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1.  Quantitative Measurement of Solvent Accessibility of Histidine Imidazole Groups in Proteins 
Biochemistry  2012;51(36):7202-7208.
We report a method to express the solvent accessibility of histidine imidazole groups in proteins. The method is based on measuring the rate of hydrogen exchange (HX) reaction of the imidazole Cε1-hydrogen. The rate profile of the HX reaction as a function of pH gives a sigmoidal curve, which reaches the maximum rate constant (kmax) on the alkaline side of the sigmoidal curve. To quantitatively describe the solvent accessibility of imidazole groups in proteins, it is necessary to compare the kmax of the imidazole groups with their intrinsic kmax (ikmax), the maximum rate constants for the given imidazole groups when they are fully exposed to the bulk solvent. However, the mechanism of HX reaction suggests that the ikmax of an imidazole group differs depending on its pKa, and no systematic study has been conducted to clarify how the ikmax is affected by pKa. We therefore investigated the relationship between ikmax and pKa using four imidazole derivatives at three different temperatures. The experimentally determined pKa-specific ikmax values allowed us to derive a general formula to estimate the ikmax value of any given imidazole group exhibiting a specific pKa at a specific temperature. Using the formula, the protection factors (PF), the ratio of ikmax to kmax, of five imidazole groups in dihydrofolate reductase were obtained and used to express the magnitude of their solvent accessibility. In this definition, the smaller the PF value, the higher the solvent accessibility, and a value of 1 indicates full exposure to the bulk solvent. The solvent accessibility expressed by the PF values agreed well with the solvent accessible surface areas (ASA) obtained from the X-ray diffraction data.
doi:10.1021/bi300911d
PMCID: PMC3462353  PMID: 22901083
2.  Genetic identification of C-fibers that detect massage-like stroking of hairy skin in vivo 
Nature  2013;493(7434):669-673.
Stroking of the skin produces pleasant sensations that can occur during social interactions with conspecifics, such as grooming1. Despite numerous physiological studies (reviewed in ref. 2), molecularly defined sensory neurons that detect pleasant stroking of hairy skin3,4 in vivo have not been reported. Previously, we identified a rare population of unmyelinated sensory neurons that express the G protein-coupled receptor (GPCR) MrgprB45,6. These neurons exclusively innervate hairy skin with large terminal arborizations7 that resemble the receptive fields of C-tactile (CT) afferents in humans8. Unlike other molecularly defined mechanosensory C-fiber subtypes9,10, MrgprB4+ neurons could not be detectably activated by sensory stimulation of the skin ex vivo. Therefore, we developed a preparation for calcium imaging in their spinal projections during stimulation of the periphery in intact animals. MrgprB4+ neurons were activated by massage-like stroking of hairy skin, but not by noxious punctate mechanical stimulation. By contrast, a different population of C-fibers expressing MrgprD11 was activated by pinching but not by stroking, consistent with previous physiological and behavioral data10,12. Pharmacogenetic activation of MrgprB4- expressing neurons in freely behaving animals promoted conditioned place preference13, suggesting that such activation is positively reinforcing and/or anxiolytic. These data open the way to understanding the function of MrgprB4 neurons during natural behaviors, and provide a general approach to functionally characterizing genetically identified subsets of somatosensory neurons in vivo.
doi:10.1038/nature11810
PMCID: PMC3563425  PMID: 23364746
3.  DISCOVERY OF NOVEL GLUCOSE-REGULATED PROTEINS IN ISOLATED HUMAN PANCREATIC ISLETS USING LC-MS/MS-BASED PROTEOMICS 
Journal of Proteome Research  2012;11(7):3520-3532.
The prevalence of diabetes mellitus is increasing dramatically throughout the world, and the disease has become a major public health issue. The most common form of the disease, type 2 diabetes, is characterized by insulin resistance and insufficient insulin production from the pancreatic beta-cell. Since glucose is the most potent regulator of beta-cell function under physiological conditions, identification of the insulin secretory defect underlying type 2 diabetes requires a better understanding of glucose regulation of human beta-cell function. To this aim, a bottom-up LC-MS/MS-based proteomics approach was used to profile pooled islets from multiple donors under basal (5 mM) or high (15 mM) glucose conditions. Our analysis discovered 256 differentially abundant proteins (~p<0.05) after 24 h of high glucose exposure from more than 4500 identified in total. Several novel glucose-regulated proteins were elevated under high glucose conditions, including regulators of mRNA splicing (Pleiotropic regulator 1), processing (Retinoblastoma binding protein 6), and function (Nuclear RNA export factor 1), in addition to Neuron navigator 1 and Plasminogen activator inhibitor 1. Proteins whose abundances markedly decreased during incubation at 15 mM glucose included Bax inhibitor 1 and Synaptotagmin-17. Up-regulation of Dicer 1 and SLC27A2 and down-regulation of Phospholipase Cβ4 were confirmed by Western blots. Many proteins found to be differentially abundant after high glucose stimulation are annotated as uncharacterized or hypothetical. These findings expand our knowledge of glucose regulation of the human islet proteome and suggest many hitherto unknown responses to glucose that require additional studies to explore novel functional roles.
