Nitric oxide in B6 mouse and nitric oxide-sensitive soluble guanylate cyclase in cat modulate acetylcholine release in pontine reticular formation.

ACh regulates arousal, and the present study was designed to provide insight into the neurochemical mechanisms modulating ACh release in the pontine reticular formation. Nitric oxide (NO)-releasing beads microinjected into the pontine reticular formation of C57BL/6J (B6) mice significantly (P < 0.0001) increased ACh release. Microdialysis delivery of the NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC-12) to the mouse pontine reticular formation also caused a concentration-dependent increase in ACh release (P < 0.001). These are the first neurochemical data showing that ACh release in the pontine reticular formation of the B6 mouse is modulated by NO. The signal transduction cascade through which NO modulates ACh release in the pontine reticular formation has not previously been characterized. Therefore, an additional series of studies quantified the effects of a soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), on ACh release in the cat medial pontine reticular formation. During naturally occurring states of sleep and wakefulness, but not anesthesia, ODQ caused a significant (P < 0.001) decrease in ACh release. These results show for the first time that NO modulates ACh in the medial pontine reticular formation of the cat via an NO-sensitive sGC signal transduction cascade. Isoflurane and halothane anesthesia have been shown to decrease ACh release in the medial pontine reticular formation. The finding that ODQ did not alter ACh release during isoflurane or halothane anesthesia demonstrates that these anesthetics disrupt the NO-sensitive sGC-cGMP pathway. Considered together, results from the mouse and cat indicate that NO modulates ACh release in arousal-promoting regions of the pontine reticular formation via an NO-sensitive sGC-cGMP pathway.     

Stimulation of the P2Y Purinergic Receptor on Type 1 Astroglia Results in Inositol Phosphate Formation and Calcium Mobilization

Cultured astroglia express purinergic receptors that initiate phosphoinositide metabolism and calcium mobilization. Experiments were conducted to characterize the purinergic receptor subtype on type 1 astroglia responsible for stimulation these second-messenger systems. Inositol phosphate (IP) accumulation and calcium mobilization were measured after stimulation with ATP or purinergic receptor subtype-selective ATP analogues. ATP (10(-5) M) increased IP accumulation severalfold. Dose-effect assays monitoring astroglial IP accumulation revealed the order of potency that defines the P2Y receptor: 2-methylthioadenosine 5'-triphosphate greater than ATP greater than alpha beta-methyleneadenosine 5'-triphosphate greater than beta gamma-methyleneadenosine 5'-triphosphate. The influence of ATP on intracellular calcium levels in individual type 1 astroglia was examined using the calcium indicator dye, fura-2. Dose-effect experiments indicated that ATP was equally potent for generating inositol phosphates and increasing cellular calcium. The most prevalent response (87% of total responses) to ATP consisted of a rapid increase in calcium to a peak level that was approximately five times greater than the prestimulation level. This peak was followed by a decline to a plateau level that was significantly above baseline. This plateau phase of the calcium increase was maintained for at least 5 min in the presence of ATP and was dependent on external calcium. Many (23%) astroglia exhibited spontaneous calcium oscillations whose frequency and magnitude increased after the addition of 10(-5) M ATP. Immunocytochemical staining indicated that the responses occurred in glial fibrillary acidic protein positive cells. We conclude that type 1 astroglia express the P2Y purinergic receptor which regulates IP production and calcium mobilization.     

Focal Plant Observations as a Standardised Method for Pollinator Monitoring: Opportunities and Limitations for Mass Participation Citizen Science

BackgroundRecently there has been increasing focus on monitoring pollinating insects, due to concerns about their declines, and interest in the role of volunteers in monitoring pollinators, particularly bumblebees, via citizen science.

Methodology / Principal Findings

The Big Bumblebee Discovery was a one-year citizen science project run by a partnership of EDF Energy, the British Science Association and the Centre for Ecology & Hydrology which sought to assess the influence of the landscape at multiple scales on the diversity and abundance of bumblebees. Timed counts of bumblebees (Bombus spp.; identified to six colour groups) visiting focal plants of lavender (Lavendula spp.) were carried out by about 13 000 primary school children (7–11 years old) from over 4000 schools across the UK. 3948 reports were received totalling 26 868 bumblebees. We found that while the wider landscape type had no significant effect on reported bumblebee abundance, the local proximity to flowers had a significant effect (fewer bumblebees where other flowers were reported to be >5m away from the focal plant). However, the rate of mis-identifcation, revealed by photographs uploaded by participants and a photo-based quiz, was high.

