Evaluation of Acute Toxicity and Antioxidant Response of Earthworm Exposed to a Lignin-Modified Crosslinked Hydrogel.

Hydrogels are polymers of great importance due to their multiple applications, which have led to an exponential increase in their production. However, once they have fulfilled their function, they become waste and their ecotoxicological effects are unknown. The aim of the present study was to evaluate the acute toxicity and total antioxidant capacity of the earthworm (Eisenia fetida) exposed to a terpolymeric hydrogel (acrylic acid, acrylamide, and 2-acrylamido-2-methyl-1-propane-sulfonic acid) crosslinked with modified kraft lignin. Four different amounts of hydrogel per unit area were evaluated (0.0924, 0.1848, 0.9242, and 1.848 mg hydrogel/cm2) plus a control, and three replicates were performed for each group. Starting from the amount of 0.1848 mg hydrogel/cm2, the earthworms showed physiological and behavioral alterations; at higher amounts, 0.9242 and 1.848 mg hydrogel/cm2, more acute signs were observed with mortality rates of 51.7% and 100%, respectively. On the other hand, the antioxidant activity assay showed that the higher the hydrogel exposure amount, the higher the oxidative stress, as evidenced by lower antioxidant activity (67.09% inhibition of the ABTS●+ radical). Therefore, we concluded that the lignin-modified hydrogel generated oxidative stress and acute lethal toxic effects in Eisenia fetida.

Comparative study of composition, antioxidant and antimicrobial activity of two adult edible insects from Tenebrionidae family.

In the case of Tenebrionidae family insects, studies focus on larval stage, leaving a lack of information regarding other stages. Therefore, this study was performed in order to understand the differences between the nutritional composition and the bioactivity of two species of this family in their adult stage, fed with a specific diet. Adult beetles of both species were defatted, lyophilized and protein extracted with buffer. Proximal and phytochemical analysis of the extracts of each insect were performed, along with protein extract and hydrolysis analysis by Tris-Tricine and Tris Glycine SDS PAGE. This analysis showed that T. molitor contained more protein and fat than U. dermestoides but contained less crude fiber. The protein extraction was made with PBS, where 130 and 45 kDa bands showed predominant for U. dermestoides, and less protein was present for T. molitor. Antioxidant and antimicrobial activities of the enzymatic protein hydrolysates and protein crude extracts were determined. Presence of protein associated with the antioxidant activity were found in both insects. Nonetheless U. dermestoides had a higher antioxidant activity with the protein extract in contrast with the higher antioxidant activity shown by U. dermestoides once the extracts were digested. After proteolysis, protein extracts showed an increasing antioxidant activity, plus, the ability to inhibit microbial growth of Proteus, Shigella and Bacillus. Insect protein hydrolysates with protease open the possibility for the use of these beetles as new sources of encrypted peptides for microbiological control once characterized.

Recombinant Hemagglutinin of Avian Influenza Virus H5 Expressed in the Chloroplast of Chlamydomonas reinhardtii and Evaluation of Its Immunogenicity in Chickens.

Globally, avian influenza (AI) is a serious problem in poultry farming. Despite vaccination, the prevalence of AI in México highlights the need for new approaches to control AI and to reduce the economic losses associated with its occurrence in susceptible birds. Recombinant proteins from avian influenza virus (AIV) have been expressed in different organisms, such as plants. The present study investigated the feasibility of designing and expressing the HA protein of AIV in the transplastomic microalga Chlamydomonas reinhardtii as a novel approach for AIV control and taking advantage of culture conditions, its reproductive range, and safe use in consideration of the generally regarded as safe food ingredient regulatory classification. The results showed that the HA protein of AIV in C. reinhardtii presents antigenic activity by western blot test and through its application in chickens, demonstrating its feasibility as a recombinant antigen against AIV.

Yeast mitochondrial RNA polymerase primes mitochondrial DNA polymerase at origins of replication and promoter sequences.

