Dissection of synaptic excitability phenotypes by using a dominant-negative Shaker K+ channel subunit

During nervous system development, synapses undergo morphological change as a function of electrical activity. In Drosophila, enhanced activity results in the expansion of larval neuromuscular junctions. We have examined whether these structural changes involve the pre- or postsynaptic partner by selectively enhancing electrical excitability with a Shaker dominant-negative (SDN) potassium channel subunit. We find that the SDN enhances neurotransmitter release when expressed in motoneurons, postsynaptic potential broadening when expressed in muscles and neurons, and selectively suppresses fast-inactivating, Shaker-mediated IA currents in muscles. SDN expression also phenocopies the canonical behavioral phenotypes of the Sh mutation. At the neuromuscular junction, we find that activity-dependent changes in arbor size occur only when SDN is expressed presynaptically. This finding indicates that elevated postsynaptic membrane excitability is by itself insufficient to enhance presynaptic arbor growth. Such changes must minimally involve increased neuronal excitability.     

Characterization of an electron conduit between bacteria and the extracellular environment

A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning β-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment.     

Molecular Characterization of a Novel Positive-Sense,Single-Stranded RNA Mycovirus Infecting the Plant Pathogenic Fungus Sclerotinia sclerotiorum

Recent studies have demonstrated that a diverse array of mycoviruses infect the plant pathogenic fungus Sclerotinia sclerotiorum. Here, we report the molecular characterization of a newly identified mycovirus, Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), which was isolated from a sclerotia-defective strain JMTJ14 of S. sclerotiorum. Excluding a poly (A) tail, the genome of SsFV1 comprises 7754 nucleotides (nts) in length with 83 and 418 nts for 5''- and 3''-untranslated regions, respectively. SsFV1 has four non-overlapping open reading frames (ORFs): ORF1 encodes a 191 kDa polyprotein (1664 amino acid residues in length) containing conserved RNA-dependent RNA polymerase (RdRp) and helicase domains; the other three ORFs encode three putative hypothetical proteins of unknown function. Phylogenetic analysis, based on RdRp and Helicase domains, indicated that SsFV1 is phylogenetically related to Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), a cluster of an independent group belonging to a newly proposed family Fusarividae. However, SsFV1 is markedly different from FgV1 and RnFV1 in genome organization and nucleotide sequence. SsFV1 was transmitted successfully to two vegetatively incompatible virus-free strains. SsFV1 is not responsible for the abnormal phenotype of strain JMTJ14.     

Detection and Characterization of a Novel Reassortant Mammalian Orthoreovirus in Bats in Europe

A renewed interest in mammalian orthoreoviruses (MRVs) has emerged since new viruses related to bat MRV type 3, detected in Europe, were identified in humans and pigs with gastroenteritis. This study reports the isolation and characterization of a novel reassortant MRV from the lesser horseshoe bat (Rhinolophus hipposideros). The isolate, here designated BatMRV1-IT2011, was first identified by electron microscopy and confirmed using PCR and virus-neutralization tests. The full genome sequence was obtained by next-generation sequencing. Molecular and antigenic characterizations revealed that BatMRV1-IT2011 belonged to serotype 1, which had not previously been identified in bats. Phylogenetic and recombination detection program analyses suggested that BatMRV1-IT2011 was a reassortant strain containing an S1 genome segment similar to those of MRV T1/bovine/Maryland/Clone23/59 and C/bovine/Indiana/MRV00304/2014, while other segments were more similar to MRVs of different hosts, origins and serotypes. The presence of neutralizing antibodies against MRVs has also been investigated in animals (dogs, pigs, bovines and horses). Preliminary results suggested that MRVs are widespread in animals and that infections containing multiple serotypes, including MRVs of serotype 1 with an S1 gene similar to BatMRV1-IT2011, are common. This paper extends the current knowledge of MRVs and stresses the importance to continue and improve MRV surveillance in bats and other mammals through the development and standardization of specific diagnostic tools.     

