Is photoisomerization required for NO photorelease in ruthenium nitrosyl complexes?

The factors that explain the competition between intramolecular NO linkage photoisomerization and NO photorelease in five ruthenium nitrosyl complexes were investigated. By applying DFT-based methods, it was possible to characterize the ground states and lowest triplet potential energy surfaces of these species, and to establish that both photoisomerization and photorelease processes can occur in the lowest triplet state of each species. This work highlights the crucial role of the sideways-bonded isomer, a metastable state also known as the MS2 isomer, in the photochemical loss of NO, while the results obtained also indicate that the population of the triplet state of this isomer is compulsory for both processes and show how photoisomerization and photorelease interfere.

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Graphical Abstract Illustration of the crucial role of the ³MS2 state in the photoreactivities of ruthenium nitrosyl complexes

     

Seed reproductive biology of the rare aquatic carnivorous plant Aldrovanda vesiculosa (Droseraceae)

Despite the unprecedented global decline in extant populations of Aldrovanda vesiculosa in the last century, little is known about the reproductive biology of this iconic aquatic carnivorous plant. This study aimed to investigate the role of seed‐based reproduction in the ecology of A. vesiculosa, with particular focus on the interplay between the regulation of seed dormancy by temperature cues and the efficacy of exogenous ethylene gas to act as a germination stimulant, the desiccation capacity and long‐term storage potential of seeds for conservation purposes. Sexual reproduction appears to be extremely limited in both natural and naturalized populations across three continents, with high variability in the success of flowering and seed set between sites and between seasons. Overall, flowering yielded few fertile fruit (6–19% of flowers producing fertile fruit) and seed viability was variable but generally low (29–88%). Fecundity appears to be influenced by seasonal climatic conditions and microhabitat characteristics. Aldrovanda vesiculosa possesses physiologically dormant seeds, with germination stimulated by exposure to ethylene gas (>90% germination) at 25 °C. Seeds appear sensitive to desiccation and sub‐zero temperature storage, with no germination and markedly reduced embryo growth after storage of seeds for >1 month at 15 °C and 15% relative humidity or after short‐term (24 h) storage at ?18 °C. In the absence of significant conservation and restoration initiatives, the continuing decline of dystrophic freshwater wetland habitats globally leaves A. vesiculosa facing extinction. As the successful long‐term storage of seeds appears unfeasible based on the approaches described in this study, other alternatives for germplasm conservation such as cryostorage of vegetative tissues or zygotic embryos must be considered for establishing long‐term ex situ collections of critical germplasm.     

Trust: The Missing Dimension in the Food Retail Transition in Thailand

Thailand has experienced dramatic growth of large national and international modern food retailers, such as supermarkets, hypermarkets and convenience stores in large cities and regional centres in the last two decades. Nevertheless, Thai consumers continue to purchase perishables (fruits, vegetables and animal products) from fresh markets (wet markets, talat sot) contradicting predictions from analysts that modern food retail chains will rapidly replace fresh markets as the preferred venue for purchasing all types of foods. This paper examines trust in food retail systems as an under-explored dimension lying behind the continued patronage by Thais of fresh markets to purchase perishable items. It derives from a research program commenced in 2005 that includes fieldwork visits, interviews and questionnaires. In the context of the Thai food retail transition, we propose that trust affects relationships between consumers and (1) individual fresh market-based vendors, (2) the food products sold at fresh markets and (3) the food retail system more broadly. If fresh markets can be maintained in the face of sustained pressure from modern national and international food retailers, Thais will continue to use them. Meanwhile, trust is a relatively unrecognised dimension that is supporting the continued existence of traditional food retail formats.     

Seed germination and dormancy traits of forbs and shrubs important for restoration of North American dryland ecosystems

• In degraded dryland systems, native plant community re‐establishment following disturbance is almost exclusively carried out using seeds, but these efforts commonly fail. Much of this failure can be attributed to the limited understanding of seed dormancy and germination traits.
• We undertook a systematic classification of seed dormancy of 26 species of annual and perennial forbs and shrubs that represent key, dominant genera used in restoration of the Great Basin ecosystem in the western United States. We examined germination across a wide thermal profile to depict species‐specific characteristics and assessed the potential of gibberellic acid (GA₃) and karrikinolide (KAR₁) to expand the thermal germination envelope of fresh seeds.
• Of the tested species, 81% produce seeds that are dormant at maturity. The largest proportion (62%) exhibited physiological (PD), followed by physical (PY, 8%), combinational (PY + PD, 8%) and morphophysiological (MPD, 4%) dormancy classes. The effects of chemical stimulants were temperature‐ and species‐mediated. In general, mean germination across the thermal profile was improved by GA₃ and KAR₁ for 11 and five species, respectively. We detected a strong germination response to temperature in freshly collected seeds of 20 species. Temperatures below 10 °C limited the germination of all except Agoseris heterophylla, suggesting that in their dormant state, the majority of these species are thermally restricted.
• Our findings demonstrate the utility of dormancy classification as a foundation for understanding the critical regenerative traits in these ecologically important species and highlight its importance in restoration planning.

     

P2X7 nucleotide receptors mediate blebbing in osteoblasts through a pathway involving lysophosphatidic acid

Extracellular nucleotides, released in response to mechanical or inflammatory stimuli, signal through P2 receptors in many cell types, including osteoblasts. P2X7 receptors are ATP-gated cation channels that can induce formation of large membrane pores. Disruption of the gene encoding the P2X7 receptor leads to decreased periosteal bone formation and insensitivity of the skeleton to mechanical stimulation. Our purpose was to investigate signaling pathways coupled to P2X7 activation in osteoblasts. Live cell imaging showed that ATP or 2 ',3 '-O-(4-benzoylbenzoyl)-ATP (BzATP), but not UTP, UDP, or 2-methylthio-ADP, induced dynamic membrane blebbing in calvarial osteoblasts. Blebbing was observed in calvarial cells from wildtype but not P2X7 knock-out mice. P2X7 receptors coupled to activation of phospholipase D and A2, inhibition of which suppressed BzATP-induced blebbing. Activation of these phospholipases leads to production of lysophosphatidic acid (LPA). LPA caused dynamic blebbing in osteoblasts from both wild-type and P2X7 knock-out mice, similar to that induced by BzATP in wildtype cells. However, LPA-induced blebbing was more rapid in onset and was not affected by inhibition of phospholipase D or A2. Blockade or desensitization of LPA receptors suppressed blebbing in response to LPA and BzATP, without affecting P2X7-stimulated pore formation. Thus, LPA functions downstream of P2X7 receptors to induce membrane blebbing. Furthermore, inhibition of Rho-associated kinase abolished blebbing induced by both BzATP and LPA. In summary, we propose a novel signaling axis that links P2X7 receptors through phospholipases to production of LPA and activation of Rho-associated kinase. This pathway may contribute to P2X7-stimulated osteogenesis during skeletal development and mechanotransduction.     

Split-enzyme fragment as a single affinity tag that enables protein expression,purification, and functional assays

Expressing, isolating, and characterizing recombinant proteins is crucial to many disciplines within the biological sciences. Different molecular tagging technologies have been developed to enable each individual step of protein production, from expression through purification and characterization. Monitoring the entire production process requires multiple tags or molecular interactions, because no individual tag has provided the comprehensive breadth of utility. An ideal molecular tag is small and does not interrupt expression, solubility, folding or function of the protein being purified and can be used throughout the production process. We adapted and integrated a split-luciferase system (NanoBiT®, Promega ®) to perform the range of techniques essential to protein production. We developed a simple method to monitor protein expression in real time to optimize expression conditions. We constructed a novel affinity chromatography system using the split-luciferase system to enable purification. We adapted western blot analysis, enzyme-linked immunosorbent assay, and cell-based bioassay to characterize the expressed proteins. Our results demonstrate that a single-tag can fulfill all aspects needed throughout protein production.     

Distribution of the flavohaemoglobin, HMP, between periplasm and cytoplasm in Escherichia coli

Abstract The subcellular distribution of the soluble flavohaemoglobin (HMP) of Escherichia coli has been determined. Cells over-expressing HMP from the cloned hmp gene on a multicopy plasmid were fractionated by osmotic shock and lysozyme treatment. Spectral analysis of subcellular fractions showed the CO-binding haemoprotein to be cytoplasmic. However, Western blotting using antibody raised to purified HMP revealed approximately 30% of the protein to be periplasmic in the over-expressing strain. Western analysis also revealed substantial levels of periplasmic HMP in a strain expressing only chromosomally encoded protein but none in an hmp mutant. The results are discussed in relation to protein function and the similar distribution reported for Vitreoscilla globin.