Elevated expression of complement factor I in lung cancer cells associates with shorter survival-Potentially via non-canonical mechanism.

While numerous membrane-bound complement inhibitors protect the body's cells from innate immunity's autoaggression, soluble inhibitors like complement factor I (FI) are rarely produced outside the liver. Previously, we reported the expression of FI in non-small cell lung cancer (NSCLC) cell lines. Now, we assessed the content of FI in cancer biopsies from lung cancer patients and associated the results with clinicopathological characteristics and clinical outcomes. Immunohistochemical staining intensity did not correlate with age, smoking status, tumor size, stage, differentiation grade, and T cell infiltrates, but was associated with progression-free survival (PFS), overall survival (OS) and disease-specific survival (DSS). Multivariate Cox analysis of low vs. high FI content revealed HR 0.55, 95 % CI 0.32-0.95, p=0.031 for PFS, HR 0.51, 95 % CI 0.25-1.02, p=0.055 for OS, and HR 0.32, 95 % CI 0.12-0.84, p=0.021 for DSS. Unfavorable prognosis might stem from the non-canonical role of FI, as the staining pattern did not correlate with C4d - the product of FI-supported degradation of active complement component C4b. To elucidate that, we engineered three human NSCLC cell lines naturally expressing FI with CRISPR/Cas9 technology, and compared the transcriptome of FI-deficient and FI-sufficient clones in each cell line. RNA sequencing revealed differentially expressed genes engaged in intracellular signaling pathways controlling proliferation, apoptosis, and responsiveness to growth factors. Moreover, in vitro colony-formation assays showed that FI-deficient cells formed smaller foci than FI-sufficient NSCLC cells, but their size increased when purified FI protein was added to the medium. We postulate that a non-canonical activity of FI influences cellular physiology and contributes to the poor prognosis of lung cancer patients.

Dissection of an impact of VDR and RXRA on the genomic activity of 1,25(OH)2D3 in A431 squamous cell carcinoma.

BACKGROUND: Human skin is the natural source, place of metabolism, and target for vitamin D3. The classical active form of vitamin D3, 1,25(OH)2D3, expresses pluripotent properties and is intensively studied in cancer prevention and therapy. To define the specific role of vitamin D3 receptor (VDR) and its co-receptor retinoid X receptor alpha (RXRA) in genomic regulation, VDR or RXRA genes were silenced in the squamous cell carcinoma cell line A431 and treated with 1,25(OH)2D3 at long incubation time points 24 h/72 h. Extending the incubation time of A431 WT (wild-type) cells with 1,25(OH)2D3 resulted in a two-fold increase in DEGs (differentially expressed genes) and a change in the amount of downregulated from 37% to 53%. VDR knockout led to a complete loss of 1,25(OH)2D3-induced genome-wide gene regulation at 24 h time point, but after 72 h, 20 DEGs were found, of which 75% were downregulated, and most of them belonged to the gene ontology group "immune response". This may indicate the existence of an alternative, secondary response to 1,25(OH)2D3. In contrast, treatment of A431 ΔRXRA cells with 1,25(OH)2D3 for 24 h only partially affected DEGs, suggesting RXRA-independent regulation. Interestingly, overexpression of classic 1,25(OH)2D3 targets, like CYP24A1 (family 24 of subfamily A of cytochrome P450 member 1) or CAMP (cathelicidin antimicrobial peptide) was found to be RXRA-independent. Also, immunofluorescence staining of A431 WT cells revealed partial VDR/RXRA colocalization after 24 h and 72 h 1,25(OH)2D3 treatment. Comparison of transcriptome changes induced by 1,25(OH)2D3 in normal keratinocytes vs. cancer cells showed high cell type specific expression pattern with only a few genes commonly regulated by 1,25(OH)2D3. Activation of the genomic pathway at least partially reversed the expression of cancer-related genes, forming a basis for anti-cancer activates of 1,25(OH)2D3. In summary, VDR or RXRA independent genomic activities of 1,25(OH)2D3 suggest the involvement of alternative factors, opening new challenges in this field.

Two distinct Fusarium graminearum populations colonized European wheat in the past two decades.

