Global Down-Regulation of Gene Expression in the Brain Using RNA Interference,with Emphasis on Monoamine Transporters and GPCRs: Implications for Target Characterization in Psychiatric and Neurological Disorders

RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.     

Metabolomics reveals herbivore‐induced metabolites of resistance and susceptibility in maize leaves and roots

Plants respond to herbivory by reprogramming their metabolism. Most research in this context has focused on locally induced compounds that function as toxins or feeding deterrents. We developed an ultra‐high‐pressure liquid chromatography time‐of‐flight mass spectrometry (UHPLC‐TOF‐MS)‐based metabolomics approach to evaluate local and systemic herbivore‐induced changes in maize leaves, sap, roots and root exudates without any prior assumptions about their function. Thirty‐two differentially regulated compounds were identified from Spodoptera littoralis‐infested maize seedlings and isolated for structure assignment by microflow nuclear magnetic resonance (CapNMR). Nine compounds were quantified by a high throughput direct nano‐infusion tandem mass spectrometry/mass spectrometry (MS/MS) method. Leaf infestation led to a marked local increase of 1,3‐benzoxazin‐4‐ones, phospholipids, N‐hydroxycinnamoyltyramines, azealic acid and tryptophan. Only few changes were found in the root metabolome, but 1,3‐benzoxazin‐4‐ones increased in the vascular sap and root exudates. The role of N‐hydroxycinnamoyltyramines in plant–herbivore interactions is unknown, and we therefore tested the effect of the dominating p‐coumaroyltyramine on S. littoralis. Unexpectedly, p‐coumaroyltyramine was metabolized by the larvae and increased larval growth, possibly by providing additional nitrogen to the insect. Taken together, this study illustrates that herbivore attack leads to the induction of metabolites that can have contrasting effects on herbivore resistance in the leaves and roots.     

Predation risk perception and food scarcity induce alterations of life-cycle traits of the mosquito Culex pipiens

Abstract.  1. Predators may affect prey populations by direct consumption, and by inducing defensive reactions of prey to the predation risk. Food scarcity frequently has effects on the inducible defences of prey, but no consistent pattern of food–predation risk interaction is known.
2. In this study the combined effect of food shortage and predation-risk perception in larvae of the mosquito Culex pipiens was investigated. Water exposed to the aquatic predator bug Notonecta glauca was used as a source of predation intimidation. Mosquito larvae were reared in three different media containing either no predator cues or the cues of N. glauca that had been fed on either C. pipiens larvae or on Daphnia magna . Food was provided in favourable or limited amount for these set-ups.
3. The results showed that chemical cues from the predators fed with prey's conspecifics caused a decreased survival, delayed pre-imaginal development, and reduction in body size of emerged mosquitoes, whereas chemical cues from predators fed with D. magna caused only delayed development. Food scarcity significantly exacerbates the negative effect of the predator cues on pre-imaginal development of C. pipiens . Effects of the cues on larval development and body size of imagoes are significantly stronger for females than for males.
4. The present study suggests that when food is limited, predators can affect population dynamics of prey not only by direct predation, but also by inducing lethal and sublethal effects due to perception of risk imposed by chemical cues. To understand the effects of predators on mosquito population dynamics, environmental parameters such as food deficiency should be considered.     

Abundance and primary production of filamentous green algae Zygogonium ericetorum in an extremely acid (pH 2.9) mining lake and its impact on alkalinity generation

1. In extremely acid mining lakes, benthic filamentous green algae (Zygnemataceae, Chlorophyta) thrive as effective competitors for limited carbon (C). These algae could supply C for microbial‐mediated benthic alkalinity generation. However, biomass, productivity and impact of the acidobiontic filamentous green algae at pH ≤3 have not previously been determined. 2. Periphytic filamentous green algae was mapped by harvesting their biomass from 85 1 × 1 m quadrats in mining lake Grünewalder Lauch. Zygogonium ericetorum colonised water depths between 1.6 and 10.5 m covering 88% of total area. Biomass peaked at 5–6 m depth. Total Zygogonium biomass amounted to 72.2 t dry weight for the whole lake (0.94 km²), which corresponds to 16.1 t C and the accumulation of primary production from 2.2 years. 3. Growth of Zygogonium is moderately N, C and extremely P deficient, and seriously stressed by high rates of Fe deposition during summer. Consequently, net primary production (NPP) of Zygogonium, calculated from measured photosynthesis versus irradiance characteristics and calculated underwater irradiance (0.13 g C m^?2 year^?1) and in situ oxygen measurements (7.8 g C m^?2 year^?1), corresponds to only 0.3% and 18.1% of pelagic NPP. 4. Neither pelagic nor benthic Zygogonium primary production can supply enough C for efficient acidity removal. However, at rates of benthic NPP in summer of 21.4 mg C m^?2 day^?1, Zygogonium contributed 26% of the C equivalents to remove acidity associated with ferric iron, contributing at least seasonally to efficient alkalinity generation.     

Ontogeny and homology of the neural complex of otophysan Ostariophysi

The development and homology of the neural complex in the ostariophysan subgroup Otophysi were studied, including for the first time representatives of all four major otophysan lineages. Previous hypotheses concerning the homology of the different elements of the neural complex have also been reviewed and these are discussed in light of our new findings. We found that supradorsal cartilages, paired autogenous elements at the medial side of the neural arches, play a previously unrecognized major role during the ontogeny and evolution of the neural complex. In most otophysan taxa, supradorsals 3 and 4 fuse with supraneurals 2 and 3 (cypriniforms) or supraneural 3 (characiforms, some siluriforms, gymnotiforms) during ontogeny to form the neural complex. In the majority of siluriforms, the neural complex is exclusively formed by supradorsals 3 and 4. In loricarioid siluriforms, a neural complex is missing, the fourth neural arch fuses to the occiput in development, and the first free vertebra is the fifth. Of the four otophysan subgroups, siluriforms are the most and cypriniforms the least diverse in the structure and development of their neural complex.  © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society , 2006, 147 , 301–330.     