Two isozymes of particulate methane monooxygenase with different methane oxidation kinetics are found in Methylocystis sp. strain SC2

Methane-oxidizing bacteria (methanotrophs) attenuate methane emission from major sources, such as wetlands, rice paddies, and landfills, and constitute the only biological sink for atmospheric methane in upland soils. Their key enzyme is particulate methane monooxygenase (pMMO), which converts methane to methanol. It has long been believed that methane at the trace atmospheric mixing ratio of 1.75 parts per million by volume (ppmv) is not oxidized by the methanotrophs cultured to date, but rather only by some uncultured methanotrophs, and that type I and type II methanotrophs contain a single type of pMMO. Here, we show that the type II methanotroph Methylocystis sp. strain SC2 possesses two pMMO isozymes with different methane oxidation kinetics. The pmoCAB1 genes encoding the known type of pMMO (pMMO1) are expressed and pMMO1 oxidizes methane only at mixing ratios >600 ppmv. The pmoCAB2 genes encoding pMMO2, in contrast, are constitutively expressed, and pMMO2 oxidizes methane at lower mixing ratios, even at the trace level of atmospheric methane. Wild-type strain SC2 and mutants expressing pmoCAB2 but defective in pmoCAB1 consumed atmospheric methane for >3 months. Growth occurred at 10-100 ppmv methane. Most type II but no type I methanotrophs possess the pmoCAB2 genes. The apparent K(m) of pMMO2 (0.11 muM) in strain SC2 corresponds well with the K(m(app)) values for methane oxidation measured in soils that consume atmospheric methane, thereby explaining why these soils are dominated by type II methanotrophs, and some by Methylocystis spp., in particular. These findings change our concept of methanotroph ecology.     

Preparation and Characterization of Lignocellulosic Oil Sorbent by Hydrothermal Treatment of Populus Fiber

This study is aimed at achieving the optimum conditions of hydrothermal treatment and acetylation of Populus fiber to improve its oil sorption capacity (OSC) in an oil-water mixture. The characteristics of the hydrolyzed and acetylated fibers were comparatively investigated by FT-IR, CP-MAS ¹³C-NMR, SEM and TGA. The optimum conditions of the hydrothermal treatment and acetylation were obtained at 170 °C for 1 h and 120 °C for 2 h, respectively. The maximum OSC of the hydrolyzed fiber (16.78 g/g) was slightly lower than that of the acetylated fiber (21.57 g/g), but they were both higher than the maximum OSC of the unmodified fiber (3.94 g/g). In addition, acetylation after hydrothermal treatment for the Populus fiber was unnecessary as the increment of the maximum OSC was only 3.53 g/g. The hydrolyzed and the acetylated Populus fibers both displayed a lumen orifice enabling a high oil entrapment. The thermal stability of the modified fibers was shown to be increased in comparison with that of the raw fiber. The hydrothermal treatment offers a new approach to prepare lignocellulosic oil sorbent.     

Probing the evolution of appendage specialization by Hox gene misexpression in an emerging model crustacean

Changes in the expression of Hox genes have been widely linked to the evolution of animal body plans, but functional demonstrations of this relationship have been impeded by the lack of suitable model organisms. A classic case study involves the repeated evolution of specialized feeding appendages, called maxillipeds, from anterior thoracic legs, in many crustacean lineages. These leg-to-maxilliped transformations correlate with the loss of Ultrabithorax (Ubx) expression from corresponding segments, which is proposed to be the underlying genetic cause. To functionally test this hypothesis, we establish tools for conditional misexpression and use these to misexpress Ubx in the crustacean Parhyale hawaiensis. Ectopic Ubx leads to homeotic transformations of anterior appendages toward more posterior thoracic fates, including maxilliped-to-leg transformations, confirming the capacity of Ubx to control thoracic (leg) versus gnathal (feeding) segmental identities. We find that maxillipeds not only are specified in the absence of Ubx, but also can develop in the presence of low/transient Ubx expression. Our findings suggest a path for the gradual evolutionary transition from thoracic legs to maxillipeds, in which stepwise changes in Hox gene expression have brought about this striking morphological and functional transformation.     

Characterization of MADS-box genes in charophycean green algae and its implication for the evolution of MADS-box genes

The MADS-box genes of land plants are extensively diverged to form a superfamily and are important in various aspects of development including the specification of floral organs as homeotic selector genes. The closest relatives of land plants are the freshwater green algae charophyceans. To study the origin and evolution of land plant MADS-box genes, we characterized these genes in three charophycean green algae: the stonewort Chara globularis, the coleochaete Coleochaete scutata, and the desmid Closterium peracerosum-strigosum-littorale complex. Phylogenetic analyses suggested that MADS-box genes diverged extensively in the land plant lineage after the separation of charophyceans from land plants. The stonewort C. globularis mRNA was specifically detected in the oogonium and antheridium together with the egg and spermatozoid during their differentiation. The expression of the C. peracerosum-strigosum-littorale-complex gene increased when vegetative cells began to differentiate into gametangial cells and decreased after fertilization. These expression patterns suggest that the precursors of land plant MADS-box genes originally functioned in haploid reproductive cell differentiation and that the haploid MADS-box genes were recruited into a diploid generation during the evolution of land plants.     

