Silencing of FAD synthase gene in Caenorhabditis elegans upsets protein homeostasis and impacts on complex behavioral patterns.

BACKGROUND: FAD synthase is a ubiquitous enzyme that catalyses the last step of FAD biosynthesis, allowing for the biogenesis of several flavoproteins. In humans different isoforms are generated by alternative splicing, isoform 1 being localized in mitochondria. Homology searching in Caenorabditis elegans leads to the identification of two human FAD synthase homologues, coded by the single copy gene R53.1. METHODS: The C. elegans R53.1 gene was silenced by feeding. The expression level of transcripts was established by semi-quantitative RT-PCR. Overall protein composition was evaluated by two-dimensional electrophoresis. Enzymatic activities were measured by spectrophotometry and oxygen consumption by polarography on isolated mitochondria. RESULTS: From R53.1 two transcripts are generated by trans-splicing. Reducing by 50% the transcription efficiency of R53.1 by RNAi results in a 50% reduction in total flavin with decrease in ATP content and increase in ROS level. Significant phenotypical changes are noticed in knock-down nematodes. Among them, a significant impairment in locomotion behaviour possibly due to altered cholinergic transmission. At biochemical level, impairment of flavoenzyme activities and of some KCN-insensitive oxygen-consuming enzymes is detected. At proteomic level, at least 15 abundant proteins are affected by R53.1 gene silencing, among which superoxide dismutases. CONCLUSION AND GENERAL SIGNIFICANCE: For the first time we addressed the existence of different isoforms of FAD-metabolizing enzymes in nematodes. A correlation between FAD synthase silencing and flavoenzyme derangement, energy shortage and redox balance impairment is apparent. In this aspect R53.1-interfered nematodes could provide an animal model system for studying human pathologies with alteration in flavin homeostasis/flavoenzyme biogenesis.

A comparative study of covariance selection models for the inference of gene regulatory networks.

MOTIVATION: The inference, or 'reverse-engineering', of gene regulatory networks from expression data and the description of the complex dependency structures among genes are open issues in modern molecular biology. RESULTS: In this paper we compared three regularized methods of covariance selection for the inference of gene regulatory networks, developed to circumvent the problems raising when the number of observations n is smaller than the number of genes p. The examined approaches provided three alternative estimates of the inverse covariance matrix: (a) the 'PINV' method is based on the Moore-Penrose pseudoinverse, (b) the 'RCM' method performs correlation between regression residuals and (c) 'ℓ(2C)' method maximizes a properly regularized log-likelihood function. Our extensive simulation studies showed that ℓ(2C) outperformed the other two methods having the most predictive partial correlation estimates and the highest values of sensitivity to infer conditional dependencies between genes even when a few number of observations was available. The application of this method for inferring gene networks of the isoprenoid biosynthesis pathways in Arabidopsis thaliana allowed to enlighten a negative partial correlation coefficient between the two hubs in the two isoprenoid pathways and, more importantly, provided an evidence of cross-talk between genes in the plastidial and the cytosolic pathways. When applied to gene expression data relative to a signature of HRAS oncogene in human cell cultures, the method revealed 9 genes (p-value<0.0005) directly interacting with HRAS, sharing the same Ras-responsive binding site for the transcription factor RREB1. This result suggests that the transcriptional activation of these genes is mediated by a common transcription factor downstream of Ras signaling. AVAILABILITY: Software implementing the methods in the form of Matlab scripts are available at: http://users.ba.cnr.it/issia/iesina18/CovSelModelsCodes.zip.

Effect of different light-emitting diode (LED) irradiation on the shelf life and phytonutrient content of broccoli (Brassica oleracea L. var. italica).

Broccoli (Brassica oleracea L. var. italica) is largely cultivated in southern Italy. It is an important source of phytonutrients, which are partially lost during postharvest storage. The aim of this work was to evaluate the overall effect of five different low-intensity light-emitting diodes (LEDs) on the quality parameters of broccoli florets over 20 d of cold storage. The level of ascorbic acid, chlorophylls, carotenoids, phenolic compounds and soluble proteins, as well as colour analysis, were evaluated. Green LED increased the chlorophyll and ascorbic acid content; white, red and yellow LEDs had a positive effect on the redox status of broccoli. Globally, only green LED had a statistically significant positive effect when considering all analysed parameters and could be proposed to prolong the shelf life of broccoli during cold storage.

Mycotoxin Biotransformation by Native and Commercial Enzymes: Present and Future Perspectives.

Worldwide mycotoxins contamination has a significant impact on animal and human health, and leads to economic losses accounted for billions of dollars annually. Since the application of pre- and post- harvest strategies, including chemical or physical removal, are not sufficiently effective, biological transformation is considered the most promising yet challenging approach to reduce mycotoxins accumulation. Although several microorganisms were reported to degrade mycotoxins, only a few enzymes have been identified, purified and characterized for this activity. This review focuses on the biotransformation of mycotoxins performed with purified enzymes isolated from bacteria, fungi and plants, whose activity was validated in in vitro and in vivo assays, including patented ones and commercial preparations. Furthermore, we will present some applications for detoxifying enzymes in food, feed, biogas and biofuel industries, describing their limitation and potentialities.

Draft Genome Sequence of Pseudomonas fluorescens Strain ITEM 17298, Associated with Cheese Spoilage.

Pseudomonas fluorescens is a genetically and phenotypically heterogeneous species that is often reported as a spoiler of fresh foods, but it has recently been implicated in clinical infection. In this study, we sequenced the genome of P. fluorescens strain ITEM 17298, isolated from mozzarella cheese and able to cause several alterations under cold storage.