Ubiquitins (polyubiquitin and ubiquitin extension protein) in marine sponges: cDNA sequence and phylogenetic analysis

The complete nucleotide sequences of two Suberites domuncula cDNAs and one Sycon raphanus cDNA, all encoding ubiquitin, have been determined. One cDNA from S. domuncula codes for polyubiquitin with four tandemly repeated monomeric units and the second cDNA encodes ubiquitin fused to a ribosomal protein of 78 amino acids (aa). S. domuncula possesses at least one additional polyubiquitin gene, from which the last two monomers were also sequenced. All analysed genes from S. domuncula encode identical ubiquitin proteins, with only one aa difference (Ala 19) to the human/higher animals ubiquitin (Pro 19). Ubiquitin in S. domuncula is identical with the ubiquitin found in another Demospongia, Geodia cydonium. The cDNA from S. raphanus encodes polyubiquitin with seven tandemly repeated units. All these gene monomers code for the same ubiquitin, which differs from the human/higher animals ubiquitin only at position 24 (Asp in Sycon, Glu in others). However, ubiquitin from S. raphanus (Calcarea) shows two aa differences (positions 19 and 24), when compared with the ubiquitin sequences from the two Demospongiae. In a phylogenetic tree constructed by multiple sequence alignment of all sponge ubiquitin gene monomers so far identified, all monomers from the same species cluster together, with the clear exception of the monomer from S. domuncula ribosomal protein fusion gene. This monomer branches off first from the tree and forms a separate line; this gives evidence for a very ancient split of ubiquitin-ribosomal-protein fusion genes from polyubiquitin encoding genes and their long separate coexistence in eukaryotes. The ubiquitin extension protein from S. domuncula is 78 aa long, displays all characteristics of 76–81 aa long ribosomal fusion proteins and shows 78% identity in the first 73 aa with the human S27a protein. However, its C-terminal sequence: 69-GLTYVYKKSD-78 is more similar to the plant consensus (69-GLTYVYQ/NK-76), than to the higher animal consensus (69-CLTYCFNK-76). This protein isolated from a sponge, belonging to the phylogenetically oldest multicellular animals, the Porifera, branches off first from the phylogenetic tree of metazoan ubiquitin extension proteins of the small ribosomal subunits.     

Interspecific differences in the response of arbuscular mycorrhizal fungi to Artemisia tridentata grown under elevated atmospheric CO2

Arbuscular mycorrhizal (AM) fungi form mutualistic symbioses with the root systems of most plant species. These mutualisms regulate nutrient exchange in the plant–soil interface and might influence the way in which plants respond to increasing atmospheric CO₂. In other experiments, mycorrhizal responses to elevated CO₂ have been variable, so in this study we test the hypothesis that different genera of AM fungi differ in their response, and in turn alter the plant's response, to elevated CO₂. Four species from three genera of AM fungi were tested. Artemisia tridentata Nutt. seedlings were inoculated with either Glomus intraradices Schenck & Smith, Glomus etunicatum Becker & Gerdemann, Acaulospora sp. or Scutellospora calospora (Nicol. & Gerd.) Walker & Sanders and grown at either ambient CO₂ (350 ppm) or elevated CO₂ (700 ppm). Several significant inter-specific responses were detected. Elevated CO₂ caused percent arbuscular and hyphal colonization to increase for the two Glomus species, but not for Acaulospora sp. or S. calospora . Vesicular colonization was not affected by elevated CO₂ for any fungal species. In the extra-radical phase, the two Glomus species produced a significantly higher number of spores in response to elevated CO₂, whereas Acaulospora sp. and S. calospora developed significantly higher hyphal lengths. These data show that AM fungal taxa differ in their growth allocation strategies and in their responses to elevated CO₂, and that mycorrhizal diversity should not be overlooked in global change research.     

Post‐Soviet farmland abandonment,forest recovery,and carbon sequestration in western Ukraine

Land use is a critical factor in the global carbon cycle, but land‐use effects on carbon fluxes are poorly understood in many regions. One such region is Eastern Europe and the former Soviet Union, where land‐use intensity decreased substantially after the collapse of socialism, and farmland abandonment and forest expansion have been widespread. Our goal was to examine how land‐use trends affected net carbon fluxes in western Ukraine (57 000 km²) and to assess the region's future carbon sequestration potential. Using satellite‐based forest disturbance and farmland abandonment rates from 1988 to 2007, historic forest resource statistics, and a carbon bookkeeping model, we reconstructed carbon fluxes from land use in the 20th century and assessed potential future carbon fluxes until 2100 for a range of forest expansion and logging scenarios. Our results suggested that the low‐point in forest cover occurred in the 1920s. Forest expansion between 1930 and 1970 turned the region from a carbon source to a sink, despite intensive logging during socialism. The collapse of the Soviet Union created a vast, but currently largely untapped carbon sequestration potential (up to～150 Tg C in our study region). Future forest expansion will likely maintain or even increase the region's current sink strength of 1.48 Tg C yr^?1. This may offer substantial opportunities for offsetting industrial carbon emissions and for rural development in regions with otherwise diminishing income opportunities. Throughout Eastern Europe and the former Soviet Union, millions of hectares of farmland were abandoned after the collapse of socialism; thus similar reforestation opportunities may exist in other parts of this region.