Proteomic profiling and characterization of differential allergens in the nematodes Anisakis simplex sensu stricto and A. pegreffii

The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In this study, the relationship of this Anisakis species complex and their allergenic capacities is assessed by studying the differences between the two most frequent species (A. simplex s.s., A. pegreffii) and their hybrid haplotype by studying active L3 larvae parasiting Merluccius merluccius. They were compared by 2D gel electrophoresis and parallel Western blot (2DE gels were hybridized with pools of sera from Anisakis allergenic patients). Unambiguous spot differences were detected and protein assignation was made by MALDI‐TOF/TOF analysis or de novo sequencing. Seventy‐five gel spots were detected and the corresponding proteins were identified. Differentially expressed proteins for A. simplex s.s., A. pegreffii, and their hybrid are described and results are statistically supported. Twenty‐eight different allergenic proteins are classified according to different families belonging to different biological functions. These proteins are described for the first time as antigenic and potentially new allergens in Anisakis. Comparative proteomic analyses of allergenic capacities are useful for diagnosis, epidemiological surveys, and clinical research. All MS data have been deposited in the ProteomeXchange with identifier PXD000662 ( http://proteomecentral.proteomexchange.org/dataset/PXD000662 ).     

Structural Characterization of a Gcn5-Related N-Acetyltransferase from Staphylococcus aureus

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphyloccocus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic β-bulge splayed at the β-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.     

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.     

Recent Findings in the Chemistry of Odorants from Four Baccharis Species and Their Impact as Chemical Markers

Baccharis is a widespread genus belonging to the Asteraceae family that includes almost 400 species exclusively from the Americas. Even when studied in detail, the taxonomic classification among species from this genus is not yet fully defined. Within the framework of our study of the volatile composition of the Baccharis genus, four species (B. trimera, B. milleflora, B. tridentata, and B. uncinella) were collected from the ‘Campos de Cima da Serra’ highlands of the Brazilian state of Rio Grande do Sul. The aerial parts were dried and extracted by the simultaneous distillation extraction (SDE) procedure. This is the first time that SDE has been applied to obtain and compare the volatile‐extract composition in the Baccharis genus. Characterization of the volatile extracts allowed the identification of 180 peaks with many coeluting components; these latter being detailed for the first time for this genus. The multivariate statistical analyses allowed separating the volatile extracts of the four populations of Baccharis into two separate groups. The first one included the B. milleflora, B. trimera, and B. uncinella volatile extracts. The three species showed a high degree of similarity in their volatile composition, which was characterized by the presence of high contents of sesquiterpene compounds, in particular of spathulenol. The second group comprised the extract of B. tridentata, which contained α‐pinene, β‐pinene, limonene, and (E)‐β‐ocimene in high amounts.     

Improved strategies for electrochemical 1,4-NAD(P)H2 regeneration: A new era of bioreactors for industrial biocatalysis

Industrial enzymatic reactions requiring 1,4-NAD(P)H₂ to perform redox transformations often require convoluted coupled enzyme regeneration systems to regenerate 1,4-NAD(P)H₂ from NAD(P) and recycle the cofactor for as many turnovers as possible. Renewed interest in recycling the cofactor via electrochemical means is motivated by the low cost of performing electrochemical reactions, easy monitoring of the reaction progress, and straightforward product recovery. However, electrochemical cofactor regeneration methods invariably produce adventitious reduced cofactor side products which result in unproductive loss of input NAD(P). We review various literature strategies for mitigating adventitious product formation by electrochemical cofactor regeneration systems, and offer insight as to how a successful electrochemical bioreactor system could be constructed to engineer efficient 1,4-NAD(P)H₂-dependent enzyme reactions of interest to the industrial biocatalysis community.     

Histones and DNA Compete for Binding Polyphosphoinositides in Bilayers

Recent discoveries on the presence and location of phosphoinositides in the eukaryotic cell nucleoplasm and nuclear membrane prompted us to study the putative interaction of chromatin components with these lipids in model membranes (liposomes). Turbidimetric studies revealed that a variety of histones and histone combinations (H1, H2AH2B, H3H4, octamers) caused a dose-dependent aggregation of phosphatidylcholine vesicles (large unilamellar vesicle or small unilamellar vesicle) containing negatively charged phospholipids. 5 mol % phosphatidylinositol-4-phosphate (PIP) was enough to cause extensive aggregation under our conditions, whereas with phosphatidylinositol (PI) at least 20 mol % was necessary to obtain a similar effect. Histone binding to giant unilamellar vesicle and vesicle aggregation was visualized by confocal microscopy. Histone did not cause vesicle aggregation in the presence of DNA, and the latter was able to disassemble the histone-vesicle aggregates. At DNA/H1 weight ratios 0.1–0.5 DNA- and PIP-bound H1 appear to coexist. Isothermal calorimetry studies revealed that the PIP-H1 association constant was one order of magnitude higher than that of PI-H1, and the corresponding lipid/histone stoichiometries were ∼0.5 and ∼1, respectively. The results suggest that, in the nucleoplasm, a complex interplay of histones, DNA, and phosphoinositides may be taking place, particularly at the nucleoplasmic reticula that reach deep within the nucleoplasm, or during somatic and nonsomatic nuclear envelope assembly. The data described here provide a minimal model for analyzing and understanding the mechanism of these interactions.     

Volatile Constituents from Baccharis spp. L. (Asteraceae): Chemical Support for the Conservation of Threatened Species in Uruguay

Chemical bioprospecting is an important tool for generating knowledge regarding local human‐threatened floras and for conservation management. For Baccharis L. (Asteraceae), several volatile components have been reported for Brazil, Argentina, Bolivia, and Chile as a result of bioprospection, but not for Uruguayan flora, which is composed of more than 50 native species. In this work, through collection of aerial parts of different species and volatile simultaneous‐distillation extraction and gas chromatography‐mass spectrometry analyses, twelve native species of Baccharis were studied (B. articulata, B. cultrata, B. genistifolia, B. gibertii, B. gnaphalioides, B. ochracea, B. phyteumoides, B. punctulata, B. crispa, B. dracunculifolia, B. linearifolia subsp. linearifolia, and B. spicata). A detailed analysis of the male and female volatile composition was conducted for the last four species. The profiles of B. cultrata, B. genistifolia, B. gibertii, and B. gnaphalioides are reported for the first time. Because half of the species analyzed in this work are in Uruguay and are threatened or potentially threatened by human economic activities, the importance of their conservation as natural, sustainable resources is highlighted.     

Circadian preference and decision-making styles

The present study aimed to explore for the first time the relationship between circadian preference and different decision-making styles. In total, 501 young adults (330 females), with a mean age of 21.07 ± 1.99 years, took part in the study. The participants completed the reduced version of the morningness–eveningness questionnaire (rMEQ) and the general decision-making style inventory (GDMS). The rMEQ enabled to assess the circadian preference, with lower rMEQ scores pointing toward eveningness preference. The GDMS measured five decision-making styles: rational, intuitive, dependent, avoidant and spontaneous. Higher scores on each GDMS decision-making style reflect a higher prevalence of the corresponding style. A set of multiple regression analyses was performed with rMEQ score, gender and age as predictors together with each GDMS decision-making style as dependent variable. rMEQ score proved to be the only significant (negative) predictor of avoidant and spontaneous decision-making styles, i.e. lower rMEQ score (tendency toward eveningness) significantly predicted higher score at these decision-making styles. The present results suggest that eveningness preference is significantly related to avoidant and spontaneous decision-making styles in young adults. Such results will be discussed with reference to the effects of decision-making styles on decision-making in different types of workers and mental health.