LIN-44/Wnt directs dendrite outgrowth through LIN-17/Frizzled in C. elegans Neurons

Nervous system function requires proper development of two functional and morphological domains of neurons, axons and dendrites. Although both these domains are equally important for signal transmission, our understanding of dendrite development remains relatively poor. Here, we show that in C. elegans the Wnt ligand, LIN-44, and its Frizzled receptor, LIN-17, regulate dendrite development of the PQR oxygen sensory neuron. In lin-44 and lin-17 mutants, PQR dendrites fail to form, display stunted growth, or are misrouted. Manipulation of temporal and spatial expression of LIN-44, combined with cell-ablation experiments, indicates that this molecule is patterned during embryogenesis and acts as an attractive cue to define the site from which the dendrite emerges. Genetic interaction between lin-44 and lin-17 suggests that the LIN-44 signal is transmitted through the LIN-17 receptor, which acts cell autonomously in PQR. Furthermore, we provide evidence that LIN-17 interacts with another Wnt molecule, EGL-20, and functions in parallel to MIG-1/Frizzled in this process. Taken together, our results reveal a crucial role for Wnt and Frizzled molecules in regulating dendrite development in vivo.     

Reassessing the Detection of B-Virus–Specific Serum Antibodies

B virus, a natural pathogen of macaques, can cause a fatal zoonotic disease in humans. Serologic screening of macaques by titration ELISA (tELISA, screening test) and by Western blot analysis (WBA, confirmatory test) is one of the principle measures to prevent human infection. Here we slightly modified these 2 tests and reevaluated their correlation. We developed a high-throughput tELISA and used it to screen 278 sera simultaneously against the homologous BV antigen and the heterologous antigens of Papiine herpesvirus 2 and Human herpesvirus 1. More sera (35.6%) were positive by the BV-ELISA than by the HVP2-ELISA (21.6%) or HSV1-ELISA (19.8%). The superiority of the homologous tELISA over the heterologous tELISA was prominent in low-titer sera. WBA confirmed only 21% of the tELISA-positive sera with low or intermediate antibody titers. These sera might have contained antibodies to conformational epitopes that could not be detected by WBA, in which denatured antigens are used, but that could be detected by tELISA, which detects both linear and conformational epitopes. WBA confirmed 82% of the tELISA high-titer sera. However, WBA defined the remaining 18% of sera, which were negative by tELISA, as nonnegative. This finding can be attributed to the difficulties encountered with the subjective interpretation of results by WBA. Together, the current results indicate the inadequacy of WBA as a confirmatory assay for sera with low antibody titers.Abbreviations: BV, B virus (Macacine herpesvirus 1); CLIA, Clinical Laboratory Improvement Amendments; EU, ELISA units; HSV, Human herpesvirus; HVL, langur herpesvirus; HVM, Herpesvirus managabey; HVP, Papiine herpesvirus; SA8, simian agent 8; tELISA, titration ELISA; UN, uninfected; WBA, Western blot analysisB virus (BV; Macacine herpesvirus 1), which is endemic in all species of macaques (natural hosts), is a member of the genus Simplexvirus, subfamily Alphaherpesvirinae and family Herpesviridae. Alphaherpesviruses are characterized by the ability to establish a neurotropic, generally asymptomatic, infection in their natural hosts. Macaques spread BV within a group by contact with macaques that are shedding virus during an acute or intermittently reactivated infection. BV is closely related to 2 well-characterized human alphaherpesviruses, Human herpesvirus (HSV) types 1 and 2, to simian agent 8 (SA8; Cercopithecine herpesvirus 2), which is endemic in African green monkeys (Cercopithecus aethiops), and to 2 recently identified simian alphaherpesviruses, HVP2 (Papiine herpesvirus 2) in baboons (Papio spp.) and langur herpesvirus (HVL), which is endemic in langur monkeys (Presbytis spp.)^5,6,7 and which has not been officially classified or named.⁹ Recently, sera from a group of sooty mangabey monkeys (Cercocebus atys) were found to crossreact serologically with other simian simplexviruses.^6,8,11 It was assumed that crossreactive antibodies were induced by a putative alphaherpesvirus that is endemic in mangabeys. This virus was provisionally named as Herpesvirus managabey (HVM) and is pending taxonomic evaluation.Each of the simplex viruses has remarkable host specificity in nature. However, cross-species infections with BV have been reported. BV is the only nonhuman primate alphaherpesvirus that infects humans. When it does so, BV causes an often-fatal zoonotic disease in 80% of untreated humans.^{7,10,21,23,32,33}BV is transmitted through bites, scratches, or contact with infected oral or genital body fluids. In addition, the virus can be transmitted via fomites and from human to human through contact with contaminated wounds. Virus replication occurs in epithelial or fibroblast cells at the epidermal or dermal site of virus entry; however, BV also enters the peripheral nervous system via axons without replicating locally in surrounding epithelial cells, as has been reported for other simplex viruses.