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) disease in wheat in Europe. To reveal population structure and to pinpoint genetic targets of selection we studied genomes of 96 strains of F. graminearum using population genomics. Bayesian and phylogenomic analyses indicated that the F. graminearum emergence in Europe could be linked to two independently evolving populations termed here as East European (EE) and West European (WE) population. The EE strains are primarily prevalent in Eastern Europe, but to a lesser extent also in western and southern areas. In contrast, the WE population appears to be endemic to Western Europe. Both populations evolved in response to population-specific selection forces, resulting in distinct localized adaptations that allowed them to migrate into their environmental niche. The detection of positive selection in genes with protein/zinc ion binding domains, transcription factors and in genes encoding proteins involved in transmembrane transport highlights their important role in driving evolutionary novelty that allow F. graminearum to increase adaptation to the host and/or environment. F. graminearum also maintained distinct sets of accessory genes showing population-specific conservation. Among them, genes involved in host invasion and virulence such as those encoding proteins with high homology to tannase/feruloyl esterase and genes encoding proteins with functions related to oxidation-reduction were mostly found in the WE population. Our findings shed light on genetic features related to microevolutionary divergence of F. graminearum and reveal relevant genes for further functional research aiming at better control of this pathogen.

Embryonic signals mediate extracellular vesicle biogenesis and trafficking at the embryo-maternal interface.

BACKGROUND: Extracellular vesicles (EVs) are membrane-coated nanoparticles secreted by almost all cell types in living organisms. EVs, as paracrine mediators, are involved in intercellular communication, immune response, and several reproductive events, including the maintenance of pregnancy. Using a domestic animal model (Sus scrofa) with an epitheliochorial, superficial type of placentation, we focused on EV biogenesis pathway at the embryo-maternal interface, when the embryonic signaling occurs for maternal recognition and the maintenance of pregnancy. RESULTS: Transmission electron microscopy was used during early pregnancy to visualize EVs and apocrine and/or merocrine pathways of secretion. Immunofluorescent staining localized proteins responsible for EV biogenesis and cell polarization at the embryo-maternal interface. The expression profiles of genes involved in biogenesis and the secretion of EVs pointed to the possible modulation of endometrial expression by embryonic signals. Further in vitro studies showed that factors of embryonic origin can regulate the expression of the ESCRT-II complex and EV trafficking within endometrial luminal epithelial cells. Moreover, miRNA-mediated rapid negative regulation of gene expression was abolished by delivered embryonic signals. CONCLUSIONS: Our findings demonstrated that embryonic signals are potent modulators of ESCRT-dependent EV-mediated secretory activity of the endometrium during the critical stages of early pregnancy. Video Abstract.

The molecular dialog between the conceptus and maternal tissues that takes place prior to and during implantation is slowly becoming better known. The need for better understanding of one of life's founding stages is even greater in light of the observation that, not only in our species, many embryos fail to implant, both in natural conception and following assisted reproductive techniques. Although implantation strategies differ among eutherian mammals, the initial stages of apposition and adhesion are common and are a foundation for successful pregnancy. In early pregnancy, as the embryo arrives in the uterus, intensive communication between the embryo and mother begins. Among the wide range of cell-to-cell communication strategies, there is one, relatively recently discovered, governed by extracellular vesicles, small membranous vesicles that contain cell-specific collections of proteins, lipids, and genetic material. The present study was undertaken to answer the question of how signaling molecules released by cells participating in the embryo-maternal dialog contribute to extracellular vesicle-mediated cell-to-cell communication. Our results shed new light on the role of hormones, non-coding RNAs, and extracellular vesicles in the early stages of mammalian pregnancy, which are contributing to species reproductive success.

PDIA3 modulates genomic response to 1,25-dihydroxyvitamin D3 in squamous cell carcinoma of the skin.