Three proteins phylogenetically grouped with proteins from the T7 replisome localize to yeast mitochondria: DNA polymerase γ (Mip1), mitochondrial RNA polymerase (Rpo41), and a single-stranded binding protein (Rim1). Human and T7 bacteriophage RNA polymerases synthesize primers for their corresponding DNA polymerases. In contrast, DNA replication in yeast mitochondria is explained by two models: a transcription-dependent model in which Rpo41 primes Mip1 and a model in which double stranded breaks create free 3' OHs that are extended by Mip1. Herein we found that Rpo41 transcribes RNAs that can be extended by Mip1 on single and double-stranded DNA. In contrast to human mitochondrial RNA polymerase, which primes DNA polymerase γ using transcripts from the light-strand and heavy-strand origins of replication, Rpo41 primes Mip1 at replication origins and promoter sequences in vitro. Our results suggest that in ori1, short transcripts serve as primers, whereas in ori5 an RNA transcript longer than 29 nucleotides is used as primer.

The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding.

Single subunit RNA polymerases have evolved 2 mechanisms to synthesize long transcripts without falling off a DNA template: binding of nascent RNA and interactions with an RNA:DNA hybrid. Mitochondrial RNA polymerases share a common ancestor with T-odd bacteriophage single subunit RNA polymerases. Herein we characterized the role of the thumb subdomain of the yeast mtRNA polymerase gene (RPO41) in complex stability, processivity, and fidelity. We found that deletion and point mutants of the thumb subdomain of yeast mtRNA polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. Mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (Mtf1). The latter suggests that the thumb subdomain is part of an extended binding surface area involved in binding Mtf1.

Yeast mitochondrial RNAP conformational changes are regulated by interactions with the mitochondrial transcription factor.

Mitochondrial RNA polymerases (MtRNAPs) are members of the single-subunit RNAP family, the most well-characterized member being the RNAP from T7 bacteriophage. MtRNAPs are, however, functionally distinct in that they depend on one or more transcription factors to recognize and open the promoter and initiate transcription, while the phage RNAPs are capable of performing these tasks alone. Since the transcriptional mechanisms that are conserved in phage and mitochondrial RNAPs have been so effectively characterized in the phage enzymes, outstanding structure-mechanism questions concern those aspects that are distinct in the MtRNAPs, particularly the role of the mitochondrial transcription factor(s). To address these questions we have used both negative staining and cryo-EM to generate three-dimensional reconstructions of yeast MtRNAP initiation complexes with and without the mitochondrial transcription factor (MTF1), and of the elongation complex. Together with biochemical experiments, these data indicate that MTF1 uses multiple mechanisms to drive promoter opening, and that its interactions with the MtRNAP regulate the conformational changes undergone by the latter enzyme as it traverses the template strand.

Conservation of promoter melting mechanisms in divergent regions of the single-subunit RNA polymerases.

The single-subunit RNA polymerases make up a widespread family of proteins found in phage, mitochondria, and chloroplasts. Unlike the phage RNAPs, the eukaryotic RNAPs require accessory factors to melt their promoters and diverge from the phage RNAPs in the regions where functions associated with promoter melting in the latter have been mapped, suggesting that promoter melting mechanisms in the eukaryotic RNAPs diverge from those in the phage enzymes. However, here we show that an element in the yeast mitochondrial RNAP, identified by sequence alignment with the T7 phage RNAP, fulfills a role in promoter melting similar to that filled by the T7RNAP "intercalating hairpin". The yeast mitochondrial RNAP intercalating hairpin appears to be as important in promoter melting as the mitochondrial transcription factor, MTF1, and both a structurally integral hairpin and MTF1 are required to achieve high levels of transcription on a duplex promoter. Deletions from the hairpin also relieve MTF1 inhibition of promoter escape on premelted promoters, likely because such deletions disrupt interactions with the upstream edge of the transcription bubble. These results are consistent with recent structural and functional studies of human mitochondrial RNAP and further reveal the surprising extent of mechanistic conservation between the eukaryotic and phage-encoded members of the single-subunit RNAP family.