Two C18 hydroxy-cyclohexenone fatty acids from mammalian epidermis: Potential relation to 12R-lipoxygenase and covalent binding of ceramides.

A key requirement in forming the water permeability barrier in the mammalian epidermis is the oxidation of linoleate esterified in a skin-specific acylceramide by the sequential actions of 12R-lipoxygenase, epidermal lipoxygenase-3, and the epoxyalcohol dehydrogenase SDR9C7 (short-chain dehydrogenase-reductase family 7 member 9). By mechanisms that remain unclear, this oxidation pathway promotes the covalent binding of ceramides to protein, forming a critical structure of the epidermal barrier, the corneocyte lipid envelope. Here, we detected, in porcine, mouse, and human epidermis, two novel fatty acid derivatives formed by KOH treatment from precursors covalently bound to protein: a "polar" lipid chromatographing on normal-phase HPLC just before omega-hydroxy ceramide and a "less polar" lipid nearer the solvent front. Approximately 100 µg of the novel lipids were isolated from porcine epidermis, and the structures were established by UV-spectroscopy, LC-MS, GC-MS, and NMR. Each is a C18 fatty acid and hydroxy-cyclohexenone with the ring on carbons C9-C14 in the polar lipid and C8-C13 in the less polar lipid. Overnight culture of [14C]linoleic acid with whole mouse skin ex vivo led to recovery of the 14C-labeled hydroxy-cyclohexenones. We deduce they are formed from covalently bound precursors during the KOH treatment used to release esterified lipids. KOH-induced intramolecular aldol reactions from a common precursor can account for their formation. Discovery of these hydroxy-cyclohexenones presents an opportunity for a reverse pathway analysis, namely to work back from these structures to identify their covalently bound precursors and relationship to the linoleate oxidation pathway.

Recombinant PNPLA1 catalyzes the synthesis of acylceramides and acyl acids with selective incorporation of linoleic acid.

Loss-of-function mutations in patatin-like phospholipase domain-containing protein 1 (PNPLA1) cause autosomal recessive congenital ichthyosis, and altered PNPLA1 activity is implicated in the pathogenesis of atopic dermatitis and other common skin diseases. To examine the hypothesis that PNPLA1 catalyzes the synthesis of acylceramides and acyl acids, we expressed and partially purified a soluble, truncated form of PNPLA1 in Escherichia coli, (PNPLA1trun) along with the related protein PNPLA2 (ATGL, adipose triglyceride lipase) and coactivator CGI-58. Liposomal substrates were incubated with recombinant enzymes for 0.5-24 h and products analyzed by HPLC-UV and LC-MS. Using trilinolein or dilinolein substrates, PNPLA1trun, like ATGLtrun, catalyzed lipolysis and acyltransferase reactions with 2-30% conversion into linoleic acid, monolinolein, and trilinolein. CGI-58 enhanced ATGL-catalyzed lipolysis as previously reported, but transacylase activity was not enhanced with ATGL or PNPLA1. In matching the proposed activity in vivo, PNPLA1 catalyzed acyl transfer from trilinolein and dilinolein donors to omega-hydroxy ceramide, omega-hydroxy glucosylceramide, and omega-hydroxy acid acceptors to form acylceramide, glucosyl-acylceramide, and acyl acid, respectively, albeit with only ∼0.05% conversion of the substrates. Notably, in experiments comparing dilinolein vs. diolein acyl donors, PNPLA1 transferred linoleate with 3:1 selectivity over oleate into acylceramide. These results support the role for PNPLA1 in the synthesis of acylceramides and acyl acids in epidermis and suggest that the enrichment of these lipids with linoleic acid could result from the substrate selectivity of PNPLA1.

FLG Deficiency in Mice Alters the Early-Life CD4+ T-Cell Response to Skin Commensal Bacteria.