Selection of a trioxaquine as an antimalarial drug candidate

Trioxaquines are antimalarial agents based on hybrid structures with a dual mode of action. One of these molecules, PA1103/SAR116242, is highly active in vitro on several sensitive and resistant strains of Plasmodium falciparum at nanomolar concentrations (e.g., IC₅₀ value = 10 nM with FcM29, a chloroquine-resistant strain) and also on multidrug-resistant strains obtained from fresh patient isolates in Gabon. This molecule is very efficient by oral route with a complete cure of mice infected with chloroquine-sensitive or chloroquine-resistant strains of Plasmodia at 26–32 mg/kg. This compound is also highly effective in humanized mice infected with P. falciparum. Combined with a good drug profile (preliminary absorption, metabolism, and safety parameters), these data were favorable for the selection of this particular trioxaquine for development as drug candidate among 120 other active hybrid molecules.     

Characterization of mononuclear phagocytic cells in medaka fish transgenic for a cxcr3a:gfp reporter

Chemokines and chemokine receptors are key evolutionary innovations of vertebrates. They are involved in morphogenetic processes and play an important role in the immune system. Based on an analysis of the chemokine receptor gene family in teleost genomes, and the expression patterns of chemokine receptor genes during embryogenesis and the wounding response in young larvae of Oryzias latipes, we identified the chemokine receptor cxcr3a as a marker of innate immune cells. Cells expressing cxcr3a were characterized in fish transgenic for a cxcr3a:gfp reporter. In embryos and larvae, cxcr3a-expressing cells are motile in healthy and damaged tissues, and phagocytic; the majority of these cells has the morphology of tissue macrophages, whereas a small fraction has a dendritic phenotype. In adults, cxcr3a-positive cells continue to specifically express myeloid-associate markers and genes related to antigen uptake and presentation. By light microscopy and ultrastructural analysis, the majority of cxcr3a-expressing cells has a dendritic phenotype, whereas the remainder resembles macrophage-like cells. After challenge of adult fish with bacteria or CpG oligonucleotides, phagocytosing cxcr3a-positive cells in the blood up-regulated il12p40 genes, compatible with their function as part of the mononuclear phagocytic system. Our results identify a marker of teleost mononuclear phagocytic cells and suggest a surprising degree of morphological and functional similarity between the innate immune systems of lower and higher vertebrates.     

Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish

Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.     

A single amino acid converts a repressor to an activator of flowering

Homologous proteins occurring through gene duplication may give rise to novel functions through mutations affecting protein sequence or expression. Comparison of such homologues allows insight into how morphological traits evolve. However, it is often unclear which changes are key to determining new functions. To address these ideas, we have studied a system where two homologues have evolved clear and opposite functions in controlling a major developmental switch. In plants, flowering is a major developmental transition that is critical to reproductive success. Arabidopsis phosphatidylethanolamine-binding protein homologues TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) are key controllers of flowering, determining when and where flowers are made, but as opposing functions: TFL1 is a repressor, FT is an activator. We have uncovered a striking molecular basis for how these homologous proteins have diverged. Although <60% identical, we have shown that swapping a single amino acid is sufficient to convert TFL1 to FT function and vice versa. Therefore, these key residues may have strongly contributed to the selection of these important functions over plant evolution. Further, our results suggest that TFL1 and FT are highly conserved in biochemical function and that they act as repressors or activators of flowering through discrimination of structurally related interactors by a single residue.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Interrogating an insect society

Insect societies such as those of ants, bees, and wasps consist of 1 or a small number of fertile queens and a large number of sterile or nearly sterile workers. While the queens engage in laying eggs, workers perform all other tasks such as nest building, acquisition and processing of food, and brood care. How do such societies function in a coordinated and efficient manner? What are the rules that individuals follow? How are these rules made and enforced? These questions are of obvious interest to us as fellow social animals but how do we interrogate an insect society and seek answers to these questions? In this article I will describe my research that was designed to seek answers from an insect society to a series of questions of obvious interest to us. I have chosen the Indian paper wasp Ropalidia marginata for this purpose, a species that is abundantly distributed in peninsular India and serves as an excellent model system. An important feature of this species is that queens and workers are morphologically identical and physiologically nearly so. How then does an individual become a queen? How does the queen suppress worker reproduction? How does the queen regulate the nonreproductive activities of the workers? What is the function of aggression shown by different individuals? How and when is the queen''s heir decided? I will show how such questions can indeed be investigated and will emphasize the need for a whole range of different techniques of observation and experimentation.     

