Concise report : teenage sexting on the rise? Results of a cohort study using a weighted sample of adolescents

Background: The vast differences in sampling techniques, cultural contexts between international studies, the differences in age groups of the samples and various definitions used to measure sexting make it hard to compare how sexting behaviour has evolved over the past years. This exploratory study aims to address this critical gap in the research by using two datasets of a biennial study on adolescents' media use. The first aim of this study is to compare the sexting prevalence and correlates (i.e. age, gender and smartphone ownership) of youth in 2015 and 2017. The second aim is to investigate the risk mitigation behaviours of youth who engage in sexting and to assess how their behaviours differ between the two time points. Methods: The data for cohort 1 were collected in October and November 2015 and comprise of 2663 students from 11 secondary schools in the Dutch-speaking community of Belgium. For the second cohort, the data were collected in October and November 2017 and comprise of 2681 students from 10 secondary schools. A weighing factor was implemented on the dataset. Results: The results show sexting behaviour has significantly increased between cohorts, with 8.3% of the respondents having sent a sext in the first cohort, compared with 12.1% in the second cohort. The sexting prevalence rates also significantly increased between cohorts when taking into account students who owned a smartphone. Furthermore, engagement in sexting was associated with being older, and no gender differences were found. In the second cohort, 36.8% of youth who had sent a sext were identifiable in those images. There were no differences between cohorts. Conclusion: The results indicate that other factors next to smartphone ownership may be associated with an increase in sexting prevalence. The findings also highlight the need for the development of age-appropriate sexting educational materials

A zone-of-inhibition assay to screen for humoral antimicrobial activity in mosquito hemolymph

In insects, antibacterial immunity largely depends on the activation of downstream signaling and effector responses, leading to the synthesis and secretion of soluble effector molecules, such as antimicrobial peptides (AMPs). AMPs are acute infection response peptides secreted into the hemolymph upon bacterial stimulation. The transcription of innate immunity genes encoding for AMPs is highly dependent on several signaling cascade pathways, such as the Toll pathway. In the African malaria mosquito, Anopheles gambiae, AMPs hold a special interest as their upregulation have been shown to limit the growth of malaria parasites, bacteria, and fungi. Most of the current knowledge on the regulation of insect AMPs in microbial infection have been obtained from Drosophila. However, largely due to the lack of convenient assays, the regulation of antimicrobial activity in mosquito hemolymph is still not completely understood. In this study, we report a zone of inhibition assay to identify the contribution of AMPs and components of the Toll pathway to the antimicrobial activity of A. gambiae hemolymph. As a proof of principle, we demonstrate that Micrococcus luteus challenge induces antimicrobial activity in the adult female mosquito hemolymph, which is largely dependent on defensin 1. Moreover, by using RNAi to silence Cactus, REL1, and MyD88, we showed that Cactus kd induces antimicrobial activity in the mosquito hemolymph, whereas the antimicrobial activity in REL1 kd and MyD88 kd is reduced after challenge. Finally, while injection itself is not sufficient to induce antimicrobial activity, our results show that it primes the response to bacterial challenge. Our study provides information that increases our knowledge of the regulation of antimicrobial activity in response to microbial infections in mosquitoes. Furthermore, this assay represents an ex vivo medium throughput assay that can be used to determine the upstream regulatory elements of antimicrobial activity in A. gambiae hemolymph

Crystal structure of native Anopheles gambiae Serpin-2, a negative regulator of melanization in mosquitoes

Serpins are the dominant group of protease inhibitors in metazoans that control a wide variety of biological processes including major innate immune reactions. One of these inhibitors, SRPN2, controls melanization in mosquitoes – a powerful, arthropod-specific innate immune response. SRPN2 depletion from the hemolymph of adult female mosquitoes significantly reduces longevity and therefore this serpin is a potential target for novel insecticides. We report here the crystal structure of SRPN2 in its native conformation from the African malaria mosquito, Anopheles gambiae to 1.75 Å resolution. SRPN2 adopts a similar fold as observed for other serpins with a core of three β-sheets surrounded by nine α-helices with an exposed reactive center loop (RCL) that extends from the protein body. Similar to other native serpin structures, several residues within the reactive center loop were disordered and could not be modeled. Intriguingly, the N-terminal hinge of the RCL in SRPN2 was found to be inserted into β-sheet A, suggesting a potential activation mechanism analogous to heparin-mediated activation of Antithrombin III