FLG variants underlie ichthyosis vulgaris and increased risk of atopic dermatitis, conditions typified by disruption of the skin microbiome and cutaneous immune response. Yet, it remains unclear whether neonatal skin barrier compromise because of FLG deficiency alters the quality of commensal-specific T cells and the functional impact of such responses. To address these questions, we profiled changes in the skin barrier and early cutaneous immune response of neonatal C57BL/6 Flg‒/‒ and wild-type mice using single-cell RNA sequencing, flow cytometry, and other modalities. Flg‒/‒ neonates showed little alteration in transepidermal water loss or lipid- or corneocyte-related gene expression. However, they showed increases in barrier disruption genes, epidermal dye penetration, and numbers of skin CD4+ T cells. Using an engineered strain of Staphylococcus epidermidis (S. epidermidis 2W) to study the response to neonatal skin colonization, we found that commensal-specific CD4+ T cells were skewed in Flg‒/‒ pups toward effector rather than regulatory T cells. This altered response persisted into adulthood, where it was typified by T helper 17 (Th17) cells and associated with increased susceptibility to imiquimod-induced skin inflammation. Thus, subtle but impactful differences in neonatal barrier function in Flg‒/‒ mice are accompanied by a skewed commensal-specific CD4+ response, with enduring consequences for skin immune homeostasis.

Engineering of Cry3Bb1 provides mechanistic insights toward countering western corn rootworm resistance.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is an economically important pest of corn (maize) in North America and Europe. Current management practices for WCR involve transgenic expression of insecticidal proteins to minimize larval feeding damage to corn roots. The evolution of resistant WCR populations to transgenic corn expressing insecticidal proteins (e.g. Cry3Bb1, Gpp34Ab1/Tpp35Ab1) necessitates efforts to discover and deploy new modes of action for WCR control. Here, we tested the hypothesis that the addition of short peptides selected for binding to the WCR gut would restore insecticidal activity of Cry3Bb1 to resistant insects. Phage display technology coupled with deep sequencing was used to identify peptides selected for binding to WCR brush border membrane vesicles and to recombinant putative receptors aminopeptidase and cadherin. The binding and specificity of selected peptides was confirmed by ELISA and pull-down assays, and candidate gut surface binding partners were identified. Although production of 284 novel Cry3Bb1 variants with these peptides did not restore activity against resistant WCR in artificial diet bioassays, 112 variants were active against susceptible insects. These results provided insights for the mechanism of Cry3Bb1 activity and toward engineering a new mode-of-action via receptor re-targeting in the context of protein structure and function.

ω-O-Acylceramides but not ω-hydroxy ceramides are required for healthy lamellar phase architecture of skin barrier lipids.

Epidermal omega-O-acylceramides (ω-O-acylCers) are essential components of a competent skin barrier. These unusual sphingolipids with ultralong N-acyl chains contain linoleic acid esterified to the terminal hydroxyl of the N-acyl, the formation of which requires the transacylase activity of patatin-like phospholipase domain containing 1 (PNPLA1). In ichthyosis with dysfunctional PNPLA1, ω-O-acylCer levels are significantly decreased, and ω-hydroxylated Cers (ω-OHCers) accumulate. Here, we explore the role of the linoleate moiety in ω-O-acylCers in the assembly of the skin lipid barrier. Ultrastructural studies of skin samples from neonatal Pnpla1+/+ and Pnpla1-/- mice showed that the linoleate moiety in ω-O-acylCers is essential for lamellar pairing in lamellar bodies, as well as for stratum corneum lipid assembly into the long periodicity lamellar phase. To further study the molecular details of ω-O-acylCer deficiency on skin barrier lipid assembly, we built in vitro lipid models composed of major stratum corneum lipid subclasses containing either ω-O-acylCer (healthy skin model), ω-OHCer (Pnpla1-/- model), or combination of the two. X-ray diffraction, infrared spectroscopy, and permeability studies indicated that ω-OHCers could not substitute for ω-O-acylCers, although in favorable conditions, they form a medium lamellar phase with a 10.8 nm-repeat distance and permeability barrier properties similar to long periodicity lamellar phase. In the absence of ω-O-acylCers, skin lipids were prone to separation into two phases with diminished barrier properties. The models combining ω-OHCers with ω-O-acylCers indicated that accumulation of ω-OHCers does not prevent ω-O-acylCer-driven lamellar stacking. These data suggest that ω-O-acylCer supplementation may be a viable therapeutic option in patients with PNPLA1 deficiency.