Enniatin and Beauvericin Biosynthesis in Fusarium Species: Production Profiles and Structural Determinant Prediction.

Members of the fungal genus Fusarium can produce numerous secondary metabolites, including the nonribosomal mycotoxins beauvericin (BEA) and enniatins (ENNs). Both mycotoxins are synthesized by the multifunctional enzyme enniatin synthetase (ESYN1) that contains both peptide synthetase and S-adenosyl-l-methionine-dependent N-methyltransferase activities. Several Fusarium species can produce ENNs, BEA or both, but the mechanism(s) enabling these differential metabolic profiles is unknown. In this study, we analyzed the primary structure of ESYN1 by sequencing esyn1 transcripts from different Fusarium species. We measured ENNs and BEA production by ultra-performance liquid chromatography coupled with photodiode array and Acquity QDa mass detector (UPLC-PDA-QDa) analyses. We predicted protein structures, compared the predictions by multivariate analysis methods and found a striking correlation between BEA/ENN-producing profiles and ESYN1 three-dimensional structures. Structural differences in the ß strand's Asn789-Ala793 and His797-Asp802 portions of the amino acid adenylation domain can be used to distinguish BEA/ENN-producing Fusarium isolates from those that produce only ENN.

Transcriptional Analysis of Acinetobacter sp. neg1 Capable of Degrading Ochratoxin A.

Ochratoxin A (OTA) is a nephrotoxic and potentially carcinogenic mycotoxin produced by several species of Aspergillus and Penicillium, contaminating grapes, wine and a variety of food products. We recently isolated from OTA contaminated soil vineyard a novel free-living strain of Acinetobacter sp. neg1, ITEM 17016, able to degrade OTA into the non-toxic catabolic product ochratoxin α. Biochemical studies suggested that the degradation reaction proceeds via peptide bond hydrolysis with phenylalanine (Phe) release. In order to identify genes responsible for OTA degradation we performed a differential gene expression analysis of ITEM 17016 grown in the presence or absence of the toxin. Among the differentially expressed genes, six peptidases up-regulated at 6 h were identified. The degrading activity of the carboxypeptidase PJ_1540 was confirmed in vitro in a heterologous system. The enrichment analysis for Gene Ontology terms confirmed that OTA degradation proceeds through peptidase activities and revealed the over-representation of pathways related to Phe catabolism. These results indicate that Phe may represent an energy source for this Acinetobacter sp. neg1 strain and that OTA degrading reaction triggers the modulation of further catabolic activities.

Aflatoxin B1 and M1 Degradation by Lac2 from Pleurotus pulmonarius and Redox Mediators.

Laccases (LCs) are multicopper oxidases that find application as versatile biocatalysts for the green bioremediation of environmental pollutants and xenobiotics. In this study we elucidate the degrading activity of Lac2 pure enzyme form Pleurotus pulmonarius towards aflatoxin B1 (AFB1) and M1 (AFM1). LC enzyme was purified using three chromatographic steps and identified as Lac2 through zymogram and LC-MS/MS. The degradation assays were performed in vitro at 25 °C for 72 h in buffer solution. AFB1 degradation by Lac2 direct oxidation was 23%. Toxin degradation was also investigated in the presence of three redox mediators, (2,2'-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) (ABTS) and two naturally-occurring phenols, acetosyringone (AS) and syringaldehyde (SA). The direct effect of the enzyme and the mediated action of Lac2 with redox mediators univocally proved the correlation between Lac2 activity and aflatoxins degradation. The degradation of AFB1 was enhanced by the addition of all mediators at 10 mM, with AS being the most effective (90% of degradation). AFM1 was completely degraded by Lac2 with all mediators at 10 mM. The novelty of this study relies on the identification of a pure enzyme as capable of degrading AFB1 and, for the first time, AFM1, and on the evidence that the mechanism of an effective degradation occurs via the mediation of natural phenolic compounds. These results opened new perspective for Lac2 application in the food and feed supply chains as a biotransforming agent of AFB1 and AFM1.

Draft genome sequence of Acinetobacter sp. neg1 capable of degrading ochratoxin A.

Ochratoxin A (OTA) is a nephrotoxic and potentially carcinogenic mycotoxin produced by several species of Aspergillus and Penicillium. It is one of the major mycotoxins contaminating grain, grapes and a variety of food products, and the development of methods for reducing pre- and post-harvest contamination has drawn considerable attention. In the current study, we isolated and sequenced the genome of a novel free-living Acinetobacter strain able to degrade OTA. Biochemical studies suggest that the degradation reaction proceeds via peptide bond hydrolysis.

Loss of connectivity in cancer co-expression networks.

Differential gene expression profiling studies have lead to the identification of several disease biomarkers. However, the oncogenic alterations in coding regions can modify the gene functions without affecting their own expression profiles. Moreover, post-translational modifications can modify the activity of the coded protein without altering the expression levels of the coding gene, but eliciting variations to the expression levels of the regulated genes. These considerations motivate the study of the rewiring of networks co-expressed genes as a consequence of the aforementioned alterations in order to complement the informative content of differential expression. We analyzed 339 mRNAomes of five distinct cancer types to find single genes that presented co-expression patterns strongly differentiated between normal and tumor phenotypes. Our analysis of differentially connected genes indicates the loss of connectivity as a common topological trait of cancer networks, and unveils novel candidate cancer genes. Moreover, our integrated approach that combines the differential expression together with the differential connectivity improves the classic enrichment pathway analysis providing novel insights on putative cancer gene biosystems not still fully investigated.