The mosquito Aedes aegypti (Ae. aegypti) is the most notorious vector of illness-causing viruses such as Dengue, Chikugunya, and Zika. Although numerous genetic expression studies utilizing quantitative real-time PCR (qPCR) have been conducted with regards to Ae. aegypti, a panel of genes to be used suitably as references for the purpose of expression-level normalization within this epidemiologically important insect is presently lacking. Here, the usability of seven widely-utilized reference genes i.e. actin (ACT), eukaryotic elongation factor 1 alpha (eEF1α), alpha tubulin (α-tubulin), ribosomal proteins L8, L32 and S17 (RPL8, RPL32 and RPS17), and glyceraldeyde 3-phosphate dehydrogenase (GAPDH) were investigated. Expression patterns of the reference genes were observed in sixteen pre-determined developmental stages and in cell culture. Gene stability was inferred from qPCR data through three freely available algorithms i.e. BestKeeper, geNorm, and NormFinder. The consensus rankings generated from stability values provided by these programs suggest a combination of at least two genes for normalization. ACT and RPS17 are the most dependably expressed reference genes and therefore, we propose an ACT/RPS17 combination for normalization in all Ae. aegypti derived samples. GAPDH performed least desirably, and is thus not a recommended reference gene. This study emphasizes the importance of validating reference genes in Ae. aegypti for qPCR based research.

ObjectiveTo determine the prevalence of vitamin D deficiency (<37.5 nmol/L) among young adolescents in Malaysia and its association with demographic characteristics, anthropometric measures and physical activity.DesignThis is a cross-sectional study among Form 1 (year 7) students from 15 schools selected using a stratified random sampling design. Information regarding sociodemographic characteristics, clinical data and environmental factors was collected and blood samples were taken for total vitamin D. Descriptive and multivariable logistic regression was performed on the data.SettingNational secondary schools in Peninsular Malaysia.Participants1361 students (mean age 12.9±0.3 years) (61.4% girls) completed the consent forms and participated in this study. Students with a chronic health condition and/or who could not understand the questionnaires due to lack of literacy were excluded.Main outcome measuresVitamin D status was determined through measurement of sera 25-hydroxyvitamin D (25(OH)D). Body mass index (BMI) was classified according to International Obesity Task Force (IOTF) criteria. Self-reported physical activity levels were assessed using the validated Malay version of the Physical Activity Questionnaire for Older Children (PAQ-C).ResultsDeficiency in vitamin D was seen in 78.9% of the participants. The deficiency was significantly higher in girls (92.6%, p<0.001), Indian adolescents (88.6%, p<0.001) and urban-living adolescents (88.8%, p<0.001). Females (OR=8.98; 95% CI 6.48 to 12.45), adolescents with wider waist circumference (OR=2.64; 95% CI 1.65 to 4.25) and in urban areas had higher risks (OR=3.57; 95% CI 2.54 to 5.02) of being vitamin D deficient.ConclusionsThe study shows a high prevalence of vitamin D deficiency among young adolescents. Main risk factors are gender, ethnicity, place of residence and obesity.

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Members of the Aedes genus of mosquitoes are widely recognized as vectors of viral diseases. Ae.albopictus is its most invasive species, and are known to carry viruses such as Dengue, Chikugunya and Zika. Its emerging importance puts Ae.albopictus on the forefront of genetic interaction and evolution studies. However, a panel of suitable reference genes specific for this insect is as of now undescribed. Nine reference genes, namely ACT, eEF1-γ, eIF2α, PP2A, RPL32, RPS17, PGK1, ILK and STK were evaluated. Expression patterns of the candidate reference genes were observed in a total of seventeen sample types, separated by stage of development and age. Gene stability was inferred from obtained quantification data through three widely cited evaluation algorithms i.e. BestKeeper, geNorm, and NormFinder. No single gene showed a satisfactory degree of stability throughout all developmental stages. Therefore, we propose combinations of PGK and ILK for early embryos; RPL32 and RPS17 for late embryos, all four larval instars, and pupae samples; eEF1-γ with STK for adult males; eEF1-γ with RPS17 for non-blood fed females; and eEF1-γ with eIF2α for both blood-fed females and cell culture. The results from this study should be able to provide a more informed selection of normalizing genes during qPCR in Ae.albopictus.

CTP synthase (CTPSyn) is an essential metabolic enzyme, synthesizing precursors required for nucleotides and phospholipids production. Previous studies have also shown that CTPSyn is elevated in various cancers. In many organisms, CTPSyn compartmentalizes into filaments called cytoophidia. In Drosophila melanogaster , only its isoform C (CTPSynIsoC) forms cytoophidia. In the fruit fly’s testis, cytoophidia are normally seen in the transit amplification regions close to its apical tip, where the stem-cell niche is located, and development is at its most rapid. Here, we report that CTPSynIsoC overexpression causes the lengthening of cytoophidia throughout the entirety of the testicular body. A bulging apical tip is found in approximately 34% of males overexpressing CTPSynIsoC . Immunostaining shows that this bulged phenotype is most likely due to increased numbers of both germline cells and spermatocytes. Through a microRNA (miRNA) overexpression screen, we found that ectopic miR-975 concurrently increases both the expression levels of CTPSyn and the length of its cytoophidia. The bulging testes phenotype was also recovered at a penetration of approximately 20%. However, qPCR assays reveal that CTPSynIsoC and miR-975 overexpression each provokes a differential response in expression of a number of cancer-related genes, indicating that the shared CTPSyn upregulation seen in either case is likely the cause of observed testicular overgrowth. This study presents the first instance of consequences of miRNA-asserted regulation upon CTPSyn in D . melanogaster , and further reaffirms the enzyme’s close ties to germline cells overgrowth.