annual trend, which became a non-negligible and a significant impediment for malaria elimination. Therefore, the need to eliminate instances of malaria reintroduction to receptive malaria-free areas should drive strategies to align with the epidemiological changes.