Methodology of a reevaluation of cardiovascular outcomes in the RECORD trial: study design and conduct

Background In 2010, after regulatory review of rosiglitazone licensing, the US Food and Drug Administration (FDA) requested a reevaluation of cardiovascular end points in the RECORD trial.<p></p> Methods Automated screening of the original clinical trial database and manual case report form review were performed to identify all potential cardiovascular and noncardiovascular deaths, and nonfatal myocardial infarction (MI) and stroke events. Search techniques were used to find participants lost to follow-up, and sites were queried for additional source documents. Suspected events underwent blinded adjudication using both original RECORD end point definitions and new FDA end point definitions, before analysis by the Duke Clinical Research Institute.<p></p> Results The reevaluation effort included an additional 328 person-years of follow-up. Automated screening identified 396 suspected deaths, 2,052 suspected MIs, and 468 suspected strokes. Manual review of documents by Duke Clinical Research Institute clinical events classification (CEC) coordinators identified an additional 31 suspected deaths, 49 suspected MIs, and 28 suspected strokes. There were 127 CEC queries issued requesting additional information on suspected deaths; 43 were closed with no site response, 61 were closed with a response that no additional data were available, and additional data were received for 23. Seventy CEC queries were issued requesting additional information for suspected MI and stroke events; 31 were closed with no site response, 20 were closed with a response that no additional data were available, and 19 resulted in additional data.<p></p> Conclusions Comprehensive procedures were used for rigorous event reascertainment and readjudication in a previously completed open-label, global clinical trial. These procedures used in this unique situation were consistent with other common approaches in the field, were enhanced to address the FDA concerns about the original RECORD trial results, and could be considered by clinical trialists designing event readjudication protocols for drug development programs that have been completed.<p></p&gt

Evaluating RNAlater® as a preservative for using near-infrared spectroscopy to predict Anopheles gambiae age and species.

Mosquito age and species identification is a crucial determinant of the efficacy of vector control programmes. Near-infrared spectroscopy (NIRS) has previously been applied successfully to rapidly, non-destructively, and simultaneously determine the age and species of freshly anesthetized African malaria vectors from the Anopheles gambiae s.l. species complex: An. gambiae s. s. and Anopheles arabiensis. However, this has only been achieved on freshly-collected specimens and future applications will require samples to be preserved between field collections and scanning by NIRS. In this study, a sample preservation method (RNAlater(®)) was evaluated for mosquito age and species identification by NIRS against scans of fresh samples. Two strains of An. gambiae s.s. (CDC and G3) and two strains of An. arabiensis (Dongola, KGB) were reared in the laboratory while the third strain of An. arabiensis (Ifakara) was reared in a semi-field system. All mosquitoes were scanned when fresh and rescanned after preservation in RNAlater(®) for several weeks. Age and species identification was determined using a cross-validation. The mean accuracy obtained for predicting the age of young (<7 days) or old (≥ 7 days) of all fresh (n = 633) and all preserved (n = 691) mosquito samples using the cross-validation technique was 83% and 90%, respectively. For species identification, accuracies were 82% for fresh against 80% for RNAlater(®) preserved. For both analyses, preserving mosquitoes in RNAlater(®) was associated with a highly significant reduction in the likelihood of a misclassification of mosquitoes as young or old using NIRS. Important to note is that the costs for preserving mosquito specimens with RNAlater(®) ranges from 3-13 cents per insect depending on the size of the tube used and the number of specimens pooled in one tube. RNAlater(®) can be used to preserve mosquitoes for subsequent scanning and analysis by NIRS to determine their age and species with minimal costs and with accuracy similar to that achieved from fresh insects. Cold storage availability allows samples to be stored longer than a week after field collection. Further study to develop robust calibrations applicable to other strains from diverse ecological settings is recommended