Phenotypic overlap between atopic dermatitis and autism.

BACKGROUND: Autism, a childhood behavioral disorder, belongs to a large suite of diseases, collectively referred to as autism spectrum disorders (ASD). Though multifactorial in etiology, approximately 10% of ASD are associated with atopic dermatitis (AD). Moreover, ASD prevalence increases further as AD severity worsens, though these disorders share no common causative mutations. We assessed here the link between these two disorders in the standard, valproic acid mouse model of ASD. In prior studies, there was no evidence of skin involvement, but we hypothesized that cutaneous involvement could be detected in experiments conducted in BALB/c mice. BALB/c is an albino, laboratory-bred strain of the house mouse and is among the most widely used inbred strains used in animal experimentation. METHODS: We performed our studies in valproic acid (VPA)-treated BALB/c hairless mice, a standard mouse model of ASD. Mid-trimester pregnant mice received a single intraperitoneal injection of either valproic acid sodium salt dissolved in saline or saline alone on embryonic day 12.5 and were housed individually until postnatal day 21. Only the brain and epidermis appeared to be affected, while other tissues remain unchanged. At various postnatal time points, brain, skin and blood samples were obtained for histology and for quantitation of tissue sphingolipid content and cytokine levels. RESULTS: AD-like changes in ceramide content occurred by day one postpartum in both VPA-treated mouse skin and brain. The temporal co-emergence of AD and ASD, and the AD phenotype-dependent increase in ASD prevalence correlated with early appearance of cytokine markers (i.e., interleukin [IL]-4, 5, and 13), as well as mast cells in skin and brain. The high levels of interferon (IFN)γ not only in skin, but also in brain likely account for a significant decline in esterified very-long-chain N-acyl fatty acids in brain ceramides, again mimicking known IFNγ-induced changes in AD. CONCLUSION: Baseline involvement of both AD and ASD could reflect concurrent neuro- and epidermal toxicity, possibly because both epidermis and neural tissues originate from the embryonic neuroectoderm. These studies illuminate the shared susceptibility of the brain and epidermis to a known neurotoxin, suggesting that the atopic diathesis could be extended to include ASD.

Unbound Corneocyte Lipid Envelopes in 12R-Lipoxygenase Deficiency Support a Specific Role in Lipid-Protein Cross-Linking.

Loss-of-function mutations in arachidonate lipoxygenase 12B (ALOX12B) are an important cause of autosomal recessive congenital ichthyosis (ARCI). 12R-lipoxygenase (12R-LOX), the protein product of ALOX12B, has been proposed to covalently bind the corneocyte lipid envelope (CLE) to the proteinaceous corneocyte envelope, thereby providing a scaffold for the assembly of barrier-providing, mature lipid lamellae. To test this hypothesis, an in-depth ultrastructural examination of CLEs was performed in ALOX12B-/- human and Alox12b-/- mouse epidermis, extracting samples with pyridine to distinguish covalently attached CLEs from unbound (ie, noncovalently bound) CLEs. ALOX12B--/- stratum corneum contained abundant pyridine-extractable (ie, unbound) CLEs, compared with normal stratum corneum. These unbound CLEs were associated with defective post-secretory lipid processing, and were specific to 12R-LOX deficiency, because they were not observed with deficiency of the related ARCI-associated proteins, patatin-like phospholipase 1 (Pnpla1) or abhydrolase domain containing 5 (Abhd5). These results suggest that 12R-LOX contributes specifically to CLE-corneocyte envelope cross-linking, which appears to be a prerequisite for post-secretory lipid processing, and provide insights into the pathogenesis of 12R-LOX deficiency in this subtype of ARCI, as well as other conditions that display a defective CLE.

Exosomes from Human Adipose Tissue-Derived Mesenchymal Stem Cells Promote Epidermal Barrier Repair by Inducing de Novo Synthesis of Ceramides in Atopic Dermatitis.

