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DEET (N, N-diethyl-meta-toluamide) is a synthetic chemical identified by the US Department of Agriculture in 1946 in a screen for repellents to protect soldiers from mosquito-borne diseases(1,2). Since its discovery, DEET has become the world's most widely used arthropod repellent and is effective against invertebrates separated by millions of years of evolution-including biting flies(3), honeybees(4), ticks(5), and land leeches(3). In insects, DEET acts on the olfactory system(5-12) and requires the olfactory receptor co-receptor Orco(7,9-12), but exactly how it works remains controversial(13). Here we show that the nematode Caenorhabditis elegans is sensitive to DEET and use this genetically tractable animal to study the mechanism of action of this chemical. We found that DEET is not a volatile repellent, but instead interferes selectively with chemotaxis to a variety of attractant and repellent molecules. In a forward genetic screen for DEET-resistant worms, we identified a gene that encodes a single G protein-coupled receptor, str-217, which is expressed in a single pair of chemosensory neurons that are responsive to DEET, called ADL neurons. Mis-expression of str-217 in another chemosensory neuron conferred responses to DEET. Engineered str-217 mutants, and a wild isolate of C. elegans that carries a str-217 deletion, are resistant to DEET. We found that DEET can interfere with behaviour by inducing an increase in average pause length during locomotion, and show that this increase in pausing requires both str-217 and ADL neurons. Finally, we demonstrated that ADL neurons are activated by DEET and that optogenetic activation of ADL neurons increased average pause length. This is consistent with the 'confusant' hypothesis, which proposes that DEET is not a simple repellent but that it instead modulates multiple olfactory pathways to scramble behavioural responses(10,11). Our results suggest a consistent motif in the effectiveness of DEET across widely divergent taxa: an effect on multiple chemosensory neurons that disrupts the pairing between odorant stimulus and behavioural response.

Clinical and animal studies show that ethanol exposure and inflammation during pregnancy cause similar behavioral disturbances in the offspring. While ethanol is shown to stimulate both neuroimmune and neurochemical systems in adults, little is known about their anatomical relationship in response to ethanol in utero and whether neuroimmune factors mediate ethanol's effects on neuronal development and behavior in offspring. Here we examined in female and male adolescent rats a specific population of neurons concentrated in lateral hypothalamus, which coexpress the inflammatory chemokine C-C motif ligand 2 (CCL2) or its receptor CCR2 with the orexigenic neuropeptide, melanin-concentrating hormone (MCH), that promotes ethanol drinking behavior. We demonstrate that maternal administration of ethanol (2 g/kg/d) from embryonic day 10 (E10) to E15, while having little impact on glia, stimulates expression of neuronal CCL2 and CCR2, increases density of both large CCL2 neurons colocalizing MCH and small CCL2 neurons surrounding MCH neurons, and stimulates ethanol drinking and anxiety in adolescent offspring. We show that these neuronal and behavioral changes are similarly produced by maternal administration of CCL2 (4 or 8 mu g/kg/d, E10-E15) and blocked by maternal administration of a CCR2 antagonist INCB3344 (1 mg/kg/d, E10-E15), and these effects of ethanol and CCL2 are sexually dimorphic, consistently stronger in females. These results suggest that this neuronal CCL2/CCR2 system closely linked to MCH neurons has a role in mediating the effects of maternal ethanol exposure on adolescent offspring and contributes to the higher levels of adolescent risk factors for alcohol use disorders described in women.

The standard reference Caenorhabditis elegans strain, N2, has evolved marked behavioral changes in social feeding behavior since its isolation from the wild. We show that the causal, laboratory-derived mutations in two genes, npr-1 and glb-5, confer large fitness advantages in standard laboratory conditions. Using environmental manipulations that suppress social/solitary behavior differences, we show the fitness advantages of the derived alleles remained unchanged, suggesting selection on these alleles acted through pleiotropic traits. Transcriptomics, developmental timing, and food consumption assays showed that N2 animals mature faster, produce more sperm, and consume more food than a strain containing ancestral alleles of these genes regardless of behavioral strategies. Our data suggest that the pleiotropic effects of glb-5 and npr-1 are a consequence of changes to O-2-sensing neurons that regulate both aerotaxis and energy homeostasis. Our results demonstrate how pleiotropy can lead to profound behavioral changes in a popular laboratory model.

Two of the most predominant features of the Alzheimer's disease (AD) brain are deposition of beta-amyloid (A beta) plaques and inflammation. The mechanism behind these pathologies remains unknown, but there is evidence to suggest that inflammation may predate the deposition of A beta. Furthermore, immune activation is increasingly being recognized as a major contributor to the pathogenesis of the disease, and disorders involving systemic inflammation, such as infection, aging, obesity, atherosclerosis, diabetes, and depression are risk factors for the development of AD. Plasminogen (PLG) is primarily a blood protein synthesized in the liver, which when cleaved into its active form, plasmin (PL), plays roles in fibrinolysis, wound healing, cell signaling, and inflammatory regulation. Here we show that PL in the blood is a regulator of brain inflammatory action and AD pathology. Depletion of PLG in the plasma of an AD mouse model through antisense oligonucleotide technology dramatically improved AD pathology and decreased glial cell activation in the brain, whereas an increase in PL activity through alpha-2-antiplasmin (A2AP) antisense oligonucleotide treatment exacerbated the brain's immune response and plaque deposition. These studies suggest a crucial role for peripheral PL in mediating neuroimmune cell activation and AD progression and could provide a link to systemic inflammatory risk factors that are known to be associated with AD development.