An active form of vitamin D3 (1,25-dihydroxyvitamin D3) acts through vitamin D receptor (VDR) initiating genomic response, but several studies described also non-genomic actions of 1,25-dihydroxyvitamin D3, implying the role of PDIA3 in the process. PDIA3 is a membrane-associated disulfide isomerase involved in disulfide bond formation, protein folding, and remodeling. Here, we used a transcriptome-based approach to identify changes in expression profiles in PDIA3-deficient squamous cell carcinoma line A431 after 1,25-dihydroxyvitamin D3 treatment. PDIA3 knockout led to changes in the expression of more than 2000 genes and modulated proliferation, cell cycle, and mobility of cells; suggesting an important regulatory role of PDIA3. PDIA3-deficient cells showed increased sensitivity to 1,25-dihydroxyvitamin D3, which led to decrease migration. 1,25-dihydroxyvitamin D3 treatment altered also genes expression profile of A431ΔPDIA3 in comparison to A431WT cells, indicating the existence of PDIA3-dependent genes. Interestingly, classic targets of VDR, including CAMP (Cathelicidin Antimicrobial Peptide), TRPV6 (Transient Receptor Potential Cation Channel Subfamily V Member 6), were regulated differently by 1,25-dihydroxyvitamin D3, in A431ΔPDIA3. Deletion of PDIA3 impaired 1,25-dihydroxyvitamin D3-response of genes, such as PTGS2, MMP12, and FOCAD, which were identified as PDIA3-dependent. Additionally, response to 1,25-dihydroxyvitamin D3 in cancerous A431 cells differed from immortalized HaCaT keratinocytes, used as non-cancerous control. Finally, silencing of PDIA3 and 1,25-dihydroxyvitamin D3, at least partially reverse the expression of cancer-related genes in A431 cells, thus targeting PDIA3 and use of 1,25-dihydroxyvitamin D3 could be considered in a prevention and therapy of the skin cancer. Taken together, PDIA3 has a strong impact on gene expression and physiology, including genomic response to 1,25-dihydroxyvitamin D3.

Effect of Chromium Nanoparticles and Switching from a High-Fat to a Low-Fat Diet on the Cecal Microenvironment in Obese Rats.

Previous studies showed that chromium nanoparticles (Cr-NPs) might be used as dietary compounds against some obesity-related disorders; however, there is little information on how these compounds influence the gut microenvironment. The aim of this study was to investigate whether the negative effects of a high-fat diet in the large intestine of rats might be mitigated by switching to a low-fat diet and supplementation with Cr-NPs. Microbiota sequencing analysis revealed that the main action of the Cr-NPs was focused on changing the gut microbiota's activity. Supplementation with nanoparticles decreased the activity of ß-glucuronidase and enzymes responsible for the hydrolysis of dietary oligosaccharides and, thus, lowered the concentration of short-chain fatty acids in the cecum. In this group, there was also an elevated level of cecal lithocholic acid. The most favorable effect on the regulation of obesity-related disorders was observed when a high-fat diet was switched to a low-fat diet. This dietary change enhanced the production of short-chain fatty acids, reduced the level of secondary bile acids, and increased the microbial taxonomic richness, microbial differences, and microbial enzymatic activity in the cecum. To conclude, supplementation of a high-fat diet with Cr-NPs primarily had an effect on intestinal microbial activity, but switching to a low-fat diet had a powerful, all-encompassing effect on the gut that improved both microbial activity and composition.

Tamoxifen decreases ovarian toxicity without compromising cancer treatment in a rat model of mammary cancer.

BACKGROUND: Premenopausal women diagnosed with breast cancer often face aggressive chemotherapy resulting in infertility. Tamoxifen (TAM) is a selective estrogen receptor modulator that was previously suggested as a protective agent against chemotherapy-induced ovarian failure. In the current study, we examined mechanisms of the protective action of TAM in the ovaries of tumor-bearing rats treated with the chemotherapy drug cyclophosphamide (CPA). RESULTS: TAM prevented CPA-induced loss of ovarian follicular reserves. The protective TAM effect in the rat ovary partially resulted from decreased apoptosis. In addition, transcriptomic and proteomic screening also implicated the importance of DNA repair pathways as well as cell adhesion and extracellular matrix remodeling in the protective ovarian actions of TAM. CONCLUSIONS: Tamoxifen shielded the ovary from the side effects of chemotherapy without lessening the tumoricidal actions of mammary cancer treatment.

