Respiratory versatility in Desulfovibrio desulfuricans ATCC 27774 – a proteomic approach

Poster presented at the Bacterial Electron Transfer Processes and their Regulation Meeting, European Federation of Biotechnology Microbial Physiology Section, 15-18 March 2015, Vimeiro, Portugal

Preparation of biodegradable materials by reactive extrusion

This work aimed to prepare biodegradable polymeric materials based on blends of a synthetic high density polyethylene (HDPE) and biodegradable polymers such as polylactic acid (PCL) and poly(caprolactone) (PLA), in a co-rotating twin-screw extruder. A polyethylene modified with maleic anhydride was used as compatibiliser. The mechanical results showed that the addition of PLA improves the blends stiffness while the addition of PCL leads to materials with a greater elongation at break and a lower Young modulus. This feature is related with the mechanical properties of each material as well as the adhesion between them. Concerning the biodegradability tests, it was found that HDPE/PCL blend presents the highest degree of biodegradability

A genetic code alteration generates a proteome of high diversity in the human pathogen Candida albicans

Background - Genetic code alterations have been reported in mitochondrial, prokaryotic, and eukaryotic cytoplasmic translation systems, but their evolution and how organisms cope and survive such dramatic genetic events are not understood. Results - Here we used an unusual decoding of leucine CUG codons as serine in the main human fungal pathogen Candida albicans to elucidate the global impact of genetic code alterations on the proteome. We show that C. albicans decodes CUG codons ambiguously and tolerates partial reversion of their identity from serine back to leucine on a genome-wide scale. Conclusion - Such codon ambiguity expands the proteome of this human pathogen exponentially and is used to generate important phenotypic diversity. This study highlights novel features of C. albicans biology and unanticipated roles for codon ambiguity in the evolution of the genetic code.publishe

Codon-triplet context unveils unique features of the Candida albicans protein coding genome

<p>Abstract</p> <p>Background</p> <p>The evolutionary forces that determine the arrangement of synonymous codons within open reading frames and fine tune mRNA translation efficiency are not yet understood. In order to tackle this question we have carried out a large scale study of codon-triplet contexts in 11 fungal species to unravel associations or relationships between codons present at the ribosome A-, P- and E-sites during each decoding cycle.</p> <p>Results</p> <p>Our analysis unveiled high bias within the context of codon-triplets, in particular strong preference for triplets of identical codons. We have also identified a surprisingly large number of codon-triplet combinations that vanished from fungal ORFeomes. <it>Candida albicans </it>exacerbated these features, showed an unbalanced tRNA population for decoding its pool of codons and used near-cognate decoding for a large set of codons, suggesting that unique evolutionary forces shaped the evolution of its ORFeome.</p> <p>Conclusion</p> <p>We have developed bioinformatics tools for large-scale analysis of codon-triplet contexts. These algorithms identified codon-triplets context biases, allowed for large scale comparative codon-triplet analysis, and identified rules governing codon-triplet context. They could also detect alterations to the standard genetic code.</p

Penicillium crustosum as a potential OTA producer - new insights from whole - genome sequencing of strain MUM 16.125

Ochratoxin A (OTA) is a well-studied mycotoxin that poses severe health risks. OTA is mainly produced by Aspergillus and Penicillium species associated with food spoilage and it is present in a wide diversity of food and feed products. Recent studies have reported the presence of OTA in food matrices where known OTA producers are not present1,2. For that reason, other species such as P. crustosum are now being considered. A recent study using comparative genomic analysis3 clarified the OTA biosynthetic gene cluster composition. In order to gain insight into the secondary metabolism of P. crustosum, this study aimed to sequence and explore the complete genome of strain MUM 16.125. This strain was isolated from cheese rind sample contaminated with OTA in which no known OTA producers were present1. The genome assembly comprises 199 contigs with a total length of 30.95 Mb and contains 10975 predicted protein-coding genes. In total, 109 gene clusters potentially related with secondary metabolism were identified, including putative gene clusters for penitrem, clavaric acid or naphthopyrones biosynthesis. Nevertheless, no evidence of an OTA biosynthetic gene cluster was found. A total of 83 complete and 49 partial protein sequences from published OTA biosynthetic genes from 11 Aspergillus and 3 Penicillium species were queried against the predicted P. crustosum proteins. Only 3 strong matches were found (to a short partial P. verrucosum PKS and 2 P. thymicola chloroperoxidases) but matches to complete key genes were absent. Considering these findings, it appears that strain MUM 16.125 lacks the most common genetic pathway to produce OTA, providing important information relevant to understand the role of P. crustosum as putative OTA producer. Nevertheless, the additional secondary metabolism gene clusters found (such as penitrem, clavaric acid or naphthopyrones) highlight the potential of this strain for metabolite production, including other mycotoxins or compounds with antioxidant, anticancer or antibiotic properties.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of CEB (UID/BIO/04469/2019) and iBiMED (UIDB/04501/2020) units; and by CANCYL (POCI-01-0145-FEDER-031849) and GenomePT (POCI-01-0145-FEDER-022184) projectsinfo:eu-repo/semantics/publishedVersio

Construction of effective disposable biosensors for point of care testing of nitrite