Characterization of the anti-Leishmania effect induced by cisplatin, an anticancer drug

The cis-diamminedichloroplatinum(II), known as cis-DDP or cisplatin is a widely used drug in cancer chemotherapy. Although a recent study has shown the anti-Leishmania activity of some cis-DDP derivatives, the cytotoxic properties were measured only on promastigotes, the insect vector form of the parasite. In this study the effect of cis-DDP on promastigotes and amastigotes, the vertebrate stage of the parasite is reported. The IC50, determined by flow cytometry, after 72 h of drug incubation was four times higher, 7.73+/-1.03 microM in the case of promastigotes compared to axenic amastigotes, 1.88+/-0.10 microM. In intracellular amastigotes the IC50, determined by counting the parasite index was 1.85+/-0.22 microM. By using flow cytometry, two patterns of cell cycle changes was observed: cis-DDP treated promastigotes and amastigotes accumulated in S phase and G2 phase, respectively. The cis-DDP response was also found to involve an "apoptosis-like" death of both promastigotes and amastigotes. However, DNA fragmentation was only detected in promastigote forms. In contrast mitochondrial transmembrane potential loss was observed for both stages of the parasite. Upon incubation of parasites with the drug an increase on GSH and GSSG levels and reactive oxygen species could be detected in the case of promastigote. Moreover, a slight increase of GSH level was detected on amastigote form. Taken together, these observations indicate that amastigotes are more sensitive to cis-DDP when compared to promastigotes. However, the signaling pathways leading to cell death could be different.     

Evolution of moth sex pheromone composition by a single amino acid substitution in a fatty acid desaturase