Super-Mitobarcoding in Plant Species Identification? It Can Work! The Case of Leafy Liverworts Belonging to the Genus Calypogeia.

Molecular identification of species is especially important where traditional taxonomic methods fail. The genus Calypogeia belongs to one of the tricky taxons. The simple morphology of these species and a tendency towards environmental plasticity make them complicated in identification. The finding of the universal single-locus DNA barcode in plants seems to be 'the Holy Grail'; therefore, researchers are increasingly looking for multiloci DNA barcodes or super-barcoding. Since the mitochondrial genome has low sequence variation in plants, species delimitation is usually based on the chloroplast genome. Unexpectedly, our research shows that super-mitobarcoding can also work! However, our outcomes showed that a single method of molecular species delimitation should be avoided. Moreover, it is recommended to interpret the results of molecular species delimitation alongside other types of evidence, such as ecology, population genetics or comparative morphology. Here, we also presented genetic data supporting the view that C. suecica is not a homogeneous species.

Insights into adaptive evolution of plastomes in Stipa L. (Poaceae).

BACKGROUND: The study presents results of research on the evolution of plastid genomes in Stipa L. which is a large genus of the Poaceae family, comprising species diverse in terms of geographic distribution, growing under highly variated habitat conditions. Complete plastome sequences of 43 taxa from Stipeae and Ampelodesmae tribes were analyzed for the variability of the coding regions against the background of phylogenetic relationships within the genus Stipa. The research hypothesis put forward in our research was that some of coding regions are affected by a selection pressure differentiated between individual phylogenetic lines of Stipa, potentially reducing the phylogenetic informativeness of these CDS. The study aimed to answer the question, which genes evolve in Stipa most rapidly and what kind of changes in the properties of encoded amino acids this entails. Another goal of this research was to find out whether individual genes are affected by positive selection and finally, whether selective pressure is uniform within the genus or does it vary between particular evolutionary lines within the genus. RESULTS: Results of our study proved the presence of selective pressure in 11 genes: ccsA, matK, ndhC, ndhF, ndhK, rbcL, rpoA rpoC1, rpoC2, rps8 and rps11. For the first time the effect of positive selection on the rps8, rps11, and ndhK genes was documented in grasses. The varied pace of evolution, different intensity and effects of selective pressure have been demonstrated between particular phylogenetic lines of the genus tested. CONCLUSIONS: Positive selection in plastid genome in Stipa mostly affects photosynthetic genes. The potential strongest adaptive pressure was observed in the rbcL gene, especially in the oldest evolutionary group comprising Central Asian high-mountain species: S. basiplumosa, S. klimesii, S. penicillata and S. purpurea, where adaptive pressure probably affected the amino acids directly related to the efficiency of CO2 assimilation.

The Role of Reduced Oxygen Supply and Transcription Factors cJUN and CREB in Progesterone Production during the Corpus Luteum Rescue in Gilts.

The corpus luteum plays a fundamental role in regulating reproduction via progesterone production. Still, there is little data on factors regulating the maintenance of luteal function during early pregnancy in gilts. Previous studies emphasize the role of hypoxia and HIF-1 in the regulation of steroidogenic and angiogenic genes expression and progesterone production by ovarian cells. Using the corpus luteum of cyclic and early pregnant gilts we analyzed: (1) the in vitro effects of reduced oxygen tension on progesterone production and mRNA expression of HIF1A and luteal function regulators, STAR and VEGFA; (2) the ex vivo profiles of mRNA and protein expression of HIF-1α, STAR, VEGFA and transcription factors-cJUN and CREB, regulating STAR expression, in the corpus luteum of cyclic and pregnant gilts. The synthesis of progesterone was gradually inhibited in cyclic or pregnant gilt luteal tissue (on day 13 of cycle or pregnancy) incubated in a decreasing concentration−20%, 10%, and 3% of oxygen (O2). Luteal tissues of pregnant gilts produced trace amounts of progesterone in 10% O2, which was similar to cyclic gilts in 3% O2. HIF1A expression increased after 24 h of incubation in tissues of cyclic gilts in 3% vs. 20% O2 (p < 0.01), whereas levels of STAR and VEGFA increased significantly in cyclic and pregnant gilt tissues incubated in 10% and 3% vs. 20% O2. The ex vivo mRNA expression of HIF1A and VEGFA was elevated (p < 0.001) on day 14 vs. day 12 of pregnancy. The protein expression of HIF-1 and VEGFA increased (p < 0.001), whereas the level of STAR (mRNA and protein) and progesterone dropped (p < 0.001) on day 14 of the estrous cycle vs. a parallel day of pregnancy and/or day 12 of the estrous cycle. The content of phosphorylated cJUN and CREB was elevated (p < 0.01) in the luteal tissue on day 12 or 14 of pregnancy vs. parallel days of the estrous cycle. These increases of phosphorylated cJUN and CREB may be involved in STAR expression in the luteal tissue during early pregnancy in gilts.