doi:10.1021/pr3002996
PMCID: PMC3391329  PMID: 22578083
human; pancreatic islet; glucose; type 2 diabetes; proteomics; mass spectrometry; LC-MS/MS
4.  Evidence for Asymmetrical Divergence-Gene Flow of Nuclear Loci, but Not Mitochondrial Loci, between Seabird Sister Species: Blue-Footed (Sula nebouxii) and Peruvian (S. variegata) Boobies 
PLoS ONE  2013;8(4):e62256.
Understanding the process of speciation requires understanding how gene flow influences divergence. Recent analyses indicate that divergence can take place despite gene flow and that the sex chromosomes can exhibit different levels of gene flow than autosomes and mitochondrial DNA. Using an eight marker dataset including autosomal, z-linked, and mitochondrial loci we tested the hypothesis that blue-footed (Sula nebouxii) and Peruvian (S. variegata) boobies diverged from their common ancestor with gene flow, paying specific attention to the differences in gene flow estimates from nuclear and mitochondrial markers. We found no gene flow at mitochondrial markers, but found evidence from the combined autosomal and z-linked dataset that blue-footed and Peruvian boobies experienced asymmetrical gene flow during or after their initial divergence, predominantly from Peruvian boobies into blue-footed boobies. This gene exchange may have occurred either sporadically between periods of allopatry, or regularly throughout the divergence process. Our results add to growing evidence that diverging species can remain distinct but exchange genes.
doi:10.1371/journal.pone.0062256
PMCID: PMC3629132  PMID: 23614045
5.  Genetic dissection of an amygdala microcircuit that gates conditioned fear 
Nature  2010;468(7321):270-276.
The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. We used molecular genetic approaches to map the functional connectivity of a subpopulation of GABAergic neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-delta (PKCδ). Channelrhodopsin-2 assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKCδ+ neurons inhibit output neurons in the medial CE (CEm), and also make reciprocal inhibitory synapses with PKCδ− neurons in CEl. Electrical silencing of PKCδ+ neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus (CS), called CEloff units (Ciocchi et al, this issue). This correspondence, together with behavioral data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing.
doi:10.1038/nature09553
PMCID: PMC3597095  PMID: 21068836
6.  OPTOGENETICS, SEX AND VIOLENCE IN THE BRAIN: IMPLICATIONS FOR PSYCHIATRY 
Biological psychiatry  2011;71(12):1081-1089.
Pathological aggression, and the inability to control aggressive impulses, takes a tremendous toll on society. Yet aggression is a normal component of the innate behavior repertoire of most vertebrate animal species, as well as of many invertebrates. Progress in understanding the etiology of disorders of aggressive behavior, whether genetic or environmental in nature, therefore requires an understanding of the brain circuitry that controls normal aggression. Efforts to understand this circuitry at the level of specific neuronal populations have been constrained by the limited resolution of classical methodologies, such as electrical stimulation and electrolytic lesion. The availability of new, genetically based tools for mapping and manipulating neural circuits at the level of specific, genetically defined neuronal subtypes provides an opportunity to investigate the functional organization of aggression circuitry with cellular resolution. However these technologies are optimally applied in the mouse, where there has been surprisingly little traditional work on the functional neuroanatomy of aggression. Here we discuss recent, initial efforts to apply optogenetics and other state-of-the-art methods to the dissection of aggression circuitry in the mouse. We find, surprisingly, that neurons necessary and sufficient for inter-male aggression are located within the ventrolateral subdivision of the ventromedial hypothalamic nucleus (VMHvl), a structure traditionally associated with reproductive behavior. These neurons are intermingled with neurons activated during male-female mating, with ~20% overlap between the populations. We discuss the significance of these findings with respect to neuroethological and neuroanatomical perspectives on the functional organization of innate behaviors, and their potential implications for psychiatry.
doi:10.1016/j.biopsych.2011.11.012
PMCID: PMC3380604  PMID: 22209636
aggression; mating; violence; hypothalamus; optogenetics; channelrhodopsin; mouse
7.  Visualizing Neuromodulation In Vivo: TANGO-Mapping of Dopamine Signaling Reveals Appeptite Control of Sugar Sensing 
Cell  2012;148(3):583-595.