^20,23 Once BV enters peripheral nerves, life-long latency is established in the dorsal root of spinal ganglia or cranial ganglia of infected hosts. BV undergoes periodic reactivation in macaques as well as in humans that survive this zoonosis. In both macaques and humans, BV can be reactivated in the ganglia, generally resulting in anterograde travel of the virus and replication at the original site of infection.^10,33 This event results in virus shedding from infected cells, an event that can be detected by PCR or virus isolation if samples are collected during this event. However, because virus shedding is unpredictable and sporadic, identification of BV infection by means of PCR or virus isolation is rare. A more practical approach to identifying infected macaques or humans is the use of serologic methods for identifying antibodies specific for BV, although the shortcomings of this approach are appreciated when screening sera from subjects that are in the midst of a primary infection but have not yet produced detectable levels of antibodies or from BV-infected subjects that lack detectable antibody because of waning levels or anergy.Because of the high lethality of BV to humans and life-long infection in survivors that lack effective strategies to clear this virus, BV antigen is produced under BSL4 conditions according to federal guidelines and under strict biosecurity regulations.³ Many laboratories in the United States, Europe, and Asia cannot produce BV antigens because of these restrictions and therefore use alternative crossreacting (heterologous) herpesvirus antigens such as HVP2 and HSV1 for the detection of antibodies to BV.^{10,19,26,29,34} Our previous studies¹⁶ indicated that using heterologous viruses in serologic assays for detecting BV antibodies contributes to increased false-negative results.Serologic diagnosis of BV infection in macaques at the National B Virus Resource Laboratory has been based on 2 principal tests that meet standards proscribed by the Clinical Laboratory Improvement Amendments:⁴ the titration ELISA (tELISA) and Western blot analysis (WBA).^15,31 tELISA detects antibody in sera from most BV-infected macaques by using the complex mixture of BV antigens that is present in lysates from infected cells and adsorbed onto polystyrene microtiter plates. These infected-cell lysates are prepared by using nondenaturing detergents. Quality-control assessment of each antigen lot is performed, including determinations of protein concentration, antigen mass, immunoreactivity with macaque serum pools, and reactivity in WBA assays.WBA was chosen as the confirmatory test for the BV-screening tELISA; in addition, WBA serves as the primary screening assay for detecting antibodies to BV, HSV1, and HSV2 in human sera collected after exposure in the workplace.^27,31 The relationship between ELISA and WBA is complex, and therefore the agreement between the tests may be lower than expected given that WBA detects only conformationally independent epitopes, whereas ELISA identifies both linear and conformational epitopes (unless the epitopes are cryptic).²⁸ In addition, the different antibodies detected by each test might be induced at distinctly different time points after infection, so that what one assay detects, the other may not.^2,8,9 Despite the difficulties encountered and due to the lack of a better alternative, both assays are required to maximize the likelihood of correctly identifying whether BV antibodies are present in a sample.Because the identification of BV-infected macaques in any colony, especially SPF colonies, is of great medical and economic significance, assays should be designed to identify low levels of antibody as soon as possible after infection.^12,31 For BV, as for other alphaherpesviruses, the presence of serum antibodies likely indicates the presence of virus that is either in an active lytic state (and actively replicating) or in an inactive, latent state. It is therefore important to have both a sensitive assay, to prevent missing low-responders, and a highly specific assay, to avoid obtaining false-negative results that can lead to costly veterinary and medical decisions.In our continuing efforts to improve the accuracy of the tELISA for BV, we recently modified and automated it.¹⁸ This process included validation experiments that enabled us to reexamine some features of the BV serologic diagnosis.Here we describe additional modifications of the tELISA and its adaptation to an automated, high-throughput 384-well format. This format enabled us to assess the sensitivities of tELISA using homologous compared with heterologous antigens in the same run. In addition, we evaluated the correlation between tELISA and WBA and discuss the effectiveness of WBA as a confirmatory test for tELISA. All assays were developed and performed inhouse in accordance with CLIA regulations,⁴ because each test either was used or could be used for identification of BV antibodies in human sera as well as macaque sera. Although CLIA does not regulate veterinary assays, we feel strongly that the tests described are superior because of the strict quality control that must be maintained for testing human samples.     

