The involvement of CYP1A2 in biodegradation of dioxins in pigs.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most harmful chemicals showing resistance to biodegradation. The majority of TCDD effects is mediated by the aryl hydrocarbon receptor (AhR) pathway. TCDD binding to AhR results in the activation of cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP1B1) involved in dioxin biodegradation. The goal of the study was to explore the potential role of CYP1A2 in the metabolism of TCDD. We investigated a molecular structure of CYP1A2 and the binding selectivity and affinity between the pig CYP1A2 and: 1/ DiCDD or TCDD (dioxins differing in toxicity and biodegradability) or 2/ their selected metabolites. pCYP1A2 demonstrated higher affinity towards DiCDD and TCDD than other pCYP1 enzymes. All dioxin-pCYP1A2 complexes were found to be stabilized by hydrophobic interactions. The calculated distances between the heme oxygen and the dioxin carbon nearest to the oxygen, reflecting the hydroxylating potential of CYP1A2, were higher than in other pCYP1 enzymes. The distances between the heme iron and the nearest dioxin carbon exceeded 5 Å, a distance sufficient to allow the metabolites to leave the active site. However, the molecular dynamics simulations revealed that two access channels of CYP1A2 were closed upon binding the majority of the examined dioxins. Moreover, the binding of dioxin metabolites did not promote opening of channel S-an exit for hydroxylated products. It appears that the undesired changes in the behavior of access channels prevail over the hydroxylating potential of CYP1A2 towards TCDD and the favorable distances, ultimately trapping the metabolites at the enzyme's active site

Patterns of Diversity of Fusarium Fungi Contaminating Soybean Grains

Soybean is an important, high protein source of food and feed. However, like other agricultural grains, soybean may pose a risk to human and animal health due to contamination of the grains with toxigenic Fusaria and associated mycotoxins. In this study, we investigated the diversity of Fusaria on a panel of 104 field isolates obtained from soybean grains during the growing seasons in 2017–2020. The results of species-specific PCR analyses showed that Fusarium avenaceum was the most common (n = 40) species associated with soybean grains in Poland, followed by F. equiseti (n = 22) and F. sporotrichioides (11 isolates). A set of isolates, which was not determined based on PCR analyses, was whole genome sequenced. Multiple sequence analyses using tef-1α, top1, rpb1, rpb2, tub2, pgk, cam and lsu genes showed that most of them belonged to Equiseti clade. Three cryptic species from this clade: F. clavum, F. flagelliforme and FIESC 31 (lacking Latin binomial) were found on soybean for the first time. This is the first report demonstrating the prevalence of Fusaria on soybean grains in Poland

Uncovering Diagnostic Value of Mitogenome for Identification of Cryptic Species Fusarium graminearum Sensu Stricto

Fungal complexes are often composed of morphologically nearly indistinguishable species with high genetic similarity. However, despite their close relationship, they can exhibit distinct phenotypic differences in pathogenicity and production of mycotoxins. Many plant pathogenic and toxigenic fungi have been shown to consist of such cryptic species. Identification of cryptic species in economically important pathogens has added value in epidemiologic studies and provides opportunities for better control. Analysis of mitochondrial genomes or mitogenomics opens up dimensions for improved diagnostics of fungi, especially when efficient recovery of DNA is problematic. In comparison to nuclear DNA, mitochondrial DNA (mtDNA) can be amplified with improved efficacy due to its multi-copy nature. However, to date, only a few studies have demonstrated the usefulness of mtDNA for identification of cryptic species within fungal complexes. In this study, we explored the value of mtDNA for identification of one of the most important cereal pathogens Fusarium graminearum sensu stricto (F.g.). We found that homing endonucleases (HEGs), which are widely distributed in mitogenomes of fungi, display small indel polymorphism, proven to be potentially species specific. The resulting small differences in their lengths may facilitate further differentiation of F.g. from the other cryptic species belonging to F. graminearum species complex. We also explored the value of SNP analysis of the mitogenome for typing F.g. The success in identifying F.g. strains was estimated at 96%, making this tool an attractive complement to other techniques for identification of F.g

Leptin signalling regulates transcriptional differences in granulosa cells from genetically obese mice but not the activation of NLRP3 inflammasome.