For sexual communication, moths primarily use blends of fatty acid derivatives containing one or more double bonds in various positions and configurations, called sex pheromones (SPs). To study the molecular basis of novel SP component (SPC) acquisition, we used the tobacco hornworm (Manduca sexta), which uses a blend of mono-, di-, and uncommon triunsaturated fatty acid (3UFA) derivatives as SP. We identified pheromone-biosynthetic fatty acid desaturases (FADs) MsexD3, MsexD5, and MsexD6 abundantly expressed in the M. sexta female pheromone gland. Their functional characterization and in vivo application of FAD substrates indicated that MsexD3 and MsexD5 biosynthesize 3UFAs via E/Z14 desaturation from diunsaturated fatty acids produced by previously characterized Z11-desaturase/conjugase MsexD2. Site-directed mutagenesis of sequentially highly similar MsexD3 and MsexD2 demonstrated that swapping of a single amino acid in the fatty acyl substrate binding tunnel introduces E/Z14-desaturase specificity to mutated MsexD2. Reconstruction of FAD gene phylogeny indicates that MsexD3 was recruited for biosynthesis of 3UFA SPCs in M. sexta lineage via gene duplication and neofunctionalization, whereas MsexD5 representing an alternative 3UFA-producing FAD has been acquired via activation of a presumably inactive ancestral MsexD5. Our results demonstrate that a change as small as a single amino acid substitution in a FAD enzyme might result in the acquisition of new SP compounds.Sex pheromones (SPs) are a diverse group of chemical compounds that are central to mate-finding behavior in insects (1). Variation in SP composition between closely related species and among populations is well documented. Despite this variation, SPs are presumed to be under strong stabilizing selection, and thus the genetic mechanisms driving SP diversification represented an enigma (2). Research on SPs in moths (Insecta: Lepidoptera) helped establish the hypothesis of asymmetric tracking as a major driving force in SP diversification. In this scenario, abrupt changes in female SP composition via a shift in component ratio or the inclusion or loss of a component result in a distinct SP that attracts males with more broadly or differentially tuned SP preference (3). Assortative mating, the preferential mating of females producing a novel SP with males attracted to this SP, restricts gene flow between subpopulations with differing SP compositions. This can ultimately lead to speciation and fixation of novel communication channels (4). Work in insect models such as wasps (5), fruit flies (6), and especially moths (7–9) is helping uncover the genetic basis of SP diversification.In the majority of moth species, females use species-specific mixtures of SP components (SPCs) consisting of volatile fatty acid (FA) derivatives to attract conspecific males at long range. These SPCs are predominantly long-chain aliphatic (C12–C18) acetates, alcohols, or aldehydes containing zero to three double bonds of various configurations at different positions along the carbon backbone (10). Pheromone biosynthesis involves modifications of fatty acyl substrates, such as chain shortening and elongation, reduction, acetylation, oxidation, and desaturation (11). SP biosynthetic enzymes [i.e., FA reductases (8), FA chain-shortening enzymes (12, 13), and particularly FA desaturases (FADs) (7, 9, 14–17)] are the most commonly discovered traits underlying SP divergence in moths.Manduca sexta females attract males by releasing an SP containing in addition to mono- and diunsaturated aldehydes, which are typical structural themes in SPs of Bombycoidea moths (10), also uncommon conjugated triunsaturated aldehydes. The production of triunsaturated SPCs represents an easily traceable phenotype, thus making M. sexta a convenient yet unexploited model organism for unraveling the mechanisms of chemical communication evolution via novel SPC recruitment. In our previous attempts to decipher the desaturation pathway leading to triunsaturated SPC FA precursors (3UFAs), we identified the MsexD2 desaturase, which exhibits Z11-desaturase and conjugase (1,4-dehydrogenase) activity and participates in stepwise production of monounsaturated (1UFA) and diunsaturated (2UFA) SPC precursors. The terminal desaturation step resulting in the third conjugated double bond remained, however, elusive (18, 19).Here, we isolated and functionally characterized FAD genes abundantly and specifically expressed in the pheromone gland (PG) capable of producing 3UFA pheromone precursors and demonstrated the biosynthesis of 3UFAs from 2UFAs. We used site-directed mutagenesis of M. sexta FADs and identified a minimal structure motif leading to acquisition of new desaturase specificities. The reconstructed evolutionary relationship of moth FADs demonstrated that the 3UFA pheromone precursors in M. sexta were acquired via (i) activation of a presumably inactive ancestral FAD gene and/or (ii) duplication of an ancestral FAD gene producing 1UFA and 2UFA SPC precursors followed by functional diversification of an FAD duplicate.     

Genetic Characterization of the Tick-Borne Orbiviruses

The International Committee for Taxonomy of Viruses (ICTV) recognizes four species of tick-borne orbiviruses (TBOs): Chenuda virus, Chobar Gorge virus, Wad Medani virus and Great Island virus (genus Orbivirus, family Reoviridae). Nucleotide (nt) and amino acid (aa) sequence comparisons provide a basis for orbivirus detection and classification, however full genome sequence data were only available for the Great Island virus species. We report representative genome-sequences for the three other TBO species (virus isolates: Chenuda virus (CNUV); Chobar Gorge virus (CGV) and Wad Medani virus (WMV)). Phylogenetic comparisons show that TBOs cluster separately from insect-borne orbiviruses (IBOs). CNUV, CGV, WMV and GIV share low level aa/nt identities with other orbiviruses, in ‘conserved’ Pol, T2 and T13 proteins/genes, identifying them as four distinct virus-species. The TBO genome segment encoding cell attachment, outer capsid protein 1 (OC1), is approximately half the size of the equivalent segment from insect-borne orbiviruses, helping to explain why tick-borne orbiviruses have a ~1 kb smaller genome.     

Characterization of a Proposed Dichorhavirus Associated with the Citrus Leprosis Disease and Analysis of the Host Response

The causal agents of Citrus leprosis are viruses; however, extant diagnostic methods to identify them have failed to detect known viruses in orange, mandarin, lime and bitter orange trees with severe leprosis symptoms in Mexico, an important citrus producer. Using high throughput sequencing, a virus associated with citrus leprosis was identified, belonging to the proposed Dichorhavirus genus. The virus was termed Citrus Necrotic Spot Virus (CNSV) and contains two negative-strand RNA components; virions accumulate in the cytoplasm and are associated with plasmodesmata—channels interconnecting neighboring cells—suggesting a mode of spread within the plant. The present study provides insights into the nature of this pathogen and the corresponding plant response, which is likely similar to other pathogens that do not spread systemically in plants.