Analysis of the ergosterol biosynthesis pathway cloning,molecular characterization and phylogeny of lanosterol 14 α-demethylase (ERG11) gene of Moniliophthora perniciosa

The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches’ broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.     

A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed In Vitro during Recombinant Baculovirus Infection

Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells.     

DROSOPHILA S2 cell culture in a WAVE Bioreactor: potential for scaling up the production of the recombinant rabies virus glycoprotein

Applied Microbiology and Biotechnology - The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal...     

Distribution of goblet and endocrine cells in the intestine: A comparative study in Amazonian freshwater Tambaqui and hybrid catfish

Goblet cells (GCs) and endocrine cells (ECs) play an important role in intestine physiology, and few studies currently exist for Amazonian fishes. This study aimed to quantify the distribution of GCs and ECs producing cholecystokinin-8 and neuropeptide Y, assessed by mucin histochemistry and peptides immunohistochemistry, in the intestine of two Amazonian species with different feeding habits Tambaqui (Colossosoma macropomum) and hybrid catfish (Pseudoplatystoma reticulatum × Leiarius marmoratus), an omnivore and carnivore, respectively. A systematic literature review correlating feeding habit and GC and EC distribution was also included to contribute to the comparative study. The results of this study provided novel information about the gut cells of Tambaqui and hybrid catfish. Both, GCs and ECs can be found sweeping the entire intestine of Tambaqui and hybrid catfish although the cells can be more concentrated in certain segments. The GCs and ECs in Tambaqui were more uniformly distributed in the midgut segments (T1, T2, and T3). Unlike, in hybrid catfish GCs were more concentrated in the hindgut (C4) and ECs mainly in the two midgut segments (C1 and C2) of hybrid catfish. Based on the comparison between Tambaqui, hybrid catfish, and other fishes in the literature review, we suggest that cell distribution can be partially explained by feeding habits, carnivorous vs. omnivorous.     

Acute effect of an amino acid mixture in the rat glycemic profile

Amino acid mixtures (AAM) are protein substitutes used for phenylketonuria treatment, but their metabolic effects have not been well characterized. The objective of this study was to compare the acute glycemic response to free amino acids (free AA) from AAM with the response to intact protein (iProtein). Male Wistar rats (n = 14) were administered by gavage a bolus of free AA (n = 7) or iProtein as albumin (n = 7) containing equivalent amounts of nitrogen. Blood glucose and insulin levels were measured at baseline and 15, 30, 60 and 120 minutes later, when gut GLP-1 content and pancreatic insulin, GLP-1 receptor and Ki67 expression were quantified at 120 minutes time point. After AAM, glucose area under the curve (free AA vs iProtein; P < 0.01), serum insulin levels at 120 minutes (free AA vs iProtein; P < 0.05), colon GLP-1 content (free AA vs iProtein; P < 0.01), pancreatic GLP-1 receptor (free AA vs iProtein; P < 0.01) and insulin expression (free AA vs iProtein; p < 0.01) were significantly lower as compared with iProtein. AAM increased Ki67 expression in pancreatic islets (free AA vs iProtein; P < 0.05). In conclusion, this study demonstrated that acute response to AAM differs from iProtein and is characterized by a lower glucose excursion, along with a decrease in gut GLP-1 and pancreatic GLP-1 receptor and insulin. This data suggests the modulation of glycemia by free AA is mediated by the incretin axis.     

Recombinant β-1,3-1,4-glucanase from Theobroma cacao impairs Moniliophthora perniciosa mycelial growth

In this work, we identified a gene from Theobroma cacao L. genome and cDNA libraries, named TcGlu2, that encodes a β-1,3-1,4-glucanase. The TcGlu2 ORF was 720 bp in length and encoded a polypeptide of 239 amino acids with a molecular mass of 25.58 kDa. TcGlu2 contains a conserved domain characteristic of β-1,3-1,4-glucanases and presented high protein identity with β-1,3-1,4-glucanases from other plant species. Molecular modeling of TcGlu2 showed an active site of 13 amino acids typical of glucanase with β-1,3 and 1,4 action mode. The recombinant cDNA TcGlu2 obtained by heterologous expression in Escherichia coli and whose sequence was confirmed by mass spectrometry, has a molecular mass of about 22 kDa (with His-Tag) and showed antifungal activity against the fungus Moniliophthora perniciosa, causal agent of the witches’ broom disease in cacao. The integrity of the hyphae membranes of M. perniciosa, incubated with protein TcGlu2, was analyzed with propidium iodide. After 1 h of incubation, a strong fluorescence emitted by the hyphae indicating the hydrolysis of the membrane by TcGlu2, was observed. To our knowledge, this is the first study of a cacao β-1,3-1,4-glucanase expression in heterologous system and the first analysis showing the antifungal activity of a β-1,3-1,4-glucanase, in particular against M. perniciosa.     

The effect of species composition dissimilarity on plant–herbivore network structure is not consistent over time

The structural organization of several antagonistic networks has been demonstrated to be largely conserved through time and space even when species beta-diversity is high. This might occur either because species are replaced by others that fulfill similar network roles or because interaction probabilities are given by species relative abundances rather than by their functional traits. Alternatively, if species-specific traits are important drivers of realized interactions, any change in species composition should promote a certain degree of network structural dissimilarity. Here, we used a spatial-temporal system comprising asteraceous plants and flower head herbivores from remnants of Brazilian Cerrado to investigate whether the relationship between spatial beta-diversity of species and network structural dissimilarity changes over time. We measured species beta-diversity using Sørensen's dissimilarity index (β_sor) and its components of species replacement (β_-3) and richness differences (β_rich). Network structural dissimilarity was estimated using three different metrics: connectance, modularity, and web asymmetry. We show that, in general, the effect of species beta-diversity on network structure was time-dependent: While some periods presented a positive relationship between spatial beta-diversity and network structural dissimilarity, others presented no significant relationship. This indicates that functionally similar species may present different turnover rates at distinct periods, and different non-exclusive processes affect plant–herbivore network organization across time.