Metallomics investigations on potential binding partners of methylmercury in tuna fish muscle tissue using complementary mass spectrometric techniques

In this study, the binding behaviour of methylmercury (MeHg(+)) towards proteins is investigated. Free sulfhydryl groups in cysteine residues are known to be the most likely binding partners, due to the high affinity of mercury to sulphur. However, detailed knowledge about discrete binding sites in living organisms has been so far scarce. A metallomics approach using different methods like size-exclusion chromatography (SEC) and liquid chromatography (LC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) as well as complementary mass spectrometric techniques (electrospray ionisation-tandem mass spectrometry, ESI-MS/MS) are combined to sequence and identify possible target proteins or peptides after enzymatic digestion. Potential targets for MeHg(+) in tuna fish muscle tissue are investigated using the certified reference material CRM464 as a model tissue. Different extraction procedures appropriate for the extraction of proteins are evaluated for their efficiency using isotope dilution analysis for the determination of total Hg in the extracts. Due to the high chemical stability of the mercury-sulphur bond, the bioconjugate can be quantitatively extracted with a combination of tris(hydroxymethyl)aminomethane (TRIS) and sodium dodecyl sulphate (SDS). Using different separation techniques such as SEC and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) it can be shown that major binding occurs to a high-molecular weight protein (M(w) > 200 kDa). A potential target protein, skeletal muscle myosin heavy chain, could be identified after tryptic digestion and capillary LC-ESI-MS/MS.     

Laterally orienting C. elegans using geometry at microscale for high-throughput visual screens in neurodegeneration and neuronal development studies

C. elegans is an excellent model system for studying neuroscience using genetics because of its relatively simple nervous system, sequenced genome, and the availability of a large number of transgenic and mutant strains. Recently, microfluidic devices have been used for high-throughput genetic screens, replacing traditional methods of manually handling C. elegans. However, the orientation of nematodes within microfluidic devices is random and often not conducive to inspection, hindering visual analysis and overall throughput. In addition, while previous studies have utilized methods to bias head and tail orientation, none of the existing techniques allow for orientation along the dorso-ventral body axis. Here, we present the design of a simple and robust method for passively orienting worms into lateral body positions in microfluidic devices to facilitate inspection of morphological features with specific dorso-ventral alignments. Using this technique, we can position animals into lateral orientations with up to 84% efficiency, compared to 21% using existing methods. We isolated six mutants with neuronal development or neurodegenerative defects, showing that our technology can be used for on-chip analysis and high-throughput visual screens.     

Molecular microcircuitry underlies functional specification in a basal ganglia circuit dedicated to vocal learning

Similarities between speech and birdsong make songbirds advantageous for investigating the neurogenetics of learned vocal communication--a complex phenotype probably supported by ensembles of interacting genes in cortico-basal ganglia pathways of both species. To date, only FoxP2 has been identified as critical to both speech and birdsong. We performed weighted gene coexpression network analysis on microarray data from singing zebra finches to discover gene ensembles regulated during vocal behavior. We found ～2,000 singing-regulated genes comprising three coexpression groups unique to area X, the basal ganglia subregion dedicated to learned vocalizations. These contained known targets of human FOXP2 and potential avian targets. We validated biological pathways not previously implicated in vocalization. Higher-order gene coexpression patterns, rather than expression levels, molecularly distinguish area X from the ventral striato-pallidum during singing. The previously unknown structure of singing-driven networks enables prioritization of molecular interactors that probably bear on human motor disorders, especially those affecting speech.     