Conclusions / Significance

Our citizen science results support recent research on the importance of local flocal resources on pollinator abundance. Timed counts of insects visiting a lure plant is potentially an effective approach for standardised pollinator monitoring, engaging a large number of participants with a simple protocol. However, the relatively high rate of mis-identifications (compared to reports from previous pollinator citizen science projects) highlights the importance of investing in resources to train volunteers. Also, to be a scientifically valid method for enquiry, citizen science data needs to be sufficiently high quality, so receiving supporting evidence (such as photographs) would allow this to be tested and for records to be verified.     

GarA is an essential regulator of metabolism in Mycobacterium tuberculosis

Alpha‐ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α‐ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non‐pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient‐dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.     

Context and connectivity in plant metapopulations and landscape mosaics: does the matrix matter?

Recent reviews of evidence for plant metapopulation prevalence in nature have concluded that most species appear not to be arranged as metapopulations – hence other frameworks may be necessary for understanding large‐scale, regional dynamics in plants. Separate but related paradigms from the disciplines of landscape ecology and metapopulation ecology exist for understanding patterns of regional population variation. The major models of both paradigms assume a binary landscape mosaic composed of “suitable habitat” and background “matrix.” An important distinction between the two approaches is that metapopulation models essentially ignore features of the matrix. A binary approach to the landscape seems inappropriate for plants for several reasons. First, plants probably do not have a binary perception of the landscape, but rather respond to gradients of resource quality. Thus properties of patches, or the matrix per se, may be less important than the nature of the landscape mosaic, in particular as this is reflected in terms of connectivity. Secondly, many plants rely on a range of other agents for dispersal of pollen and seed, all of which are also affected by their environment in terms of connectivity. Furthermore the various components of the mosaic, including physical, spatial and functional elements can significantly influence plant movements. We review important effects of the matrix – via composition and configuration of habitat patches, extent of edges, patterns of land use, etc., upon plant populations. We describe evidence supporting a general integration of metapopulation and landscape ecological approaches for understanding regional dynamics in plants, emphasizing notions of connectivity (traditionally measured in very different ways by metapopulation and landscape ecologists), and context, an emerging concept describing components of variability in the landscape from a species‐specific perspective. Finally, we describe a functional landscape mosaic approach that treats structural and functional features of the landscape and show how these interact to determine the fate of plant populations.     

Do Hybrid Trees Inherit Invasive Characteristics? Fruits of Corymbia torelliana X C. citriodora Hybrids and Potential for Seed Dispersal by Bees

Tree invasions have substantial impacts on biodiversity and ecosystem functioning, and trees that are dispersed by animals are more likely to become invasive. In addition, hybridisation between plants is well documented as a source of new weeds, as hybrids gain new characteristics that allow them to become invasive. Corymbia torelliana is an invasive tree with an unusual animal dispersal mechanism: seed dispersal by stingless bees, that hybridizes readily with other species. We examined hybrids between C. torelliana and C. citriodora subsp. citriodora to determine whether hybrids have inherited the seed dispersal characteristics of C. torelliana that allow bee dispersal. Some hybrid fruits displayed the characteristic hollowness, resin production and resin chemistry associated with seed dispersal by bees. However, we did not observe bees foraging on any hybrid fruits until they had been damaged. We conclude that C. torelliana and C. citriodora subsp. citriodora hybrids can inherit some fruit characters that are associated with dispersal by bees, but we did not find a hybrid with the complete set of characters that would enable bee dispersal. However, around 20,000 hybrids have been planted in Australia, and ongoing monitoring is necessary to identify any hybrids that may become invasive.     

Direct anabolic metabolism of three-carbon propionate to a six-carbon metabolite occurs in vivo across tissues and species

Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this reaction pathway using purified protein extracts provided limited substrates and verified the product via LC-MS using a synthetic standard. In whole-body in vivo stable isotope tracing following infusion of ¹³C-labeled valine at steady state, 2M2PE-CoA was found to form via propionyl-CoA in multiple murine tissues, including heart, kidney, and to a lesser degree, in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA also formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three- to six-carbon reaction conserved in humans and mice that utilizes propionate.