Assay of inorganic and organic phosphorus in the 0.1–5 nanomole range

Climate change-driven increases in aridity will lead to dryland expansion worldwide. In the Atlantic and Mediterranean biogeographic regions, coastal dunes are priority conservation areas highly sensitive to aridification where plants and biological soil crusts may play a key role maintaining ecosystem services. However, we still need further insights into the balance between the effects of increasing aridity and the influence of local vegetation on the soil attributes and functioning of these systems, particularly in transitional areas between humid and arid climatic zones. We sampled 24 dune systems distributed along a natural climate gradient including humid, dry-subhumid and arid environments in the Atlantic coastline of the Iberian Peninsula and investigated (i) how aridity drives the vegetation community structure, soil biotic and abiotic properties and functions in dune ecosystems at a regional scale; and (ii) to what extent soil attributes differ locally under diverse surface microsites (i.e. plants, biocrust and bare soil). Increasing aridity in coastal dunes decreased total biotic cover and favoured the formation of shrub-dominated patches. Vegetation shifts were accompanied by declines in soil microbial diversity, organic matter, nutrient contents and reduced rates of N mineralization. Soil bacterial communities were more sensitive to aridity than fungal communities. Microsites were essential regulators of soil attributes, and their effects were dependent on the degree of aridity. In drylands, shrub microsites were associated to higher soil organic C, microbial (bacterial and fungal) abundance, fungal diversity and potential heterotrophic respiration than bare microsites, likely due to enhanced litter and sediment redistribution. However, at humid and transition systems, microsites controlled soil N pools and microbial diversity, suggesting tight linkages between vegetation patches and soil microorganisms responsible for N availability. These findings provide evidence of the strong negative impacts of aridification on vegetation attributes and nutrient cycling in coastal dunes and highlight the role of the biotic cover in preserving soil fertility, microbial diversity and functioning, particularly in the most arid sites.

The ClpB–DnaK bichaperone system reactivates aggregated cellular proteins and is essential for survival of bacteria, fungi, protozoa, and plants under stress. AAA+ ATPase ClpB is a promising target for the development of antimicrobials because a loss of its activity is detrimental for survival of many pathogens and no apparent ClpB orthologs are found in metazoans. We investigated ClpB activity in the presence of several compounds that were previously described as inhibitor leads for the human AAA+ ATPase p97, an antitumor target. We discovered that N²,N⁴-dibenzylquinazoline-2,4-diamine (DBeQ), the least potent among the tested p97 inhibitors, binds to ClpB with a K_d∼60 μM and inhibits the casein-activated, but not the basal, ATPase activity of ClpB with an IC₅₀∼5 μM. The remaining p97 ligands, which displayed a higher affinity toward p97, did not affect the ClpB ATPase. DBeQ also interacted with DnaK with a K_d∼100 μM and did not affect the DnaK ATPase but inhibited the DnaK chaperone activity in vitro. DBeQ inhibited the reactivation of aggregated proteins by the ClpB–DnaK bichaperone system in vitro with an IC₅₀∼5 μM and suppressed the growth of cultured Escherichia coli. The DBeQ-induced loss of E. coli proliferation was exacerbated by heat shock but was nearly eliminated in a ClpB-deficient E. coli strain, which demonstrates a significant selectivity of DBeQ toward ClpB in cells. Our results provide chemical validation of ClpB as a target for developing novel antimicrobials. We identified DBeQ as a promising lead compound for structural optimization aimed at selective targeting of ClpB and/or DnaK.

Integrated analytical approaches can be used to determine the trace concentrations of analytes in water matrices. Here we designed and compared two novel and facile, integrated polysaccharide adsorbent SPE-Self assembled MEPS (solid phase extraction and self-assembled microextraction by packed sorbent) protocols based on chitin, and cellulose acetate respectively, for simultaneously determining the ultralow concentrations of three mononitrophenols (MNPs) in water. In addition, we reported computational, and empirical, interaction profiling, for providing insights into the possible interactions among chitin-MNP, and cellulose acetate-MNP, respectively. The integrated SPE-MEPS analytical platform enhances the MNPs detection limits, scalability and recoveries from individual water matrices with the additional ease of use. Aside from offering improved control over lower scalability range (0.05–5 ng/mL) with both adsorbents Chitin and CA as compared to previous analogues reported methods, limits of detection (LODs) were as low as 0.01–0.03 ng/mL with Chitin and 0.008–0.025 ng/mL with CA, in quality control samples. By integrating SPE with MEPS, and chromatographic separations with high performance liquid chromatography-ultra violet detector (HPLC-UV), we were able to determine three MNPs at ppt (pg/mL) levels in different water matrices. This approach may find diverse applications in water monitoring at trace levels. Finally, and most importantly, to the best of authors knowledge, this study marks the first time that integrated SPE-MEPS analytical platform has been used for the efficient trace analysis in analytical chemistry.

Melittin (ML) is a small peptide of 26 residues rich in arginine (Arg) and lysine (Lys). Several studies have been done to understand the mechanism of interaction with neutral and negatively charged lipids. However, it is not known with certainty how this interaction depends on the electrostatic or hydrophobic forces according to the composition of the membrane, nor with the different organization of lipids on the membrane, such as the microdomains that could take place in it. Therefore, comparative studies of the interaction and the effect of ML with respect to cationic peptides (Arg-7 and Lys-5) were conducted to get a deeper insight of the ML interaction mechanism with membranes. In this regard, measurements of zeta potential of different model membranes (DOPC, DMPC and DMPE liposomes) in the presence of the peptides, and molecular dynamics simulations were performed. In the special case of DMPC, we worked in its gel like-ripple phase, in order to analyzed defects of packing that potentially expose hydrocarbon regions.

In relation with experimental results, molecular analysis of ML interaction with zwiterionic lipid membrane in its ripple phase was performed by unbiased molecular dynamics simulations.

The results allow us to remark that ML penetration is favored in the gel-liquid crystalline phase transition in zwitterionic lipids. The importance of this study lies in the understanding of the first stages of action of the ML in eukaryotic membranes, in model systems of its main lipid composition.

Membrane proteins are notoriously difficult to investigate in isolation. The focus of this chapter is the key step following extraction and purification of membrane proteins; namely reconstitution. The process of reconstitution re-inserts proteins into a lipid bilayer that partly resembles their native environment. This native environment is vital to the stability of membrane proteins, ensuring that they undergo vital conformational transitions and maintain optimal interaction with their substrates. Reconstitution may take many forms and these have been classified into two broad categories. Symmetric systems enable unfettered access to both sides of a bilayer. Compartment containing systems contain a lumen and are ideally suited to measurement of transport processes. The investigator is encouraged to ascertain what aspects of protein function will be undertaken and to apply the most advantageous reconstitution system or systems. It is important to note that the process of reconstitution is not subject to defined protocols and requires empirical optimisation to specific targets.

The herbicide Fusilade Forte^® (FF) is widely applied in agricultural weed management and in the management of the invasive saltmarsh grass, Spartina anglica (ricegrass or cordgrass). FF (active ingredient fluazifop-P acid, FPA) is selective for poaceous grasses. Its primary mode of action is inhibition of the acetyl coenzyme-A carboxylase (ACCase) specific to this taxonomic group, and its secondary mode is by promotion of oxidative stress. FF is applied to S. anglica infestations in the intertidal zone, in proximity to seagrass meadows. Despite the potential for vital seagrass ecosystems to be exposed to FF, there is limited knowledge of any potential impacts. We investigated impacts of FPA on the endemic Australian seagrass, Zostera nigricaulis, measuring ACCase activity and parameters that reflect oxidative stress: photosynthetic performance, lipid peroxidation and photosynthetic pigment content. Seagrass was exposed to FF (0.01–10 mg L⁻¹ FPA and a control) for 7 d, followed by a 7-d recovery in uncontaminated seawater. An enzyme assay demonstrated that FPA ≤10 mg L⁻¹ did not inhibit the activity of ACCase isolated from Z. nigricaulis, demonstrating that this seagrass is resistant to FF's primary mode of action. However, physiological impacts occurred following 7 days exposure to ≥0.1 mg L⁻¹ FPA, including up to a 72% reduction in photosynthetic pigment concentration. After 7-d recovery, photosynthetic pigment content improved in treatment plants; however, treated plants exhibited higher levels of lipid peroxidation. This study demonstrates that while Z. nigricaulis is resistant to FF's primary mode of action, significant physiological impacts occur following 7 days exposure to ≥0.1 mg L⁻¹ FPA. This study provides valuable information on the effects of FF on a non-target species that can better inform approaches to Spartina management in coastal seagrass ecosystems.