Lipid bilayers and lipid-associated proteins play crucial roles in biology. As in vivo studies and manipulation are inherently difficult, membrane-mimetic systems are useful for the investigation of lipidic phases, lipid-protein interactions, membrane protein function and membrane structure in vitro. In this work, we describe a route to leverage the programmability of DNA nanotechnology and create DNA-encircled bilayers (DEBs). DEBs are made of multiple copies of an alkylated oligonucleotide hybridized to a single-stranded minicircle, in which up to two alkyl chains per helical turn point to the inside of the toroidal DNA ring. When phospholipids are added, a bilayer is observed to self-assemble within the ring such that the alkyl chains of the oligonucleotides stabilize the hydrophobic rim of the bilayer to prevent formation of vesicles and support thermotropic lipid phase transitions. The DEBs are completely free of protein and can be synthesized from commercially available components using routine equipment. The diameter of DEBs can be varied in a predictable manner. The well-established toolbox from structural DNA nanotechnology, will ultimately enable the rational design of DEBs so that their size, shape or functionalization can be adapted to the specific needs of biophysical investigations of lipidic phases and the properties of membrane proteins embedded into DEB nanoparticle bilayers.

The cell wall of Gram-positive bacteria contains abundant surfaceexposed carbohydrate structures that are highly conserved. While these properties make surface carbohydrates ideal targets for immunotherapy, carbohydrates elicit a poor immune response that results primarily in low-affinity IgM antibodies. In a previous publication, we introduced the lysibody approach to address this shortcoming. Lysibodies are engineered molecules that combine a high-affinity carbohydrate-binding domain of bacterial or bacteriophage origin and an Fc effector portion of a human IgG antibody, thus directing effective immunity to conserved bacterial surface carbohydrates. Here, we describe the first example of a lysibody containing the binding domain from a bacteriocin, lysostaphin. We also describe the creation of five lysibodies with binding domains derived from phage lysins, directed against Staphylococcus aureus. The lysostaphin and LysK lysibodies showed the most promise and were further characterized. Both lysibodies bound a range of clinically important staphylococcal strains, fixed complement on the staphylococcal surface, and induced phagocytosis of S. aureus by macrophages and human neutrophils. The lysostaphin lysibody had superior in vitro activity compared to that of the LysK lysibody, as well as that of the previously characterized ClyS lysibody, and it effectively protected mice in a kidney abscess/bacteremia model. These results further demonstrate that the lysibody approach is a reproducible means of creating antibacterial antibodies that cannot be produced by conventional means. Lysibodies therefore are a promising solution for opsonic antibodies that may be used passively to both treat and prevent infection by drug-resistant pathogens.

The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed "mouse kidney parvovirus" (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and estab- lish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.

Chemokines and some chemical analogs of chemokines prevent cellular HIV-1 entry when bound to the HIV-1 coreceptors C-C chemokine receptor 5 (CCR5) or C-X-C chemokine receptor 4 (CXCR4), which are G protein-coupled receptors (GPCRs). The ideal HIV-1 entry blocker targeting the coreceptors would display ligand bias and avoid activating G protein-mediated pathways that lead to inflammation. We compared CCR5-dependent activation of second messenger pathways in a single cell line. We studied two endogenous chemokines [ RANTES (also known as CCL5) and MIP-1. (also known as CCL3)] and four chemokine analogs of RANTES (5P12-, 5P14-, 6P4-, and PSC-RANTES). We found that CCR5 signaled through both G(i/o) and G(q/11). IP1 accumulation and Ca2+ flux arose from G(q/11) activation, rather than from G beta gamma subunit release after Gi/o activation as had been previously proposed. The 6P4-and PSC-RANTES analogs were superagonists for G(q/11) activation, whereas the 5P12- and 5P14-RANTES analogs displayed a signaling bias for G(i/o). These results demonstrate that RANTES analogs elicit G protein subtype-specific signaling bias and can cause CCR5 to couple preferentially to G(q/11) rather than to Gi/o signaling pathways. We propose that G protein subtype-specific signaling bias may be a general feature of GPCRs that can couple to more than one G protein family.

RNA viruses are a major threat to animals and plants. RNA interference (RNAi) and the interferon response provide innate antiviral defense against RNA viruses. Here, we performed a large-scale screen using Caenorhabditis elegans and its natural pathogen the Orsay virus (OrV), and we identified cde-1 as important for antiviral defense. CDE-1 is a homolog of the mammalian TUT4 and TUT7 terminal uridylyltransferases (collectively called TUT4(7)); its catalytic activity is required for its antiviral function. CDE-1 uridylates the 3' end of the OrV RNA genome and promotes its degradation in a manner independent of the RNAi pathway. Likewise, TUT4(7) enzymes uridylate influenza A virus (IAV) mRNAs in mammalian cells. Deletion of TUT4(7) leads to increased IAV mRNA and protein levels. Collectively, these data implicate 3'-terminal uridylation of viral RNAs as a conserved antiviral defense mechanism.