A Noninvasive, Orally Stable, Mucosa-Penetrating Polyvalent Vaccine Platform Based on Hepatitis E Virus Nanoparticle

Hepatitis E virus nanoparticle (HEVNP) is an orally stable, mucosa-penetrating delivery platform for noninvasive, targeted delivery of therapeutic and diagnostic agents. HEVNP does not carry HEV genomic RNA and is incapable of replication. The key characteristics that make HEVNP an ideal and unique vehicle for diagnostic and therapeutic delivery include surface plasticity, resistance to the harsh environment of the gastrointestinal (GI) tract, significant payload capacity, platform sustainability, and safety. Furthermore, HEVNP is easily produced using currently available expression/purification technologies; can be easily formulated as a liquid, powder, or solid; and can be distributed (and stored) without the need for a temperature-controlled supply chain

Juvenile Hormone (JH) Esterase of the Mosquito Culex quinquefasciatus Is Not a Target of the JH Analog Insecticide Methoprene

Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (kcat/KM ratio) and Vmax values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE

Quantification of GST and esterase activities in pyrethrin-resistant mosquitoes using pyrethroid-like fluorescent substrates

Botanical pyrethrins and synthetic pyrethroids are highly potent and environmentally safe insecticides that are used to control a wide range of disease vector and pest arthropods. Unfortunately, resistance to these insecticides has been demonstrated in numerous medically important mosquito species. In this study adult Culex pipiens sensu lato were captured in agricultural and urban locations in Fresno County, California, and subsequently exposed to a commercial formulation of pyrethrin insecticide by ultra low volume spraying. Following insecticide exposure, two pyrethroid-like, fluorescent substrates (cis-DCVC and cis-TFMCVC) and CDNB were used to measure esterase and glutathione S-transferase (GST) activities in surviving mosquitoes. Elevated esterase activity (2.5-fold) was found in surviving urban mosquitoes at 12 h post pyrethrin exposure (in comparison to non-insecticide exposed control mosquitoes) when cis-TFMCVC was used as a substrate. Additionally, when CDNB was used as a substrate, 2.8-fold higher GST activity was found. A simple assay was established using our pyrethroid-like, fluorescent substrates that was able to detect low-level esterase activities in homogenates made from individual mosquitoes. The cis-TFMCVC-based assay suggested that esterase activity plays a role in pyrethrin resistance in urban mosquitoes in California

Development of amide-based fluorescent probes for selective measurement of carboxylesterase 1 activity in tissue extracts

Carboxylesterases are well known for their role in the metabolism of xenobiotics. However, recent studies have also implicated carboxylesterases in regulating a number of physiological processes including metabolic homeostasis and macrophage development, underlying the need to quantify them individually. Unfortunately, current methods for selectively measuring the catalytic activity of individual carboxylesterases are not sufficiently sensitive to support many biological studies. In order to develop a more sensitive and selective method to measure the activity of human carboxylesterase 1 (hCE1), we generated and tested novel substrates with a fluorescent aminopyridine leaving group. hCE1 showed at least a 10-fold higher preference for the optimized substrate 4-MOMMP than the 13 other esterases tested. Because of the high stability of 4-MOMMP and its hydrolysis product, this substrate can be used to measure esterase activity over extended incubation periods yielding a low picogram (femtomol) limit of detection. This sensitivity is comparable to current ELISA methods; however, the new assay quantifies only the catalytically active enzyme facilitating direct correlation to biological processes. The method described herein may allow hCE1 activity to be used as a biomarker for predicting drug pharmacokinetics, early detection of hepatocellular carcinoma, and other disease states where the activity of hCE1 is altered

Sequencing and<i>De Novo</i> Assembly of the Transcriptome of the Glassy-Winged Sharpshooter (<i>Homalodisca vitripennis</i>)