Atopic dermatitis (AD) is a multifactorial, heterogeneous disease associated with epidermal barrier disruption and intense systemic inflammation. Previously, we showed that exosomes derived from human adipose tissue-derived mesenchymal stem cells (ASC-exosomes) attenuate AD-like symptoms by reducing multiple inflammatory cytokine levels. Here, we investigated ASC-exosomes' effects on skin barrier restoration by analyzing protein and lipid contents. We found that subcutaneous injection of ASC-exosomes in an oxazolone-induced dermatitis model remarkably reduced trans-epidermal water loss, while enhancing stratum corneum (SC) hydration and markedly decreasing the levels of inflammatory cytokines such as IL-4, IL-5, IL-13, TNF-α, IFN-γ, IL-17, and TSLP, all in a dose-dependent manner. Interestingly, ASC-exosomes induced the production of ceramides and dihydroceramides. Electron microscopic analysis revealed enhanced epidermal lamellar bodies and formation of lamellar layer at the interface of the SC and stratum granulosum with ASC-exosomes treatment. Deep RNA sequencing analysis of skin lesions demonstrated that ASC-exosomes restores the expression of genes involved in skin barrier, lipid metabolism, cell cycle, and inflammatory response in the diseased area. Collectively, our results suggest that ASC-exosomes effectively restore epidermal barrier functions in AD by facilitating the de novo synthesis of ceramides, resulting in a promising cell-free therapeutic option for treating AD.

Endoplasmic Reticulum Calcium Regulates Epidermal Barrier Response and Desmosomal Structure.

Ca(2+) fluxes direct keratinocyte differentiation, cell-to-cell adhesion, migration, and epidermal barrier homeostasis. We previously showed that intracellular Ca(2+) stores constitute a major portion of the calcium gradient especially in the stratum granulosum. Loss of the calcium gradient triggers epidermal barrier homeostatic responses. In this report, using unfixed ex vivo epidermis and human epidermal equivalents we show that endoplasmic reticulum (ER) Ca(2+) is released in response to barrier perturbation, and that this release constitutes the major shift in epidermal Ca(2+) seen after barrier perturbation. We find that ER Ca(2+) release correlates with a transient increase in extracellular Ca(2+). Lastly, we show that ER calcium release resulting from barrier perturbation triggers transient desmosomal remodeling, seen as an increase in extracellular space and a loss of the desmosomal intercellular midline. Topical application of thapsigargin, which inhibits the ER Ca(2+) ATPase activity without compromising barrier integrity, also leads to desmosomal remodeling and loss of the midline structure. These experiments establish the ER Ca(2+) store as a master regulator of the Ca(2+) gradient response to epidermal barrier perturbation, and suggest that ER Ca(2+) homeostasis also modulates normal desmosomal reorganization, both at rest and after acute barrier perturbation.

Comparative pharmacological characterization of D1-like dopamine receptors from Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus suggests pleiotropic signaling in mosquito vector lineages.

BACKGROUND: Small molecule antagonists of mosquito dopamine receptors (DARs) are under investigation as a new class of vector-selective insecticides. Antagonists that inhibit the D1-like DARs AaDOP2 and CqDOP2 from the mosquitoes Aedes aegypti L. and Culex quinquefasciatus Say, respectively, also cause larval mortality in bioassays. Here, we report on the orthologous DAR, AgDOP2, from the malaria mosquito Anopheles gambiae Giles that was cloned and pharmacologically characterized in HEK293 cells. Larval bioassays were then conducted to examine the potential of DAR antagonist insecticides against Anopheles vectors. FINDINGS: Previous in vitro cAMP accumulation assays demonstrated Gαs coupling for AaDOP2 and CqDOP2 and dose-dependent inhibition by DAR antagonists. We observed a negligible response of AgDOP2 in the cAMP assay, which prompted an investigation of alternative coupling for mosquito DARs. In an in vitro IP-One Gαq second messenger assay of calcium signaling, dopamine stimulation increased IP1 accumulation in AaDOP2-, CqDOP2- and AgDOP2-expressing cells, and DAR antagonists inhibited IP1 signaling in a dose-dependent manner. In larval bioassays, DAR antagonists caused considerable mortality of An. gambiae larvae within 24 h post-exposure. CONCLUSIONS: In vitro data reveal pleiotropic coupling of AaDOP2 and CqDOP2 to Gαq and Gαs. In contrast, AgDOP2 appeared to selectively couple to Gαq signaling. In vitro antagonist studies revealed general conservation in pharmacology between mosquito DARs. In vivo data suggest potential for DAR antagonist insecticides against An. gambiae. Sequence conservation among the DOP2 receptors from 15 Anopheles species indicates utility of antagonists to control residual malaria transmission. AgDOP2 Gαq-dependent signaling could be exploited for An. gambiae control via pathway specific antagonists.

Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Dopamine receptor antagonists as new mode-of-action insecticide leads for control of Aedes and Culex mosquito vectors.

BACKGROUND: New mode-of-action insecticides are sought to provide continued control of pesticide resistant arthropod vectors of neglected tropical diseases (NTDs). We previously identified antagonists of the AaDOP2 D1-like dopamine receptor (DAR) from the yellow fever mosquito, Aedes aegypti, with toxicity to Ae. aegypti larvae as leads for novel insecticides. To extend DAR-based insecticide discovery, we evaluated the molecular and pharmacological characteristics of an orthologous DAR target, CqDOP2, from Culex quinquefasciatus, the vector of lymphatic filariasis and West Nile virus. METHODS/RESULTS: CqDOP2 has 94.7% amino acid identity to AaDOP2 and 28.3% identity to the human D1-like DAR, hD1. CqDOP2 and AaDOP2 exhibited similar pharmacological responses to biogenic amines and DAR antagonists in cell-based assays. The antagonists amitriptyline, amperozide, asenapine, chlorpromazine and doxepin were between 35 to 227-fold more selective at inhibiting the response of CqDOP2 and AaDOP2 in comparison to hD1. Antagonists were toxic to both C. quinquefasciatus and Ae. aegypti larvae, with LC50 values ranging from 41 to 208 µM 72 h post-exposure. Orthologous DOP2 receptors identified from the African malaria mosquito, Anopheles gambiae, the sand fly, Phlebotomus papatasi and the tsetse fly, Glossina morsitans, had high sequence similarity to CqDOP2 and AaDOP2. CONCLUSIONS: DAR antagonists represent a putative new insecticide class with activity against C. quinquefasciatus and Ae. aegypti, the two most important mosquito vectors of NTDs. There has been limited change in the sequence and pharmacological properties of the DOP2 DARs of these species since divergence of the tribes Culicini and Aedini. We identified antagonists selective for mosquito versus human DARs and observed a correlation between DAR pharmacology and the in vivo larval toxicity of antagonists. These data demonstrate that sequence similarity can be predictive of target potential. On this basis, we propose expanded insecticide discovery around orthologous DOP2 targets from additional dipteran vectors.

Evaluation of AaDOP2 receptor antagonists reveals antidepressants and antipsychotics as novel lead molecules for control of the yellow fever mosquito, Aedes aegypti.

The yellow fever mosquito, Aedes aegypti, vectors disease-causing agents that adversely affect human health, most notably the viruses causing dengue and yellow fever. The efficacy of current mosquito control programs is challenged by the emergence of insecticide-resistant mosquito populations, suggesting an urgent need for the development of chemical insecticides with new mechanisms of action. One recently identified potential insecticide target is the A. aegypti D1-like dopamine receptor, AaDOP2. The focus of the present study was to evaluate AaDOP2 antagonism both in vitro and in vivo using assay technologies with increased throughput. The in vitro assays revealed AaDOP2 antagonism by four distinct chemical scaffolds from tricyclic antidepressant or antipsychotic chemical classes, and elucidated several structure-activity relationship trends that contributed to enhanced antagonist potency, including lipophilicity, halide substitution on the tricyclic core, and conformational rigidity. Six compounds displayed previously unparalleled potency for in vitro AaDOP2 antagonism, and among these, asenapine, methiothepin, and cis-(Z)-flupenthixol displayed subnanomolar IC50 values and caused rapid toxicity to A. aegypti larvae and/or adults in vivo. Our study revealed a significant correlation between in vitro potency for AaDOP2 antagonism and in vivo toxicity, suggesting viability of AaDOP2 as an insecticidal target. Taken together, this study expanded the repertoire of known AaDOP2 antagonists, enhanced our understanding of AaDOP2 pharmacology, provided further support for rational targeting of AaDOP2, and demonstrated the utility of efficiency-enhancing in vitro and in vivo assay technologies within our genome-to-lead pipeline for the discovery of next-generation insecticides.

Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation.

Scavenger receptor-mediated uptake of oxidized LDL (oxLDL) is thought to be the major mechanism of foam cell generation in atherosclerotic lesions. Recent data has indicated that native LDL is also capable of contributing to foam cell formation via low-affinity receptor-independent LDL particle pinocytosis and selective cholesteryl ester (CE) uptake. In the current investigation, Cu(2+)-induced LDL oxidation was found to inhibit macrophage selective CE uptake. Impairment of selective CE uptake was significant with LDL oxidized for as little as 30 min and correlated with oxidative fragmentation of apoB. In contrast, LDL aggregation, LDL CE oxidation, and the enhancement of scavenger receptor-mediated LDL particle uptake required at least 3 h of oxidation. Selective CE uptake did not require expression of the LDL receptor (LDL-R) and was inhibited similarly by LDL oxidation in LDL-R(-/-) versus WT macrophages. Inhibition of selective uptake was also observed when cells were pretreated or cotreated with minimally oxidized LDL, indicating a direct inhibitory effect of this oxLDL on macrophages. Consistent with the effect on LDL CE uptake, minimal LDL oxidation almost completely prevented LDL-induced foam cell formation. These data demonstrate a novel inhibitory effect of mildly oxidized LDL that may reduce foam cell formation in atherosclerosis.

New developments in selective cholesteryl ester uptake.

PURPOSE OF REVIEW: Selective lipid uptake (SLU) is known to be a major pathway of lipoprotein cholesterol metabolism in experimental animals and humans, but remains poorly understood. This review provides a brief overview of SLU mediated by the HDL receptor scavenger receptor B-type I (SR-BI), and highlights several surprising new findings related to the impact of SLU pathways in cholesterol homeostasis. RECENT FINDINGS: Under certain conditions, SR-BI-mediated SLU contributes to reverse cholesterol transport (RCT) independently of ABCG5/G8-mediated biliary cholesterol secretion, implying a novel trafficking mechanism. Hepatic SR-BI expression and RCT are decreased in diabetic mice. Farnesoid X receptor (FXR) and the microRNAs miR-185, miR-96 and miR-223 are emerging therapeutic targets for increasing SR-BI expression. SR-BI-independent selective cholesteryl ester uptake is a newly characterized pathway in macrophage foam cells. SUMMARY: New findings underscore the importance of SR-BI-mediated SLU in hepatic SLU and RCT, while indicating that further investigation is needed to define SLU pathways, including SR-BI-independent macrophage selective cholesteryl ester uptake. The intracellular trafficking of cholesterol in these pathways appears to be critical to their normal function and is a major subject of ongoing studies.

The Impairment of Macrophage-to-Feces Reverse Cholesterol Transport during Inflammation Does Not Depend on Serum Amyloid A.