Ophiuroid remains from the Toarcian of Sainte-Verge (Deux-Sèvres, France): paleobiological perspectives

The Toarcian sediments exposed at Sainte-Verge (Deux-Sèvres, France) are especially rich in echinoderm remains. The present paper describes and illustrates the ophiuroids. On the basis of lateral arm plates, 13 species are distinguished, including two new ones: Sinosura fasciata sp. nov. and Sinosura extensa sp. nov. Most of the recognized species have been recorded previously from the Late Toarcian and Aalenian in Germany and, to a lesser extent, from the late Early Jurassic of England and Switzerland. High similarities between the faunas of northwest Europe suggest a boreal provincialism of ophiuroids. The recognition of 13 species is comparable to the diversity known from other stratigraphic levels (Jurassic and Cretaceous) or in the richest stations of recent oceans. The species association of the Toarcian of Sainte-Verge, with two Ophiolepididae, one Ophiacanthidae, four Ophioleucidae, two Ophiodermatidae, two Ophiuridae, and one Hemieuryalidae may be compared with species associations of recent shelf, offshore environments. Such persistence of components of diversity and ecological affinities of species suggests strong evolutionary conservatism of the ophiuroids, after a rapid radiation during the earliest Jurassic. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.     

The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling

Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein-protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.     

Synthesis and antibacterial activity of C-6 carbamate ketolides, a novel series of orally active ketolide antibiotics

A new series of antibacterial ketolides is reported, which features the use of a C-6 carbamate for tethering the arylalkyl sidechain to the macrolide core. The best members of this series display in vitro and in vivo activity comparable to telithromycin. Partial epimerization at C-2, unobserved in previously reported ketolides, was noted for this series.     

The synthesis and antimicrobial evaluation of a new series of isoxazolinyl oxazolidinones

A series of oxazolidinone antibacterial agents containing a 5-substituted isoxazol-3-yl moiety were synthesized via a nitrile oxide [3+2] dipolar cycloaddition reaction. These compounds were screened against a panel of susceptible and resistant Gram-positive organisms. Several analogs from this series were comparable to or more potent than linezolid in vitro.     

A mass spectrometric determination of the conformation of dimeric apolipoprotein A-I in discoidal high density lipoproteins

Discoidal forms of high density lipoproteins (HDL) are critical intermediates between lipid-poor apolipoprotein A-I (apo A-I), the major protein constituent of HDL, and the mature spherical forms that comprise the bulk of circulating particles. Thus, many studies have focused on understanding apoA-I structure in discs reconstituted in vitro. Recent theoretical and experimental work supports a "belt" model for apoA-I in which repeating amphipathic helical domains run parallel to the plane of the lipid disc. However, disc-associated apoA-I can adopt several tertiary arrangements that are consistent with a belt orientation. To distinguish among these, we cross-linked near-neighbor Lys groups in homogeneous 96 A discs containing exactly two molecules of apoA-I. After delipidation and tryptic digestion, mass spectrometry was used to identify 9 intermolecular and 11 intramolecular cross-links. The cross-linking pattern strongly suggests a "double-belt" molecular arrangement for apoA-I in which two apoA-I molecules wrap around the lipid bilayer disc forming two stacked rings in an antiparallel orientation with helix 5 of each apoA-I in juxtaposition (LL5/5 orientation). The data also suggests the presence of an additional double-belt orientation with a shifted helical registry (LL5/2 orientation). Furthermore, a 78 A particle with two molecules of apoA-I fit a similar double-belt motif with evidence for conformational changes in the N-terminus and the region near helix 5. A comparison of this work to a previous study is suggestive that a third molecule of apoA-I can form a hairpin in larger particles containing three molecules of apoA-I.