Direct and indirect gene repression by the ecdysone cascade during mosquito reproductive cycle.

SignificanceHematophagous Aedes aegypti mosquitoes spread devastating viral diseases. Upon blood feeding, a steroid hormone, 20-hydroxyecdysone (20E), initiates a reproductive program during which thousands of genes are differentially expressed. While 20E-mediated gene activation is well known, repressive action by this hormone remains poorly understood. Using bioinformatics and molecular biological approaches, we have identified the mechanisms of 20E-dependent direct and indirect transcriptional repression by the ecdysone receptor (EcR). While indirect repression involves E74, EcR binds to an ecdysone response element different from those utilized in 20E-mediated gene activation to exert direct repressive action. Moreover, liganded EcR recruits a corepressor Mi2, initiating chromatin compaction. This study advances our understanding of the 20E-EcR repression mechanism and could lead to improved vector control approaches.

MicroRNA-275 targets sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA) to control key functions in the mosquito gut.

The yellow fever mosquito Aedes aegypti is the major vector of arboviruses, causing numerous devastating human diseases, such as dengue and yellow fevers, Chikungunya and Zika. Female mosquitoes need vertebrate blood for egg development, and repeated cycles of blood feeding are tightly linked to pathogen transmission. The mosquito's posterior midgut (gut) is involved in blood digestion and also serves as an entry point for pathogens. Thus, the mosquito gut is an important tissue to investigate. The miRNA aae-miR-275 (miR-275) has been shown to be required for normal blood digestion in the female mosquito; however, the mechanism of its action has remained unknown. Here, we demonstrate that miR-275 directly targets and positively regulates sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase, which is implicated in active transport of Ca2+ from the cytosol to the sarco/endoplasmic reticulum. We utilized a combination of the gut-specific yeast transcription activator protein Gal4/upstream activating sequence (Gal4/UAS) system and miRNA Tough Decoy technology to deplete the endogenous level of miR-275 in guts of transgenic mosquitoes. This gut-specific reduction of miR-275 post blood meal decreased SERCA mRNA and protein levels of the digestive enzyme late trypsin. It also resulted in a significant reduction of gut microbiota. Moreover, the decrease of miR-275 and SERCA correlated with defects in the Notch signaling pathway and assembly of the gut actin cytoskeleton. The adverse phenotypes caused by miR-275 silencing were rescued by injections of miR-275 mimic. Thus, we have discovered that miR-275 directly targets SERCA, and the maintenance of its level is critical for multiple gut functions in mosquitoes.

MicroRNA-8 targets the Wingless signaling pathway in the female mosquito fat body to regulate reproductive processes.

Female mosquitoes require a blood meal for reproduction, and this blood meal provides the underlying mechanism for the spread of many important vector-borne diseases in humans. A deeper understanding of the molecular mechanisms linked to mosquito blood meal processes and reproductive events is of particular importance for devising innovative vector control strategies. We found that the conserved microRNA miR-8 is an essential regulator of mosquito reproductive events. Two strategies to inhibit miR-8 function in vivo were used for functional characterization: systemic antagomir depletion and spatiotemporal inhibition using the miRNA sponge transgenic method in combination with the yeast transcriptional activator gal4 protein/upstream activating sequence system. Depletion of miR-8 in the female mosquito results in defects related to egg development and deposition. We used a multialgorithm approach for miRNA target prediction in mosquito 3' UTRs and experimentally verified secreted wingless-interacting molecule (swim) as an authentic target of miR-8. Our findings demonstrate that miR-8 controls the activity of the long-range Wingless (Wg) signaling by regulating Swim expression in the female fat body. We discovered that the miR-8/Wg axis is critical for the proper secretion of lipophorin and vitellogenin by the fat body and subsequent accumulation of these yolk protein precursors by developing oocytes.

Regulation of Gene Expression Patterns in Mosquito Reproduction.

In multicellular organisms, development, growth and reproduction require coordinated expression of numerous functional and regulatory genes. Insects, in addition to being the most speciose animal group with enormous biological and economical significance, represent outstanding model organisms for studying regulation of synchronized gene expression due to their rapid development and reproduction. Disease-transmitting female mosquitoes have adapted uniquely for ingestion and utilization of the huge blood meal required for swift reproductive events to complete egg development within a 72-h period. We investigated the network of regulatory factors mediating sequential gene expression in the fat body, a multifunctional organ analogous to the vertebrate liver and adipose tissue, of the female Aedes aegypti mosquito. Transcriptomic and bioinformatics analyses revealed that ~7500 transcripts are differentially expressed in four sequential waves during the 72-h reproductive period. A combination of RNA-interference gene-silencing and in-vitro organ culture identified the major regulators for each of these waves. Amino acids (AAs) regulate the first wave of gene activation between 3 h and 12 h post-blood meal (PBM). During the second wave, between 12 h and 36 h, most genes are highly upregulated by a synergistic action of AAs, 20-hydroxyecdysone (20E) and the Ecdysone-Receptor (EcR). Between 36 h and 48 h, the third wave of gene activation-regulated mainly by HR3-occurs. Juvenile Hormone (JH) and its receptor Methoprene-Tolerant (Met) are major regulators for the final wave between 48 h and 72 h. Each of these key regulators also has repressive effects on one or more gene sets. Our study provides a better understanding of the complexity of the regulatory mechanisms related to temporal coordination of gene expression during reproduction. We have detected the novel function of 20E/EcR responsible for transcriptional repression. This study also reveals the previously unidentified large-scale effects of HR3 and JH/Met on transcriptional regulation during the termination of vitellogenesis and remodeling of the fat body.