Studies suggest that inflammation impairs reverse cholesterol transport (RCT). We investigated whether serum amyloid A (SAA) contributes to this impairment using an established macrophage-to-feces RCT model. Wild-type (WT) mice and mice deficient in SAA1.1 and SAA2.1 (SAAKO) were injected intraperitoneally with (3)H-cholesterol-labeled J774 macrophages 4 hr after administration of LPS or buffered saline. (3)H-cholesterol in plasma 4 hr after macrophage injection was significantly reduced in both WT and SAAKO mice injected with LPS, but this was not associated with a reduced capacity of serum from LPS-injected mice to promote macrophage cholesterol efflux in vitro. Hepatic accumulation of (3)H-cholesterol was unaltered in either WT or SAAKO mice by LPS treatment. Radioactivity present in bile and feces of LPS-injected WT mice 24 hr after macrophage injection was reduced by 36% (P < 0.05) and 80% (P < 0.001), respectively. In contrast, in SAAKO mice, LPS did not significantly reduce macrophage-derived (3)H-cholesterol in bile, and fecal excretion was reduced by only 45% (P < 0.05). Injection of cholesterol-loaded allogeneic J774 cells, but not syngeneic bone-marrow-derived macrophages, transiently induced SAA in C57BL/6 mice. Our study confirms reports that acute inflammation impairs steps in the RCT pathway and establishes that SAA plays only a minor role in this impairment.

Transcriptome sequencing and annotation of the predatory mite Metaseiulus occidentalis (Acari: Phytoseiidae): a cautionary tale about possible contamination by prey sequences.

Next-generation sequencing was applied to the transcriptome of the phytoseiid Metaseiulus occidentalis to characterize gene expression in all life stages reared under different conditions to optimize the recovery of as many genes as possible. One production and one titration run produced a total of 862,069 reads (average size: 314.87 bp), which generated 255.6 Mbp of sequences on the GS-FLX Titanium sequencing platform. After removal of putative prey sequences 850,543 reads were used in NewBler and PTA assemblies to produce 74,172 non-redundant sequences, including 30,691 contigs and 43,481 singlets with 11,994 contigs consisting of more than 500 bp and 37,278 sequences >300 bp, constituting 48.7 % of all sequences. There were 25,888 hits with the NCBI non-redundant database and 15,376 unique transcripts. There were 26,225 hits with the Ixodes scapularis genome and 6,634 unique transcripts. There were 22,225 hits with the RefSeq of Homo sapiens with 6,465 unique transcripts, and 23,656 hits with the RefSeq of Drosophila melanogaster with 9,216 unique transcripts. Selected ESTs corresponding to genes of interest were analyzed including those related to transposable elements, GPCRs, Sox transcription factors, diapause and foraging behavior, and pesticide resistances. Novel and important genes appear to have been discovered that provide insight into the evolution, biology, and physiology of this important predator of pest mites in agriculture and will be useful in analyzing complete genome sequences of this natural enemy.

Discovery of antagonists of tick dopamine receptors via chemical library screening and comparative pharmacological analyses.

Ticks transmit a wide variety of disease causing pathogens to humans and animals. Considering the global health impact of tick-borne diseases, there is a pressing need to develop new methods for vector control. We are exploring arthropod dopamine receptors as novel targets for insecticide/acaricide development because of their integral roles in neurobiology. Herein, we developed a screening assay for dopamine receptor antagonists to further characterize the pharmacological properties of the two D1-like dopamine receptors (Isdop1 and Isdop2) identified in the Lyme disease vector, Ixodes scapularis, and develop a screening assay for receptor antagonists. A cell-based, cyclic AMP luciferase reporter assay platform was implemented to screen the LOPAC(1280) small molecule library for Isdop2 receptor antagonists, representing the first reported chemical library screen for any tick G protein-coupled receptor. Screening resulted in the identification of 85 "hit" compounds with antagonist activity at the Isdop2 receptor. Eight of these chemistries were selected for confirmation assays using a direct measurement of cAMP, and the effects on both Isdop1 and Isdop2 were studied for comparison. Each of these eight compounds showed antagonistic activity at both Isdop1 and Isdop2, although differences were observed regarding their relative potencies. Furthermore, comparison of the pharmacological properties of the tick dopamine receptors with that of the AaDOP2 receptor from the yellow fever mosquito and the human dopamine D1 receptor (hD1) revealed species-specific pharmacological profiles of these receptors. Compounds influencing dopaminergic functioning, such as the dopamine receptor antagonists discovered here, may provide lead chemistries for discovery of novel acaricides useful for vector control