SUMMARY
Behavior cannot be predicted from a “connectome,” because the brain contains a chemical “map” of neuromodulation superimposed upon its synaptic connectivity map. Neuromodulation changes how neural circuits process information in different states, such as hunger or arousal. Here we describe a novel, genetically based method to map, in an unbiased and brain-wide manner, sites of neuromodulation under different conditions in the Drosophila brain. This method, and genetic perturbations, reveal that the well-known effect of hunger to enhance behavioral sensitivity to sugar is mediated, at least in part, by the release of dopamine onto primary gustatory sensory neurons, which enhances sugar-evoked calcium influx. These data reinforce the concept that sensory neurons constitute an important locus for state-dependent gain-control of behavior, and introduce a new methodology that can be extended to other neuromodulators and model organisms.
doi:10.1016/j.cell.2011.12.022
PMCID: PMC3295637  PMID: 22304923
8.  A CRE-DEPENDENT, ANTEROGRADE TRANS-SYNAPTIC VIRAL TRACER FOR MAPPING OUTPUT PATHWAYS OF GENETICALLY MARKED NEURONS 
Neuron  2011;72(6):938-950.
SUMMARY
Neurotropic viruses that conditionally infect or replicate in molecularly defined neuronal subpopulations, and then spread trans-synaptically, are powerful tools for mapping neural pathways. Genetically targetable retrograde trans-synaptic tracer viruses are available to map the inputs to specific neuronal subpopulations, but an analogous tool for mapping synaptic outputs is not yet available. Here we describe a Cre recombinase-dependent, anterograde trans-neuronal tracer, based on the H129 strain of herpes simplex virus (HSV). Application of this virus to transgenic or knock-in mice expressing Cre in peripheral neurons of the olfactory epithelium or the retina reveals widespread, polysynaptic labeling of higher-order neurons in the olfactory and visual systems, respectively. Polysynaptic pathways were also labeled from cerebellar Purkinje cells. In each system, the pattern of labeling was consistent with classical circuit-tracing studies, restricted to neurons and anterograde-specific. These data provide proof-of-principle for a conditional, non-diluting anterograde trans-synaptic tracer for mapping synaptic outputs from genetically marked neuronal subpopulations.
doi:10.1016/j.neuron.2011.12.002
PMCID: PMC3275419  PMID: 22196330
9.  Hierarchical chemosensory regulation of male-male social interactions in Drosophila 
Nature neuroscience  2011;14(6):757-762.
Pheromones regulate male social behaviors in Drosophila, but the identities and behavioral role(s) of these chemosensory signals, and how they interact, are incompletely understood. Here we show that (Z)-7-tricosene (7-T), a male-enriched cuticular hydrocarbon (CH) previously shown to inhibit male-male courtship, is also essential for normal levels of aggression. The opposite influences of 7-T on aggression and courtship are independent, but both require the gustatory receptor Gr32a. Surprisingly, sensitivity to 7-T is required for the aggression-promoting effect of 11-cis-vaccenyl acetate (cVA), an olfactory pheromone, but 7-T sensitivity is independent of cVA. 7-T and cVA therefore regulate aggression in a hierarchical manner. Furthermore, the increased courtship caused by depletion of male CHs is suppressed by a mutation in the olfactory receptor Or47b. Thus, male social behaviors are controlled by gustatory pheromones that promote and suppress aggression and courtship, respectively, and whose influences are dominant to olfactory pheromones that enhance these behaviors.
doi:10.1038/nn.2800
PMCID: PMC3102769  PMID: 21516101
10.  The ABL switch control inhibitor DCC-2036 is active against the chronic myeloid leukemia mutant BCR-ABLT315I and exhibits a narrow resistance profile 
Cancer research  2011;71(9):3189-3195.
