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.     

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.     

Determination of organic acids in seven wheat varieties by capillary gas chromatography

Fresh wheat tops were extracted with acidic 90% ethanol, and the ethanol was evaporated and a portion of the aqueous residue loaded onto DEAE-Sephadex. Organic acids were eluted with pyridinium formate and then lyophilized and the dried residue was derivatized with 1% trimethylchlorosilane in bis(trimethylsilyl)trifluoroacetamide. The acids were then quantitatively determined using capillary gas chromatography and identified using capillary gas chromatography-mass spectrometry. The acidic ethanol extraction of fresh plant tissue was quantitative for all acids except citric while losses in the remaining procedures were controlled by using an internal standard. The ion exchange chromatography made the greatest contribution to experimental error, imposing a minimum loading requirement of 0.1 mumol of each acid for adequate precision. Organic acid profiles were determined for seven wheat cultivars (Triticum aestivum cv Carazinho, Teal, Lance, Warigal, Isis, Maringa, and BH1146) grown on gravel in solution culture for 30 days. Profiles were simple, consisting of only malic, aconitic, and citric acids, with levels of each acid for all varieties falling within the range 2-5 mumol/g fresh tissue. Storage of samples led to a large increase in sampling error and increased the amount of extractable citric acid.     

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.     

KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1^−/− colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.     

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.