Mosquito-specific microRNA-1174 targets serine hydroxymethyltransferase to control key functions in the gut.

Lineage-specific microRNAs (miRNAs) may contribute to functions specific to hematophagous mosquitoes and, as such, have potential for contributing to the development of future mosquito control approaches. Here we report that the mosquito- and gut-specific miRNA, miR-1174, is required for proper sugar absorption, fluid excretion, blood intake, and, consequently, egg maturation and survival in female mosquitoes. miR-1174 is highly expressed and localized in the posterior midgut, the blood-digesting portion of the mosquito alimentary canal. Depletion of miR-1174 results in severe defects in sugar absorption and blood intake. We identified serine hydroxymethyltransferase (SHMT) is a direct miR-1174 target. The adverse phenotypes caused by miR-1174 silencing were rescued by SHMT RNA interference. Our results suggest that miR-1174 is essential for fine-tuning the SHMT transcript to levels necessary for normal mosquito gut functions.

Firefighters' medical use and Korean Medicine experience in Korea: A qualitative study protocol.

INTRODUCTION: Firefighters, compared to other occupational groups, are exposed more frequently in their working environment not only to physical issues, such as musculoskeletal disease, respiratory disease, and burns but also to mental health issues, such as PTSD and depression. Specifically, Korean firefighters experience significantly higher rates of work-related injuries compared to those in other countries. Recent statistics from the Korea National Fire Agency indicate a steady increase in the number of firefighting work-related injuries. However, there is a shortage of measures in place to address these issues. This study aims to investigate the health needs, overall healthcare usage, and unmet needs of firefighters in Korea. We also aim to investigate, through in-depth interviews, perceptions and hindering factors for integrative medicine approaches to fulfilling unmet needs. METHOD: This study was conducted in accordance with the consolidated criteria for reporting qualitative research. Convenience and snowball sampling methods will be used to recruit firefighters to participate in the study, and interviews will be conducted using a semi-structured interview guide. The data will be analyzed in four stages using the qualitative analysis method of Krippendorff. DISCUSSION: In this study, we examine the state of health issues and healthcare usage among Korean firefighters and investigate their perceptions of and needs for integrative medicine. In this way, we aim to explore how integrative medicine and Korean medicine approaches could improve and assist healthcare services for firefighters. Furthermore, our findings will provide policymakers and healthcare providers with the necessary basic information to develop integrative medicine systems suited to firefighters.

Single-Cell Analysis of the Transcriptome and Epigenome.

Epigenome regulation has emerged as an important mechanism for the maintenance of organ function in health and disease. Dissecting epigenomic alterations and resultant gene expression changes in single cells provides unprecedented resolution and insight into cellular diversity, modes of gene regulation, transcription factor dynamics and 3D genome organization. In this chapter, we summarize the transformative single-cell epigenomic technologies that have deepened our understanding of the fundamental principles of gene regulation. We provide a historical perspective of these methods, brief procedural outline with emphasis on the computational tools used to meaningfully dissect information. Our overall goal is to aid scientists using these technologies in their favorite system of interest.

XL-DNase-seq: improved footprinting of dynamic transcription factors.

BACKGROUND: As the cost of high-throughput sequencing technologies decreases, genome-wide chromatin accessibility profiling methods such as the assay of transposase-accessible chromatin using sequencing (ATAC-seq) are employed widely, with data accumulating at an unprecedented rate. However, accurate inference of protein occupancy requires higher-resolution footprinting analysis where major hurdles exist, including the sequence bias of nucleases and the short-lived chromatin binding of many transcription factors (TFs) with consequent lack of footprints. RESULTS: Here we introduce an assay termed cross-link (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. Mild cross-linking improved the detection of DNase-based footprints of dynamic TFs but interfered with ATAC-based footprinting of the same TFs. CONCLUSIONS: XL-DNase-seq may help extract novel gene regulatory circuits involving previously undetectable TFs. The DNase-seq and ATAC-seq data generated in our systematic comparison of various cross-linking conditions also represent an unprecedented-scale resource derived from activated mouse macrophage-like cells which share many features of inflammatory macrophages.