Acquired point mutations within the BCR-ABL kinase domain represent a common mechanism of resistance to ABL inhibitor therapy in patients with chronic myeloid leukemia (CML). The BCR-ABLT315I mutant is highly resistant to imatinib, nilotinib, and dasatinib and is frequently detected in relapsed patients. This critical gap in resistance coverage drove development of DCC-2036, an ABL inhibitor which binds the switch control pocket involved in conformational regulation of the kinase domain. We evaluated the efficacy of DCC-2036 against BCR-ABLT315I and other mutants in cellular and biochemical assays and conducted cell-based mutagenesis screens. DCC-2036 inhibited autophosphorylation of ABL and ABLT315I enzymes, and this activity was consistent with selective efficacy against Ba/F3 cells expressing BCR-ABL (IC50: 19 nmol/L), BCR-ABLT315I (IC50: 63 nmol/L), and most kinase domain mutants. Ex vivo exposure of CML cells from patients harboring BCR-ABL or BCR-ABLT315I to DCC-2036 revealed marked inhibition of colony formation and reduced phosphorylation of the direct BCR-ABL target CrkL. Cell-based mutagenesis screens identified a resistance profile for DCC-2036 centered around select P-loop mutations (G250E, Q252H, Y253H, E255K/V), although a concentration of 750 nmol/L DCC-2036 suppressed the emergence of all resistant clones. A decreased concentration of DCC-2036 (160 nmol/L) in dual-combination with either nilotinib or dasatinib achieved the same zero outgrowth result. Further screens for resistance due to BCR-ABL compound mutations (two mutations in the same clone) identified BCR-ABLE255V / T315I as the most resistant mutant. Taken together, these findings support continued evaluation of DCC-2036 as an important new agent for treatment-refractory CML.
doi:10.1158/0008-5472.CAN-10-3224
PMCID: PMC3206627  PMID: 21505103
BCR-ABL; imatinib resistance; DCC-2036
11.  Pain behavior in the formalin test persists after ablation of the great majority of C-fiber nociceptors 
Pain  2010;151(2):422-429.
Although the formalin test is a widely used model of persistent pain, the primary afferent fiber types that underlie the cellular and behavioral responses to formalin injection are largely unknown. Here we used a combined genetic and pharmacological approach to investigate the effect of ablating subsets of primary afferent nociceptors on formalin-induced nocifensive behaviors and spinal cord Fos protein expression. Intrathecal capsaicin-induced ablation of the central terminals of TRPV1+ neurons greatly reduced the behavioral responses and Fos elicited by low-dose (0.5%) formalin. In contrast, genetic ablation of the MrgprD-expressing subset of nonpeptidergic unmyelinated afferents, which constitute a largely non-overlapping population, altered neither the behavior nor the Fos induced by low-dose formalin. Remarkably, nocifensive behavior following high-dose (2%) formalin was unchanged in mice lacking either afferent population, or even in mice lacking both populations, which together make up the great majority of C-fiber nociceptors. Thus, at high doses, which are routinely used in the formalin test, formalin-induced “pain” behavior persists in the absence of the vast majority of C-fiber nociceptors, which points to a contribution of a large spectrum of afferents secondary to non-specific formalin-induced tissue and nerve damage.
doi:10.1016/j.pain.2010.08.001
PMCID: PMC2955806  PMID: 20832171
12.  Functional identification of an aggression locus in the mouse hypothalamus 
Nature  2011;470(7333):221-226.
Electrical stimulation of certain hypothalamic regions in cats and rodents can elicit attack behavior, but the exact location of relevant cells within these regions, their requirement for naturally occurring aggression and their relationship to mating circuits have not been clear. Genetic methods for neural circuit manipulation in mice provide a potentially powerful approach to this problem, but brain stimulation-evoked aggression has never been demonstrated in this species. Here we show that optogenetic, but not electrical, stimulation of neurons in the ventromedial hypothalamus, ventrolateral subdivision (VMHvl) causes male mice to attack both females and inanimate objects, as well as males. Pharmacogenetic silencing of VMHvl reversibly inhibits inter-male aggression. Immediate early gene analysis and single unit recordings from VMHvl during social interactions reveal overlapping but distinct neuronal subpopulations involved in fighting and mating. Neurons activated during attack are inhibited during mating, suggesting a potential neural substrate for competition between these behaviors.
doi:10.1038/nature09736
PMCID: PMC3075820  PMID: 21307935
13.  Identification of an aggression-promoting pheromone and its receptor neurons in Drosophila 
Nature  2009;463(7278):227-231.