Results of a reevaluation of cardiovascular outcomes in the RECORD trial

Background The US Food and Drug Administration (FDA) required a reevaluation of cardiovascular (CV) outcomes in the RECORD trial. This provided an opportunity to assess the implications of event adjudication by 2 groups and quantify the differences as well as to use new FDA end point definitions in development.&lt;p&gt;&lt;/p&gt; Methods Original data were used to systematically identify all potential deaths, myocardial infarctions (MIs), and strokes. Site investigators were approached for additional source documents and information about participants lost to follow-up. Suspected events were adjudicated using standard procedures, and the results were compared with the original trial outcomes.&lt;p&gt;&lt;/p&gt; Results Follow-up for mortality was 25,833 person-years, including an additional 328 person-years identified during the reevaluation effort. A total of 184 CV or unknown-cause deaths (88 rosiglitazone, 96 metformin/sulfonylurea), 128 participants with an MI (68 rosiglitazone, 60 metformin/sulfonylurea), and 113 participants with a stroke (50 rosiglitazone, 63 metformin/sulfonylurea) were included. The hazard ratio (HR) for rosiglitazone versus metformin/sulfonylurea for the end point of CV (or unknown cause) death, MI, or stroke was 0.95 (95% CI 0.78-1.17) compared with 0.93 (95% CI 0.74-1.15) for the original RECORD results. Treatment comparisons for MI (HR 1.13, 95% CI 0.80-1.59) and mortality (HR 0.86, 95% CI 0.68-1.08) were also the same compared with the original RECORD results. Sensitivity analyses were also consistent with the original RECORD results. Analyses using the FDA definitions showed similar results.&lt;p&gt;&lt;/p&gt; Conclusions Only a modest number of additional person-years of follow-up were ascertained from this reevaluation of CV end points in RECORD. Observed HRs and CIs from these analyses using the original RECORD or new FDA end point definitions showed similar treatment effects of rosiglitazone compared with the original RECORD results.&lt;p&gt;&lt;/p&gt

Biochemical Characterization of Anopheles gambiae SRPN6, a Malaria Parasite Invasion Marker in Mosquitoes

Serine proteinase inhibitors of the serpin family are well known as negative regulators of hemostasis, thrombolysis and innate immune responses. Additionally, non-inhibitory serpins serve functions as chaperones, hormone transporters, or anti-angiogenic factors. In the African malaria mosquito, Anopheles gambiae s.s., at least three serpins (SRPNs) are implicated in the innate immune response against malaria parasites. Based on reverse genetic and cell biological analyses, AgSRPN6 limits parasite numbers and transmission and has been postulated to control melanization and complement function in mosquitoes. This study aimed to characterize AgSRPN6 biophysically and determine its biochemical mode of action. The structure model of AgSRPN6, as predicted by I-Tasser showed the protein in the native serpin fold, with three central β-sheets, nine surrounding α-helices, and a protruding reactive center loop. This structure is in agreement with biophysical and functional data obtained from recombinant (r) AgSRPN6, produced in Escherichia coli. The physical properties of purified rAgSRPN6 were investigated by means of analytical ultracentrifugation, circular dichroism, and differential scanning calorimetry tools. The recombinant protein exists predominantly as a monomer in solution, is composed of a mixture of α-helices and β-sheets, and has a mid-point unfolding temperature of 56°C. Recombinant AgSRPN6 strongly inhibited porcine pancreatic kallikrein and to a lesser extent bovine pancreatic trypsin in vitro. Furthermore, rAgSRPN6 formed inhibitory, SDS-stable, higher molecular weight complexes with prophenoloxidase-activating proteinase (PAP)1, PAP3, and Hemolymph protein (HP)6, which are required for melanization in the lepidopteran model organism, Manduca sexta. Taken together, our results strongly suggest that AgSRPN6 takes on a native serpin fold and is an inhibitor of trypsin-like serine proteinases.This work was supported by the National Institutes of Health through 3P20RR017708-07S1 and P20RR017686 sub-awards and 1R01AI095842 to K.M. This is contribution 12-098-J from the Kansas Agricultural Experiment Station

RNAi Trigger Delivery into Anopheles gambiae Pupae

Citation: Regna, K., Harrison, R. M., Heyse, S. A., Chiles, T. C., Michel, K., & Muskavitch, M. A. T. (2016). RNAi Trigger Delivery into Anopheles gambiae Pupae. Jove-Journal of Visualized Experiments(109), 9. doi:10.3791/53738RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel