Modulating O2 reactivity in a fungal flavoenzyme: involvement of aryl-alcohol oxidase Phe-501 contiguous to catalytic histidine

Aryl-alcohol oxidase (AAO) is a flavoenzyme responsible for activation of O₂ to H₂O₂ in fungal degradation of lignin. The AAO crystal structure shows a buried active site connected to the solvent by a hydrophobic funnel-shaped channel, with Phe-501 and two other aromatic residues forming a narrow bottleneck that prevents the direct access of alcohol substrates. However, ligand diffusion simulations show O₂ access to the active site following this channel. Site-directed mutagenesis of Phe-501 yielded a F501A variant with strongly reduced O₂ reactivity. However, a variant with increased reactivity, as shown by kinetic constants and steady-state oxidation degree, was obtained by substitution of Phe-501 with tryptophan. The high oxygen catalytic efficiency of F501W, ∼2-fold that of native AAO and ∼120-fold that of F501A, seems related to a higher O₂ availability because the turnover number was slightly decreased with respect to the native enzyme. Free diffusion simulations of O₂ inside the active-site cavity of AAO (and several in silico Phe-501 variants) yielded >60% O₂ population at 3–4 Å from flavin C4a in F501W compared with 44% in AAO and only 14% in F501A. Paradoxically, the O₂ reactivity of AAO decreased when the access channel was enlarged and increased when it was constricted by introducing a tryptophan residue. This is because the side chain of Phe-501, contiguous to the catalytic histidine (His-502 in AAO), helps to position O₂ at an adequate distance from flavin C4a (and His-502 Nϵ). Phe-501 substitution with a bulkier tryptophan residue resulted in an increase in the O₂ reactivity of this flavoenzyme.     

Transport according to GARP: receiving retrograde cargo at the trans-Golgi network

Tethering factors are large protein complexes that capture transport vesicles and enable their fusion with acceptor organelles at different stages of the endomembrane system. Recent studies have shed new light on the structure and function of a heterotetrameric tethering factor named Golgi-associated retrograde protein (GARP), which promotes fusion of endosome-derived, retrograde transport carriers to the trans-Golgi network (TGN). X-ray crystallography of the Vps53 and Vps54 subunits of GARP has revealed that this complex is structurally related to other tethering factors such as the exocyst, the conserved oligomeric Golgi (COG) and Dsl1 (dependence on SLY1-20) complexes, indicating that they all might work by a similar mechanism. Loss of GARP function compromises the growth, fertility and/or viability of the defective organisms, emphasizing the essential nature of GARP-mediated retrograde transport.     

Immunological Characterization of the Teleost Adipose Tissue and Its Modulation in Response to Viral Infection and Fat-Content in the Diet

The immune response of the adipose tissue (AT) has been neglected in most animal models until recently, when the observations made in human and mice linking obesity to chronic inflammation and diabetes highlighted an important immune component of this tissue. In the current study, we have immunologically characterized the AT for the first time in teleosts. We have analyzed the capacity of rainbow trout (Oncorhynchus mykiss) AT to produce different immune mediators and we have identified the presence of local populations of B lymphocytes expressing IgM, IgD or IgT, CD8α⁺ cells and cells expressing major histocompatibility complex II (MHC-II). Because trout AT retained antigens from the peritoneal cavity, we analyzed the effects of intraperitoneal infection with viral hemorrhagic septicemia virus (VHSV) on AT functionality. A wide range of secreted immune factors were modulated within the AT in response to VHSV. Furthermore, the viral infection provoked a significant decrease in the number of IgM⁺ cells which, along with an increased secretion of IgM in the tissue, suggested a differentiation of B cells into plasmablasts. The virus also increased the number of CD8α⁺ cells in the AT. Finally, when a fat-enriched diet was fed to the fish, a significant modulation of immune gene expression in the AT was also observed. Thus, we have demonstrated for the first time in teleost that the AT functions as a relevant immune tissue; responsive to peritoneal viral infections and that this immune response can be modulated by the fat-content in the diet.     

8-Carbon oxylipins inhibit germination and growth, and stimulate aerial conidiation in Aspergillus nidulans

Germination of Aspergillus nidulans conidia in liquid cultures was progressively inhibited at inoculum loads above 1 × 10⁵ conidia mL^?1. High conidial densities also inhibited growth of neighbouring mycelia. The eight-carbon oxylipin 1-octen-3-ol was identified as the main inhibitor in a fraction also containing 3-octanone and 3-octanol.These three oxylipins also increased the conidiation rate of dark-grown surface cultures, but had no effect on liquid cultures. 3-octanone was the most conidiogenic compound. The action of 3-octanone required functional forms of developmental activators fluG, flbB-D and brlA, and was not additive to the conidiogenic effect of stress stimuli such as osmotic stress or carbon starvation.Oxylipins were produced shortly after hyphae made contact with the atmosphere and were most effective on aerial mycelia, indicating that they perform their signalling function in the gas phase.     

A state-of-the-art review on nitrous oxide control from waste treatment and industrial sources

This review aims at holistically analyzing the environmental problems associated with nitrous oxide (N₂O) emissions by evaluating the most important sources of N₂O and its environmental impacts. Emissions from wastewater treatment processes and the industrial production of nitric and adipic acid represent nowadays the most important anthropogenic point sources of N₂O. Therefore, state-of-the-art strategies to mitigate the generation and release to the atmosphere of this greenhouse and O₃-depleting gas in the waste treatment and industrial sectors are also reviewed. An updated review of the end-of-the-pipe technologies for N₂O abatement, both in the waste treatment and industrial sectors, is herein presented and critically discussed for the first time. Despite the consistent efforts recently conducted in the development of cost-efficient and eco-friendly N₂O abatement technologies, physical/chemical technologies still constitute the most popular treatments for the control of industrial N₂O emissions at commercial scale. The recent advances achieved on biological N₂O abatement based on heterotrophic denitrification have opened new opportunities for the development of eco-friendly alternatives for the treatment of N₂O emissions. Finally, the main limitations and challenges faced by these novel N₂O abatement biotechnologies are identified in order to pave the way for market implementation.     

Grassland connectivity explains entomophilous plant species assemblages in an agricultural landscape of the Pampa Region,Argentina

The Pampa grassland of Argentina is one of the most highly threatened biomes in the world. A high proportion of the original grassland cover has been transformed into land for agriculture or degraded. In the southern part of the region, fragmented semi‐natural grasslands over exposed rock still persist and connectivity between them is assumed to be crucial for maintaining viable populations. We quantified overall connectivity of grassland patches in a sector of the Southern Pampa region, and investigated the degree to which landscape connectivity explains entomophilous plant species assemblages in a subset of patches. We characterized each of the 301 patches in the landscape by their degree of intra‐patch and inter‐patch connectivity based on graph theory, and considering threshold dispersal distances from 100 to 1000 m. We surveyed entomophilous plant species in 39 grassland patches and classified the species in three categories (annual herbs, perennial herbs and shrubs) considering their different growth form and longevity. The influence of connectivity variables on entomophilous plant species assemblages variation was explored using Canonical Correspondence Analysis. Although grassland patches were poorly connected at all threshold distances, some of them were found to be critical for global connectivity. Connectivity significantly explained total, annual‐biennial and shrub assemblages for all threshold dispersal distances (6–13% of total variation). Variation in annual species assemblages was associated with intra‐patch and inter‐patch connectivity at short distance (100 m), while variation in shrub species assemblages was explained by intra‐patch and inter‐patch connectivity for distances between 100 m and 1000 m. This study evidenced the low connectivity of the study system, allowed the identification of critical areas for conservation, and provided valuable information to develop management strategies in increasingly human‐dominated landscapes.     

USP30 deubiquitylates mitochondrial Parkin substrates and restricts apoptotic cell death

Mitochondria play a pivotal role in the orchestration of cell death pathways. Here, we show that the control of ubiquitin dynamics at mitochondria contributes to the regulation of apoptotic cell death. The unique mitochondrial deubiquitylase, USP30, opposes Parkin-dependent ubiquitylation of TOM20, and its depletion enhances depolarization-induced cell death in Parkin-overexpressing cells. Importantly, USP30 also regulates BAX/BAK-dependent apoptosis, and its depletion sensitizes cancer cells to BH3-mimetics. These results provide the first evidence for a fundamental role of USP30 in determining the threshold for mitochondrial cell death and suggest USP30 as a potential target for combinatorial anti-cancer therapy.