Aggression is regulated by pheromones in many animal species1,2,3. However in no system have aggression pheromones, their cognate receptors and corresponding sensory neurons been identified. Here we show that 11-cis-vaccenyl acetate (cVA), a male-specific volatile pheromone, robustly promotes male-male aggression in the vinegar fly Drosophila melanogaster. The aggression-promoting effect of synthetic cVA requires olfactory sensory neurons (OSNs) expressing the receptor Or67d4,5,6, as well as the receptor itself. Activation of Or67d-expressing OSNs, either by genetic manipulation of their excitability or by exposure to male pheromones in the absence of other classes of OSNs, is sufficient to promote aggression. High densities of male flies can promote aggression through release of volatile cVA. In turn, cVA-promoted aggression can promote male fly dispersal from a food resource, in a manner dependent upon Or67d-expressing OSNs. These data suggest that cVA may mediate negative feedback control of male population density, through its effect on aggression. Identification of a pheromone-OSN pair controlling aggression in a genetic organism opens the way to unraveling the neurobiology of this evolutionarily conserved behavior.
doi:10.1038/nature08678
PMCID: PMC2999963  PMID: 19966787
14.  Sensory neuron-specific GPCRs Mrgprs are itch receptors mediating chloroquine-induced pruritus 
Cell  2009;139(7):1353-1365.
SUMMARY
The cellular and molecular mechanisms mediating histamine-independent itch in primary sensory neurons are largely unknown. Itch induced by chloroquine (CQ) is a common side-effect of this widely used anti-malarial drug. Here we show that Mrgprs, a family of G protein-coupled receptors expressed exclusively in peripheral sensory neurons, function as itch receptors. Mice lacking a cluster of Mrgpr genes display significant deficits in itch induced by CQ but not histamine. CQ directly excites sensory neurons in an Mrgpr-dependent manner. CQ specifically activates mouse MrgprA3 and human MrgprX1. Loss- and gain-of-function studies demonstrate that MrgprA3 is required for CQ responsiveness in mice. Furthermore, MrgprA3-expressing neurons respond to histamine and co-express Gastrin-Releasing Peptide, a peptide involved in itch sensation, and MrgprC11. Activation of these neurons with MrgprC11-specific agonist BAM8-22 induces itch in wild-type but not mutant mice. Therefore, Mrgprs may provide molecular access to itch-selective neurons and constitute novel targets for itch therapeutics.
doi:10.1016/j.cell.2009.11.034
PMCID: PMC2989405  PMID: 20004959
15.  Two Different Forms of Arousal in Drosophila are Independently and Oppositely Regulated by the Dopamine D1 Receptor DopR via Distinct Neural Circuits 
Neuron  2009;64(4):522-536.
SUMMARY
Arousal is fundamental to many behaviors, but whether it is unitary, or whether there are different types of behavior-specific arousal, has not been clear. In Drosophila, dopamine promotes sleep-wake arousal. However there is conflicting evidence regarding its influence on environmentally stimulated arousal. Here we show that loss-of-function mutations in the D1 dopamine receptor DopR enhance repetitive startle-induced arousal, while decreasing nocturnal arousal (i.e., increasing sleep). These two types of arousal are also inversely influenced by cocaine, whose effects in each case are opposite to, and abrogated by, the DopR mutation. Selective restoration of DopR function in the central complex rescues the enhanced stimulated arousal but not the increased sleep phenotype of DopR mutants. These data provide evidence for at least two different forms of arousal, which are independently regulated by dopamine in opposite directions, via distinct neural circuits.
doi:10.1016/j.neuron.2009.09.031
PMCID: PMC2908595  PMID: 19945394
16.  Distinct sensory representations of wind and near-field sound in the Drosophila brain 
Nature  2009;458(7235):201-205.
Behavioral responses to wind are thought to play a critical role in controlling the dispersal and population genetics of wild Drosophila species1,2, as well as their navigation in flight3, but their underlying neurobiological basis is unknown. We show that Drosophila melanogaster, like wild-caught Drosophila strains4, exhibits robust wind-induced suppression of locomotion (WISL), in response to air currents delivered at speeds normally encountered in nature1,2. Here we identify wind-sensitive neurons in Johnston’s Organ (JO), an antennal mechanosensory structure previously implicated in near-field sound detection (reviewed in5,6). Using Gal4 lines targeted to different subsets of JO neurons7, and a genetically encoded calcium indicator8, we show that wind and near-field sound (courtship song) activate distinct populations of JO neurons, which project to different regions of the antennal and mechanosensory motor center (AMMC) in the central brain. Selective genetic ablation of wind-sensitive JO neurons in the antenna abolishes WISL behavior, without impairing hearing. Different neuronal subsets within the wind-sensitive population, moreover, respond to different directions of arista deflection caused by airflow and project to different regions of the AMMC, providing a rudimentary map of wind-direction in the brain. Importantly, sound- and wind-sensitive JO neurons exhibit different intrinsic response properties: the former are phasically activated by small, bi-directional, displacements of the aristae, while the latter are tonically activated by unidirectional, static deflections of larger magnitude. These different intrinsic properties are well suited to the detection of oscillatory pulses of near-field sound and laminar airflow, respectively. These data identify wind-sensitive neurons in JO, a structure that has been primarily associated with hearing, and reveal how the brain can distinguish different types of air particle movements, using a common sensory organ.
doi:10.1038/nature07843
PMCID: PMC2755041  PMID: 19279637
17.  Automated Monitoring and Analysis of Social Behavior in Drosophila 
Nature methods  2009;6(4):297-303.
We introduce a method based on machine vision for automatically measuring aggression and courtship in Drosophila melanogaster. The genetic and neural circuit bases of these innate social behaviors are poorly understood. High-throughput behavioral screening in this genetically tractable model organism is a potentially powerful approach, but it is currently very laborious. Our system monitors interacting pairs of flies, and computes their location, orientation and wing posture. These features are used for detecting behaviors exhibited during aggression and courtship. Among these, wing threat, lunging and tussling are specific to aggression; circling, wing extension (courtship “song”) and copulation are specific to courtship; locomotion and chasing are common to both. Ethograms may be constructed automatically from these measurements, saving considerable time and effort. This technology should enable large-scale screens for genes and neural circuits controlling courtship and aggression.
doi:10.1038/nmeth.1310
PMCID: PMC2679418  PMID: 19270697
18.  Allocrine Modulation of Feeding Behavior by the Sex Peptide of Drosophila 
Current biology : CB  2006;16(7):692-696.
Summary
Mating elicits a dramatic reprogramming of female behavior in numerous insect species. In Drosophila, this postmating response (PMR) comprises increased egg-laying rate and reduced sexual receptivity and is controlled by the products of the male accessory glands, a family of ∼80 small peptides transferred in the male seminal fluid [1–9]. Here, we show that copulation strongly stimulates female food intake. Remarkably, this change is abolished if the males lack a single, small seminal protein, the Sex Peptide (SP). Ectopic expression of SP in virgin females mimics the effect of mating on feeding behavior, demonstrating that SP is the main agent controlling this behavioral paradigm. Our observations identify enhanced feeding behavior as a novel component of the Drosophila PMR and suggest that SP represents a molecular link between energy acquisition and reproductive investment.
doi:10.1016/j.cub.2006.02.064
PMCID: PMC2745344  PMID: 16581515
19.  Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells 
Development (Cambridge, England)  2004;131(22):5599-5612.
Summary
Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Cre-recombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation.
doi:10.1242/dev.01429
PMCID: PMC2638001  PMID: 15496445
Neural crest stem cell; Peripheral nerve development; Fate-mapping
20.  Bi-Phasic Intensity-Dependent Opioid-Mediated Neural Amplitude Changes in the Chinchilla Cochlea: Partial Blockade by an N-Methyl-d-Aspartate (NMDA)-Receptor Antagonist 
European journal of pharmacology  2007;580(1-2):100-115.