Acknowledgements: We would like to thank Dr Leslie Paul Kozak and Dr Magdalena Jura for their support in the validation and characterisation of the mouse obese phenotype and Dr Katarzyna Jankowska for the support with imaging and microscopy analysis.Obesity is associated with increased ovarian inflammation and the establishment of leptin resistance. We presently investigated the role of impaired leptin signalling on transcriptional regulation in granulosa cells (GCs) collected from genetically obese mice. Furthermore, we characterised the association between ovarian leptin signalling, the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and macrophage infiltration in obese mice. After phenotype characterisation, ovaries were collected from distinct group of animals for protein and mRNA expression analysis: (i) mice subjected to a diet-induced obesity (DIO) protocol, where one group was fed a high-fat diet (HFD) and another a standard chow diet (CD) for durations of 4 or 16 weeks; (ii) mice genetically deficient in the long isoform of the leptin receptor (ObRb; db/db); (iii) mice genetically deficient in leptin (ob/ob); and (iv) mice rendered pharmacologically hyperleptinemic (LEPT). Next, GCs from antral follicles isolated from db/db and ob/ob mice were subjected to transcriptome analysis. Transcriptional analysis revealed opposing profiles in genes associated with steroidogenesis and prostaglandin action between the genetic models, despite the similarities in body weight. Furthermore, we observed no changes in the mRNA and protein levels of NLRP3 inflammasome components in the ovaries of db/db mice or in markers of M1 and M2 macrophage infiltration. This contrasted with the downregulation of NLRP3 inflammasome components and M1 markers in ob/ob and 16-wk HFD-fed mice. We concluded that leptin signalling regulates NLRP3 inflammasome activation and the expression of M1 markers in the ovaries of obese mice in an ObRb-dependent and ObRb-independent manner. Furthermore, we found no changes in the expression of leptin signalling and NLRP3 inflammasome genes in GCs from db/db and ob/ob mice, which was associated with no effects on macrophage infiltration genes, despite the dysregulation of genes associated with steroidogenesis in homozygous obese db/db. Our results suggest that: (i) the crosstalk between leptin signalling, NLRP3 inflammasome and macrophage infiltration takes place in ovarian components other than the GC compartment; and (ii) transcriptional changes in GCs from homozygous obese ob/ob mice suggest structural rearrangement and organisation, whereas in db/db mice the impairment in steroidogenesis and secretory activity

Proteomic changes of aryl hydrocarbon receptor (AhR)-silenced porcine granulosa cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical compound contaminating the environment and affecting human/animal health and reproduction. Intracellular TCDD action usually involves the activation of aryl hydrocarbon receptor (AhR). The aim of the current study was to examine TCDD-induced changes in the proteome of AhR-silenced porcine granulosa cells. The AhR-silenced cells were treated with TCDD (100 nM) for 3, 12 or 24 h. Total protein was isolated, labeled with cyanines and next, the samples were separated by isoelectric focusing and SDS-PAGE. Proteins of interest were identified by MALDI-TOF/TOF mass spectrometry (MS) analysis and confirmed by western blotting and fluorescence immunocytochemistry. The AhR-targeted siRNA transfection reduced the granulosal expression level of AhR by 60-70%. In AhR-silenced porcine granulosa cells, TCDD influenced the abundance of only three proteins: annexin V, protein disulfide isomerase and ATP synthase subunit beta. The obtained results revealed the ability of TCDD to alter protein abundance in an AhR-independent manner. This study offers a new insight into the mechanism of TCDD action and provide directions for future functional studies focused on molecular effects exerted by TCDD

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the transcriptome of aryl hydrocarbon receptor (AhR) knock-down porcine granulosa cells

Background 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical, adversely affecting reproductive processes. The well-characterized canonical mechanism of TCDD action involves the activation of aryl hydrocarbon receptor (AhR) pathway, but AhR-independent mechanisms were also suggested. By applying RNA interference technology and Next Generation Sequencing (NGS) we aimed to identify genes involved in the mechanism of TCDD action in AhR knock-down porcine granulosa cells. Methods Porcine granulosa cells were transfected with small interfering RNAs targeting mRNA of AhR. After transfection, medium was exchanged and the AhR knock-down cells were treated with TCDD (100 nM) for 3, 12 or 24 h, total cellular RNA was isolated and designated for NGS. Following sequencing, differentially expressed genes (DEGs) were identified. To analyze functions and establish possible interactions of DEGs, the Gene Ontology (GO) database and the Search Tool for the Retrieval of Interacting Genes (STRING) database were used, respectively. Results The AhR gene expression level and protein abundance were significantly decreased after AhR-targeted siRNAs transfection of the cells. In TCDD-treated AhR knock-down cells we identified 360 differentially expressed genes (DEGs; P-adjusted < 0.05 and log2 fold change [log2FC] ≥ 1.0). The functional enrichment analysis of DEGs revealed that TCDD influenced the expression of genes involved, among other, in the metabolism of vitamin A, follicular development and oocyte maturation, proliferation and differentiation as well as inflammation, stress response, apoptosis and oncogenesis. The three-time point study demonstrated that TCDD-induced changes in the transcriptome of AhR knock-down porcine granulosa cells were especially pronounced during the early stages of the treatment (3 h). Conclusions TCDD affected the transcriptome of AhR knock-down porcine granulosa cells. The molecules involved in the AhR-independent action of TCDD were indicated in the study. The obtained data contribute to better understanding of molecular processes induced by xenobiotics in the ovary

The potential role of miRNAs and regulation of their expression in the development of mare endometrial fibrosis