Stimulation of the intestinal microbiota with prebiotics enhances hepatic levels of dietary polyphenolic compounds, lipid metabolism and antioxidant status in healthy rats.

How stimulation of the intestinal microbiota may regulate the health-promoting effects of dietary polyphenols on liver function has not been fully elucidated. Therefore, in this study, we examined whether a combination of fructooligosaccharides (FOSs) and a polyphenol preparation (PP) might stimulate the intestinal microbiota to increase liver levels of polyphenol-derived bioactive compounds. We also investigated how this supplementation affected lipid metabolism and antioxidative status in Wistar rats without diet-induced metabolic disturbances. Addition of FOSs to the diet with the PP (CPF group) considerably reduced the caecal activity of microbial enzymes and elevated the production of short-chain fatty acids, mostly propionic acid. The CPF group exhibited increased relative abundances of caecal bacteria that were able to metabolize polyphenols from the examined preparation. Considerably elevated concentrations of ellagic acid, ellagic acid dimethyl ether glucuronide, 4-hydroxybenzoic acid and methyl cyanidin-3-sophoroside were found in the liver in the CPF group. In addition, hepatic downregulation of HIF1α, AHR and SREBP1c, which are molecular factors that regulate lipid metabolism and antioxidative status, was also found in this group. These changes might explain the considerably reduced levels of fat, triglycerides and lipid peroxidation in the liver in the CPF group. Moreover, the combination of FOSs and the PP considerably elevated the total antioxidant status and activity of superoxide dismutase in the blood. To conclude, stimulation of the intestinal microbial composition and activity with FOSs increased the health-promoting effects of the PP by elevating polyphenolic metabolite levels in the liver and improving lipid metabolism and antioxidative status in healthy rats. Therefore, the combination of FOSs and polyphenols might be a valuable dietary supplement that is able to support preventive healthcare.

Early steps of embryo implantation are regulated by exchange of extracellular vesicles between the embryo and the endometrium.

In early pregnancy, as the embryo arrives in the uterus, intensive communication between the embryo and uterus begins. Hundreds of molecules are known to be involved, but despite numerous findings, full understanding of the complexity of the embryo-maternal dialog remains elusive. Recently, extracellular vesicles, nanoparticles able to transfer functionally active cargo between cells, have emerged as important players in cell-cell communication, and as such, they have gained great attention over the past decade also in reproductive biology. Here, we use a domestic animal model (Sus scrofa) with an epitheliochorial, superficial type of placentation because of its advantage in studding uterine luminal fluid extracellular vesicles. We show that during early pregnancy, the uterine lumen is abundant with extracellular vesicles that carry a plethora of miRNAs able to target genes involved in embryonic and organismal development. These extracellular vesicles, upon the delivery to primary trophoblast cells, affect genes governing development as well as cell-to-cell signaling and interactions, consequently having an impact on trophoblast cell proliferation, migration, and invasion. We conclude that the exchange of a unique population of extracellular vesicles and their molecular cargo at the maternal-embryo interface is the key to the success of embryo implantation and pregnancy.

Application of two-dimensional difference gel electrophoresis to identify protein changes between center, margin, and adjacent non-tumor tissues obtained from non-small-cell lung cancer with adenocarcinoma or squamous cell carcinoma subtype.