Dynorphins, glutamate, and glutamate-sensitive N-Methyl-d-Aspartate (NMDA) receptors exist in the mammalian cochlea. Dynorphins produce neural excitation and excitotoxic effects in the spinal cord through a κ-opioid facilitation of NMDA receptor sensitivity to glutamate. The κ-opioid receptor drug agonists N-dimethylallyl-normetazocine [(-)-pentazocine (50 mmol)] and trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide [U-50488H (100 mmol)] were administered across the cochlear round window membrane in the chinchilla. Each drug produced significant post-baseline amplitude changes in the click-evoked auditory nerve compound action potential. Amplitude changes at threshold amounted to increases in sensitivity that ranged from 4-8 decibels, measured in sound pressure level (dB SPL). The large neural amplitude increases at threshold were accompanied by progressively smaller amplitude changes at 5 and 10 dB above threshold (dB SL). However, at stimulus intensities ≥ 20dB SL, post-baseline neural amplitudes were suppressed to levels below baseline and control values. These bi-phasic intensity-dependent neural amplitude changes have never before been observed following i.v. administered (-)-pentazocine in this species. Finally, the bi-phasic neural amplitude changes in U-50488H-treated (100 mmol) animals were partially blocked (except at 20dB SL), following a round window pretreatment with the NMDA receptor drug antagonist, dizocilpine hydrogen maleate [(+)-MK-801 (8 mmol)]. Our data suggests that endogenous dynorphins within lateral efferent olivocochlear neurons differentially modulate auditory neural excitation, possibly through cochlear NMDA receptors and glutamate. The role played by lateral efferent opioid neuromodulation at cochlear NMDA receptors, is discussed.
doi:10.1016/j.ejphar.2007.10.038
PMCID: PMC2292842  PMID: 18036588
(+)-MK-801 (dizocilpine hydrogen maleate); U-50488H; (-)-pentazocine; N-Methyl-d-Aspartate (NMDA) receptors; lateral efferent olivocochlear system; κ-opioid receptors; dynorphins; glutamate; auditory nerve; compound action potential
21.  Wind, Waves, and Wing Loading: Morphological Specialization May Limit Range Expansion of Endangered Albatrosses 
PLoS ONE  2008;3(12):e4016.
Among the varied adaptations for avian flight, the morphological traits allowing large-bodied albatrosses to capitalize on wind and wave energy for efficient long-distance flight are unparalleled. Consequently, the biogeographic distribution of most albatrosses is limited to the windiest oceanic regions on earth; however, exceptions exist. Species breeding in the North and Central Pacific Ocean (Phoebastria spp.) inhabit regions of lower wind speed and wave height than southern hemisphere genera, and have large intrageneric variation in body size and aerodynamic performance. Here, we test the hypothesis that regional wind and wave regimes explain observed differences in Phoebastria albatross morphology and we compare their aerodynamic performance to representatives from the other three genera of this globally distributed avian family. In the North and Central Pacific, two species (short-tailed P. albatrus and waved P. irrorata) are markedly larger, yet have the smallest breeding ranges near highly productive coastal upwelling systems. Short-tailed albatrosses, however, have 60% higher wing loading (weight per area of lift) compared to waved albatrosses. Indeed, calculated aerodynamic performance of waved albatrosses, the only tropical albatross species, is more similar to those of their smaller congeners (black-footed P. nigripes and Laysan P. immutabilis), which have relatively low wing loading and much larger foraging ranges that include central oceanic gyres of relatively low productivity. Globally, the aerodynamic performance of short-tailed and waved albatrosses are most anomalous for their body sizes, yet consistent with wind regimes within their breeding season foraging ranges. Our results are the first to integrate global wind and wave patterns with albatross aerodynamics, thereby identifying morphological specialization that may explain limited breeding ranges of two endangered albatross species. These results are further relevant to understanding past and potentially predicting future distributional limits of albatrosses globally, particularly with respect to climate change effects on basin-scale and regional wind fields.
doi:10.1371/journal.pone.0004016
PMCID: PMC2602987  PMID: 19107200
22.  The spinal cord contains positionally distinct astrocyte subtypes whose identities are specified by a homeodomain transcriptional code 
Cell  2008;133(3):510-522.
SUMMARY
Astrocytes constitute the most abundant cell type in the CNS, and play diverse functional roles, but the ontogenetic origins of this phenotypic diversity are poorly understood. We have investigated whether positional identity, a fundamental organizing principle governing the generation of neuronal subtype diversity, is also relevant to astrocyte diversification. We identified three positionally distinct subtypes of white matter astrocytes in the spinal cord, which can be distinguished by the combinatorial expression of Reelin and Slit1. These astrocyte subtypes derive from progenitor domains expressing the homeodomain transcription factors Pax6 and Nkx6.1, respectively. Loss- and gain-of-function experiments indicate that the positional identity of these astrocyte subtypes is controlled by Pax6 and Nkx6.1, in a combinatorial manner. Thus, positional identity is an organizing principle underlying astrocyte, as well as neuronal, subtype diversification, and is controlled by a homeodomain transcriptional code whose elements are re-utilized following the specification of neuronal identity earlier in development.
doi:10.1016/j.cell.2008.02.046
PMCID: PMC2394859  PMID: 18455991
23.  Applying a Targeted Label-free Approach using LC-MS AMT Tags to Evaluate Changes in Protein Phosphorylation Following Phosphatase Inhibition 
Journal of proteome research  2007;6(11):4489-4497.