© 2015. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/"In this paper we aim to demonstrate, as a proof-of-concept, the feasibility of the mass production of effective point of care tests for nitrite quantification in environmental, food and clinical samples. Following our previous work on the development of third generation electrochemical biosensors based on the ammonia forming nitrite reductase (ccNiR), herein we reduced the size of the electrodes’ system to a miniaturized format, solved the problem of oxygen interference and performed simple quantification assays in real samples. In particular, carbon paste screen printed electrodes (SPE) were coated with a ccNiR/carbon ink composite homogenized in organic solvents and cured at low temperatures. The biocompatibility of these chemical and thermal treatments was evaluated by cyclic voltammetry showing that the catalytic performance was higher with the combination acetone and a 40 °C curing temperature. The successful incorporation of the protein in the carbon ink/solvent composite, while remaining catalytically competent, attests for ccNiR’s robustness and suitability for application in screen printed based biosensors. Because the direct electrochemical reduction of molecular oxygen occurs when electroanalytical measurements are performed at the negative potentials required to activate ccNiR (ca. -0.4 V vs Ag/AgCl), an oxygen scavenging system based on the coupling of glucose oxidase and catalase activities was successfully used. This enabled the quantification of nitrite in different samples (milk, water, plasma and urine) in a straightforward way and with small error (1 – 6%). The sensitivity of the biosensor towards nitrite reduction under optimized conditions was 0.55 A M-1 cm-2 with a linear response range 0.7 – 370 μM.

The Yeast PNC1 Longevity Gene Is Up-Regulated by mRNA Mistranslation

Translation fidelity is critical for protein synthesis and to ensure correct cell functioning. Mutations in the protein synthesis machinery or environmental factors that increase synthesis of mistranslated proteins result in cell death and degeneration and are associated with neurodegenerative diseases, cancer and with an increasing number of mitochondrial disorders. Remarkably, mRNA mistranslation plays critical roles in the evolution of the genetic code, can be beneficial under stress conditions in yeast and in Escherichia coli and is an important source of peptides for MHC class I complex in dendritic cells. Despite this, its biology has been overlooked over the years due to technical difficulties in its detection and quantification. In order to shed new light on the biological relevance of mistranslation we have generated codon misreading in Saccharomyces cerevisiae using drugs and tRNA engineering methodologies. Surprisingly, such mistranslation up-regulated the longevity gene PNC1. Similar results were also obtained in cells grown in the presence of amino acid analogues that promote protein misfolding. The overall data showed that PNC1 is a biomarker of mRNA mistranslation and protein misfolding and that PNC1-GFP fusions can be used to monitor these two important biological phenomena in vivo in an easy manner, thus opening new avenues to understand their biological relevance

Large Scale Comparative Codon-Pair Context Analysis Unveils General Rules that Fine-Tune Evolution of mRNA Primary Structure

BACKGROUND: Codon usage and codon-pair context are important gene primary structure features that influence mRNA decoding fidelity. In order to identify general rules that shape codon-pair context and minimize mRNA decoding error, we have carried out a large scale comparative codon-pair context analysis of 119 fully sequenced genomes. METHODOLOGIES/PRINCIPAL FINDINGS: We have developed mathematical and software tools for large scale comparative codon-pair context analysis. These methodologies unveiled general and species specific codon-pair context rules that govern evolution of mRNAs in the 3 domains of life. We show that evolution of bacterial and archeal mRNA primary structure is mainly dependent on constraints imposed by the translational machinery, while in eukaryotes DNA methylation and tri-nucleotide repeats impose strong biases on codon-pair context. CONCLUSIONS: The data highlight fundamental differences between prokaryotic and eukaryotic mRNA decoding rules, which are partially independent of codon usage

Dre-miR-2188 Targets Nrp2a and Mediates Proper Intersegmental Vessel Development in Zebrafish Embryos

BACKGROUND: MicroRNAs (miRNAs) are a class of small RNAs that are implicated in the control of eukaryotic gene expression by binding to the 3'UTR of target mRNAs. Several algorithms have been developed for miRNA target prediction however, experimental validation is still essential for the correct identification of miRNA targets. We have recently predicted that Neuropilin2a (Nrp2a), a vascular endothelial growth factor receptor which is essential for normal developmental angiogenesis in zebrafish, is a dre-miR-2188 target. METHODOLOGY: Here we show that dre-miR-2188 targets the 3'-untranslated region (3'UTR) of Nrp2a mRNA and is implicated in proper intersegmental vessel development in vivo. Over expression of miR-2188 in zebrafish embryos down regulates Nrp2a expression and results in intersegmental vessel disruption, while its silencing increases Nrp2a expression and intersegmental vessel sprouting. An in vivo GFP sensor assay based on a fusion between the GFP coding region and the Nrp2a 3'UTR confirms that miR-2188 binds to the 3'UTR of Nrp2a and inhibits protein translation. CONCLUSIONS: We demonstrate that miR-2188 targets Nrp2a and affects intersegmental vessel development in zebrafish embryos

Conserved and highly expressed tRNA derived fragments in zebrafish

Background: Small non-coding RNAs (sncRNAs) are a class of transcripts implicated in several eukaryotic regulatory mechanisms, namely gene silencing and chromatin regulation. Despite significant progress in their identification by next generation sequencing (NGS) we are still far from understanding their full diversity and functional repertoire. Results: Here we report the identification of tRNA derived fragments (tRFs) by NGS of the sncRNA fraction of zebrafish. The tRFs identified are 18–30 nt long, are derived from specific 5′ and 3′ processing of mature tRNAs and are differentially expressed during development and in differentiated tissues, suggesting that they are likely produced by specific processing rather than random degradation of tRNAs. We further show that a highly expressed tRF (5′tRF-ProCGG) is cleaved in vitro by Dicer and has silencing ability, indicating that it can enter the RNAi pathway. A computational analysis of zebrafish tRFs shows that they are conserved among vertebrates and mining of publicly available datasets reveals that some 5′tRFs are differentially expressed in disease conditions, namely during infection and colorectal cancer. Conclusions: tRFs constitute a class of conserved regulatory RNAs in vertebrates and may be involved in mechanisms of genome regulation and in some diseases. Keywords: tRNA derived fragments, Zebrafish, Small non coding RNAs, tRNAspublishe