Abstract Mare endometrial fibrosis (endometrosis), is one of the main causes of equine infertility. Despite the high prevalence, both ethology, pathogenesis and the nature of its progression remain poorly understood. Recent studies have shown that microRNAs (miRNAs) are important regulators in multiple cellular processes and functions under physiological and pathological circumstances. In this article, we reported changes in miRNA expression at different stages of endometrosis and the effect of transforming growth factor (TGF)-β1 on the expression of the most dysregulated miRNAs. We identified 1, 26, and 5 differentially expressed miRNAs (DEmiRs), in categories IIA (mild fibrosis), IIB (moderate fibrosis), and III (severe fibrosis) groups compared to category I (no fibrosis) endometria group, respectively (Padjusted < 0.05, log2FC ≥ 1.0/log2FC ≤  − 1.0). This study indicated the potential involvement of miRNAs in the regulation of the process associated to the development and progression of endometrosis. The functional enrichment analysis revealed, that DEmiRs target genes involved in the mitogen-activated protein kinases, Hippo, and phosphoinositide-3-kinase (PI3K)-Akt signalling pathways, focal adhesion, and extracellular matrix-receptor interaction. Moreover, we demonstrated that the most potent profibrotic cytokine—TGF-β1—downregulated novel-eca-miR-42 (P < 0.05) expression in fibroblasts derived from endometria at early-stage endometrosis (category IIA)

Bioinformatic analysis of endometrial miRNA expression profile at day 26–28 of pregnancy in the mare

Abstract The establishment of the fetomaternal interface depends on precisely regulated communication between the conceptus and the uterine environment. Recent evidence suggests that microRNAs (miRNAs) may play an important role in embryo-maternal dialogue. This study aimed to determine the expression profile of endometrial miRNAs during days 26–28 of equine pregnancy. Additionally, the study aimed to predict target genes for differentially expressed miRNAs (DEmiRs) and their potential role in embryo attachment, adhesion, and implantation. Using next-generation sequencing, we identified 81 DEmiRs between equine endometrium during the pre-attachment period of pregnancy (day 26–28) and endometrium during the mid-luteal phase of the estrous cycle (day 10–12). The identified DEmiRs appear to have a significant role in regulating the expression of genes that influence cell fate and properties, as well as endometrial receptivity formation. These miRNAs include eca-miR-21, eca-miR-126-3p, eca-miR-145, eca-miR-451, eca-miR-491-5p, members of the miR-200 family, and the miRNA-17-92 cluster. The target genes predicted for the identified DEmiRs are associated with ion channel activity and sphingolipid metabolism. Furthermore, it was noted that the expression of mucin 1 and leukemia inhibitory factor, genes potentially regulated by the identified DEmiRs, was up-regulated at day 26–28 of pregnancy. This suggests that miRNAs may play a role in regulating specific genes to create a favorable uterine environment that is necessary for proper attachment, adhesion, and implantation of the embryo in mares

Whole-genome single nucleotide polymorphism analysis for typing the pandemic pathogen Fusarium graminearum sensu stricto

Recent improvements in microbiology and molecular epidemiology were largely stimulated by whole- genome sequencing (WGS), which provides an unprecedented resolution in discriminating highly related genetic backgrounds. WGS is becoming the method of choice in epidemiology of fungal diseases, but its application is still in a pioneer stage, mainly due to the limited number of available genomes. Fungal pathogens often belong to complexes composed of numerous cryptic species. Detecting cryptic diversity is fundamental to understand the dynamics and the evolutionary relationships underlying disease outbreaks. In this study, we explore the value of whole-genome SNP analyses in identification of the pandemic pathogen Fusarium graminearum sensu stricto (F.g.). This species is responsible for cereal diseases and negatively impacts grain production worldwide. The fungus belongs to the monophyletic fungal complex referred to as F. graminearum species complex including at least sixteen cryptic species, a few among them may be involved in cereal diseases in certain agricultural areas. We analyzed WGS data from a collection of 99 F.g. strains and 33 strains representing all known cryptic species belonging to the FGSC complex. As a first step, we performed a phylogenomic analysis to reveal species-specific clustering. A RAxML maximum likelihood tree grouped all analyzed strains of F.g. into a single clade, supporting the clustering-based identification approach. Although, phylogenetic reconstructions are essential in detecting cryptic species, a phylogenomic tree does not fulfill the criteria for rapid and cost-effective approach for identification of fungi, due to the time-consuming nature of the analysis. As an alternative, analysis of WGS information by mapping sequence data from individual strains against reference genomes may provide useful markers for the rapid identification of fungi. We provide a robust framework for typing F.g. through the web-based PhaME workflow available at EDGE bioinformatics. The method was validated through multiple comparisons of assembly genomes to F.g. reference strain PH-1. We showed that the difference between intra- and interspecies variability was at least two times higher than intraspecific variation facilitating successful typing of F.g. This is the first study which employs WGS data for typing plant pathogenic fusaria