<div><p>Background</p><p>The glassy-winged sharpshooter <i>Homalodisca vitripennis</i> (Hemiptera: Cicadellidae), is a xylem-feeding leafhopper and important vector of the bacterium <i>Xylella fastidiosa;</i> the causal agent of Pierce’s disease of grapevines. The functional complexity of the transcriptome of <i>H. vitripennis</i> has not been elucidated thus far. It is a necessary blueprint for an understanding of the development of <i>H. vitripennis</i> and for designing efficient biorational control strategies including those based on RNA interference.</p><p>Results</p><p>Here we elucidate and explore the transcriptome of adult <i>H. vitripennis</i> using high-throughput paired end deep sequencing and <i>de novo</i> assembly. A total of 32,803,656 paired-end reads were obtained with an average transcript length of 624 nucleotides. We assembled 32.9 Mb of the transcriptome of <i>H. vitripennis</i> that spanned across 47,265 loci and 52,708 transcripts. Comparison of our non-redundant database showed that 45% of the deduced proteins of <i>H. vitripennis</i> exhibit identity (<i>e</i>-value ≤1⁻⁵) with known proteins. We assigned Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, and potential Pfam domains to each transcript isoform. In order to gain insight into the molecular basis of key regulatory genes of <i>H. vitripennis</i>, we characterized predicted proteins involved in the metabolism of juvenile hormone, and biogenesis of small RNAs (Dicer and Piwi sequences) from the transcriptomic sequences. Analysis of transposable element sequences of <i>H</i>. <i>vitripennis</i> indicated that the genome is less expanded in comparison to many other insects with approximately 1% of the transcriptome carrying transposable elements.</p><p>Conclusions</p><p>Our data significantly enhance the molecular resources available for future study and control of this economically important hemipteran. This transcriptional information not only provides a more nuanced understanding of the underlying biological and physiological mechanisms that govern <i>H. vitripennis</i>, but may also lead to the identification of novel targets for biorationally designed control strategies.</p></div

Relative Importance of Soluble and Microsomal Epoxide Hydrolases for the Hydrolysis of Epoxy-Fatty Acids in Human Tissues

Epoxy-fatty acids (EpFAs) are endogenous lipid mediators that have a large breadth of biological activities, including the regulation of blood pressure, inflammation, angiogenesis, and pain perception. For the past 20 years, soluble epoxide hydrolase (sEH) has been recognized as the primary enzyme for degrading EpFAs in vivo. The sEH converts EpFAs to the generally less biologically active 1,2-diols, which are quickly eliminated from the body. Thus, inhibitors of sEH are being developed as potential drug therapeutics for various diseases including neuropathic pain. Recent findings suggest that other epoxide hydrolases (EHs) such as microsomal epoxide hydrolase (mEH) and epoxide hydrolase-3 (EH3) can contribute significantly to the in vivo metabolism of EpFAs. In this study, we used two complementary approaches to probe the relative importance of sEH, mEH, and EH3 in 15 human tissue extracts: hydrolysis of 14,15-EET and 13,14-EDP using selective inhibitors and protein quantification. The sEH hydrolyzed the majority of EpFAs in all of the tissues investigated, mEH hydrolyzed a significant portion of EpFAs in several tissues, whereas no significant role in EpFAs metabolism was observed for EH3. Our findings indicate that residual mEH activity could limit the therapeutic efficacy of sEH inhibition in certain organs

RT-PCR validation of H.vitripennis transcripts.

<p>The figure shows the RT-PCR gel picture validating the transcripts of <i>actin</i>, <i>cuticle</i>, <i>chitin deacetilase</i>, <i>juvenile hormone esterase</i> (<i>jhe</i>), <i>juvenile hormone epoxide hydrolase</i> (<i>jheh</i>), <i>juvenile hormone acid methyl transferase</i> (<i>jhamt</i>), <i>serpin b6</i>, Sugar transporter, <i>Vacuolar ATPase</i>, <i>Dicer</i>, <i>argonaute-2</i> and <i>Zinc metalloproteinase</i> from <i>H. vitripennis</i> insects. Psyllid adults (<i>Diaphorina citri</i>) insects were used as negative controls.</p