In order to identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation site and protein. These data can be used to define the underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.
doi:10.1021/pr070068e
PMCID: PMC2516346  PMID: 17929957
Label-free quantitation; Targeted MS/MS; AMT tag pipeline; Comparative phosphoproteomics; Immobilized metal ion affinity chromatography (IMAC); Mass spectrometry; 20 μm ID monolithic column; Phosphoprotein phosphatase (PPP) family; Ser/Thr protein phosphatase; Calyculin A
24.  Serial monogamy and sex ratio bias in Nazca boobies 
Biased operational sex ratios (OSRs) can drive sexual selection on members of the over-represented sex via competition for mates, causing higher variance and skew in reproductive success (RS) among them if an individual's quality is a persistent characteristic. Alternatively, costs of reproduction may degrade breeding performance, creating the opportunity for members of the limiting sex to switch mates adaptively, effectively homogenizing variance and skew in RS among the sex in excess. We tested these two contrasting models in a male-biased population of the Nazca booby (Sula granti) with demonstrated costs of reproduction with data on total RS over a 14-year period. Variances and skews in RS were similar, and males changed from breeder to non-breeder more frequently than females. Under the persistent individual quality model, females should mate only with high quality males, and non-breeding males should seldom enter the breeding pool, yet 45% of non-breeding males (re)entered the breeding pool each year on average. Many Nazca booby females apparently exchange a depleted male for a new mate from the pool of current non-breeder males. Our evidence linking serial monogamy to costs of reproduction is novel and suggests selection on female mating preferences based on an interaction between at least two life-history components (OSR and reproductive effort).
doi:10.1098/rspb.2007.0578
PMCID: PMC2275186  PMID: 17567557
mate choice; operational sex ratio; cost of reproduction; serial monogamy; divorce; multi-state mark-recapture models
25.  Perinatal Androgens and Adult Behavior Vary with Nestling Social System in Siblicidal Boobies 
PLoS ONE  2008;3(6):e2460.
Background
Exposure to androgens early in development, while activating adaptive aggressive behavior, may also exert long-lasting effects on non-target components of phenotype. Here we compare these organizational effects of perinatal androgens in closely related Nazca (Sula granti) and blue-footed (S. nebouxii) boobies that differ in neonatal social system. The older of two Nazca booby hatchlings unconditionally attacks and ejects the younger from the nest within days of hatching, while blue-footed booby neonates lack lethal aggression. Both Nazca booby chicks facultatively upregulate testosterone (T) during fights, motivating the prediction that baseline androgen levels differ between obligately siblicidal and other species.
Methodology/Principal Findings
We show that obligately siblicidal Nazca boobies hatch with higher circulating androgen levels than do facultatively siblicidal blue-footed boobies, providing comparative evidence of the role of androgens in sociality. Although androgens confer a short-term benefit of increased aggression to Nazca booby neonates, exposure to elevated androgen levels during this sensitive period in development can also induce long-term organizational effects on behavior or morphology. Adult Nazca boobies show evidence of organizational effects of early androgen exposure in aberrant adult behavior: they visit unattended non-familial chicks in the colony and direct mixtures of aggression, affiliative, and sexual behavior toward them. In a longitudinal analysis, we found that the most active Non-parental Adult Visitors (NAVs) were those with a history of siblicidal behavior as a neonate, suggesting that the tendency to show social interest in chicks is programmed, in part, by the high perinatal androgens associated with obligate siblicide. Data from closely related blue-footed boobies provide comparative support for this interpretation. Lacking obligate siblicide, they hatch with a corresponding low androgen level, and blue-footed booby adults show a much lower frequency of NAV behavior and a lower probability of behaving aggressively during NAV interactions. This species difference in adult social behavior appears to have roots in both pleiotropic and experiential effects of nestling social system.
Conclusions/Significance
Our results indicate that Nazca boobies experience life-long consequences of androgenic preparation for an early battle to the death.
doi:10.1371/journal.pone.0002460
PMCID: PMC2413419  PMID: 18560542

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