Mechanism of Action of Secreted Newt Anterior Gradient Protein

Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.     

MyROOT: a method and software for the semiautomatic measurement of primary root length in Arabidopsis seedlings

Root analysis is essential for both academic and agricultural research. Despite the great advances in root phenotyping and imaging, calculating root length is still performed manually and involves considerable amounts of labor and time. To overcome these limitations, we developed MyROOT, a software for the semiautomatic quantification of root growth of seedlings growing directly on agar plates. Our method automatically determines the scale from the image of the plate, and subsequently measures the root length of the individual plants. To this aim, MyROOT combines a bottom‐up root tracking approach with a hypocotyl detection algorithm. At the same time as providing accurate root measurements, MyROOT also significantly minimizes the user intervention required during the process. Using Arabidopsis, we tested MyROOT with seedlings from different growth stages and experimental conditions. When comparing the data obtained from this software with that of manual root measurements, we found a high correlation between both methods (R² = 0.997). When compared with previous developed software with similar features (BRAT and EZ‐Rhizo), MyROOT offered an improved accuracy for root length measurements. Therefore, MyROOT will be of great use to the plant science community by permitting high‐throughput root length measurements while saving both labor and time.     

Morphogenesis and in vitro production of caffeoylquinic and caffeic acids in Baccharis conferta Kunth

We established a protocol for the in vitro propagation of Baccharis conferta Kunth. This plant is used to treat gastrointestinal problems, cramps, pain, respiratory problems, and insect bites. A high rate of shoot multiplication was obtained from nodal segments on Murashige and Skoog (MS) culture medium. The shoots regenerated roots without exogenous plant growth regulators (PGRs). All explants of wild leaves on MS medium containing 5 μM of thidiazuron (TDZ) produced friable callus. An organogenic response was achieved after 3 wk of culture when callus segments were transferred to MS medium containing a combination of plant growth regulators (PGRs): either (i) 5 μM indole butyric acid (IBA) + 5 μM kinetin (KIN) or (ii) 0.5 μM IBA + 1.10 μM benzylaminopurine (BAP). The morphogenetic responses of callus were characterized by scanning electron microscopy. Shoots regenerated from callus and formed roots on MS medium without PGRs. The micropropagated plantlets and the organogenic callus showed similar chemical profiles in HPLC-mass spectrometry analyses. The main compounds present in the cultures were caffeoylquinic acids. Only plantlets contained small amounts of triterpenes (erythrodiol and ursolic acid). These findings will be useful for the micropropagation of this important native resource, and for further studies on its biology.

     

Leaf litter stoichiometry affects decomposition rates and nutrient dynamics in tropical forests under restoration in Costa Rica

Active restoration strategies increase the production of leaf litter in tropical forests, but little is known about their effect on litter decomposition and subsequent nutrient release. We quantified changes in leaf litter stoichiometry during decomposition in former pasture sites under contrasting restoration strategies (natural regeneration, applied nucleation/islands tree planting and plantation), as well as in nearby primary forest. Litterbags were employed to evaluate decomposition. We used a leaf mixture of either the four planted tree species in the plantation and island treatments or the nearby primary forest and compared them under a factorial design. Decomposition rates were similar between restoration treatments (p > 0.5), but leaves decomposed faster in the forest mixture than in the plantation mixture (p < 0.01). The content of Ca, Mg, K, P, and the C:N ratio were higher in the forest mixture at the beginning and during decomposition (p < 0.05); the N content in the plantation mixture was higher at the beginning but lower during decomposition (p < 0.05), which meant greater mobilization of nitrogen per unit of carbon lost. K and P had a strong initial release, while Mg was released more gradually. N and Ca had an irregular pattern of initial fast release, immobilization, and re‐release in the later stages. We conclude that the differences in rates of decomposition and nutrient release in these systems under restoration were at least partly determined by the floristic heterogeneity and chemical quality of the leaf litter that reaches the soil.     

Organization Environmental Footprint through Input‐Output Analysis: A Case Study in the Construction Sector

The implementation of global sustainability has gained worldwide attention in recent years. The Organization Environmental Footprint, which encompasses 14 impact categories, is a multicriteria measure of the environmental performance of goods and services provided by an organization from a life cycle perspective. In this article, the focus is on quantifying the Organization Environmental Footprint of a construction company in Spain. By applying an environmentally extended input‐output approach, its total footprint and impacts along the supply chain from two consecutive years were calculated. The results show that the environmental impacts from the second year of implementation were significantly higher than those from the first year. The impact category climate change was found to have experienced the greatest increase from one year to the other, with a 31% increase. This work provides an overview of 14 environmental impact categories of the company assessed, as well as recommendations for the implementation of this indicator in companies and public procurement. This approach could pave the way to shape organizations’ action plans and meet the European environmental challenges.     

Enhanced preparation of adeno-associated viral vectors by using high hydrostatic pressure to selectively inactivate helper adenovirus

Gene delivery vectors based on adeno-associated virus (AAV) have significant therapeutic potential, but much room for improvement remains in the areas of vector engineering and production. AAV production requires complementation with either helper virus, such as adenovirus, or plasmids containing helper genes, and helper virus-based approaches have distinct advantages in the use of bioreactors to produce large quantities of AAV vectors for clinical applications. However, helper viruses must eventually be inactivated and removed from AAV preparations to ensure safety. The current practice of thermally inactivating adenovirus is problematic as it can also inactivate AAV. Here, we report a novel method using high hydrostatic pressure (HHP) to selectively and completely inactivate helper adenovirus without any detectable loss of functional AAV vectors. The pressure inactivation kinetics of human adenovirus serotype 5 and the high-pressure stabilities of AAV serotypes 2 and 5 (AAV2, AAV5), which were previously unknown, were characterized. Adenovirus was inactivated beyond detection at 260 MPa or higher, whereas AAV2 was stable up to approximately 450 MPa, and surprisingly, AAV5 was stable up to at least 700 MPa. The viral genomic DNA of pressure-inactivated AAV2 was made sensitive to DNAse I digestion, suggesting that gross changes in particle structure had occurred, and this hypothesis was further supported by transmission electron microscopy. This approach should be useful in the laboratory- and clinical-scale production of AAV gene delivery vectors. Moreover, HHP provides a tool for probing the biophysical properties of AAV, which may facilitate understanding and improving the functions of this important virus.