Lung cancer is responsible for the most cancer-related mortality worldwide and the mechanism of its development is poorly understood. Proteomics has become a powerful tool offering vital knowledge related to cancer development. Using a two-dimensional difference gel electrophoresis (2D-DIGE) approach, we sought to compare tissue samples from non-small-cell lung cancer (NSCLC) patients taken from the tumor center and tumor margin. Two subtypes of NSCLC, adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were compared. Data are available via ProteomeXchange with identifier PXD032736 and PXD032962 for ADC and SCC, respectively. For ADC proteins, 26 significant canonical pathways were identified, including Rho signaling pathways, a semaphorin neuronal repulsive signaling pathway, and epithelial adherens junction signaling. For SCC proteins, nine significant canonical pathways were identified, including hypoxia-inducible factor-1α signaling, thyroid hormone biosynthesis, and phagosome maturation. Proteins differentiating the tumor center and tumor margin were linked to cancer invasion and progression, including cell migration, adhesion and invasion, cytoskeletal structure, protein folding, anaerobic metabolism, tumor angiogenesis, EMC transition, epithelial adherens junctions, and inflammatory responses. In conclusion, we identified several proteins that are important for the better characterization of tumor development and molecular specificity of both lung cancer subtypes. We also identified proteins that may be important as biomarkers and/or targets for anticancer therapy.

Breaking the limits - multichromosomal structure of an early eudicot Pulsatilla patens mitogenome reveals extensive RNA-editing, longest repeats and chloroplast derived regions among sequenced land plant mitogenomes.

BACKGROUND: The mitogenomes of vascular plants are one of the most structurally diverse molecules. In the present study we characterize mitogenomes of a rare and endangered species Pulsatilla patens. We investigated the gene content and its RNA editing potential, repeats distribution and plastid derived sequences. RESULTS: The mitogenome structure of early divergent eudicot, endangered Pulsatilla patens does not support the master chromosome hypothesis, revealing the presence of three linear chromosomes of total length 986 613 bp. The molecules are shaped by the presence of extremely long, exceeding 87 kbp repeats and multiple chloroplast-derived regions including nearly complete inverted repeat. Since the plastid IR content of Ranunculales is very characteristic, the incorporation into mitogenome could be explained rather by intracellular transfer than mitochondrial HGT. The mitogenome contains almost a complete set of genes known from other vascular plants with exception of rps10 and sdh3, the latter being present but pseudogenized. Analysis of long ORFs enabled the identification of genes which are rarely present in plant mitogenomes, including RNA and DNA polymerases, albeit their presence even at species level is variable. Mitochondrial transcripts of P. patens were edited with a high frequency, which exceeded the level known in other analyzed angiosperms, despite the strict qualification criteria of counting the editing events and taking into analysis generally less frequently edited leaf transcriptome. The total number of edited sites was 902 and nad4 was identified as the most heavily edited gene with 65 C to U changes. Non-canonical, reverse U to C editing was not detected. Comparative analysis of mitochondrial genes of three Pulsatilla species revealed a level of variation comparable to chloroplast CDS dataset and much higher infrageneric differentiation than in other known angiosperm genera. The variation found in CDS of mitochondrial genes is comparable to values found among Pulsatilla plastomes. Despite the complicated mitogenome structure, 14 single copy regions of 329 kbp, not splitted by repeats or plastid-derived sequences (MTPT), revealed the potential for phylogenetic, phylogeographic and population genetics studies by revealing intra- and interspecific collinearity. CONCLUSIONS: This study provides valuable new information about mitochondrial genome of early divergent eudicots, Pulsatilla patens, revealed multi-chromosomal structure and shed new light on mitogenomics of early eudicots.

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.

Transcriptional profiling of Chinese hamster ovary (CHO) cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a man-made chemical compound contaminating the environment. An exposure of organisms to TCDD results in numerous disorders. The main mechanism of TCDD action involves the induction of the aryl hydrocarbon receptor (AhR) pathway followed by the increase in the expression and activity of cytochrome P450 family 1 (CYP1) enzymes. The main aim of the present study was to identify, by means of RNA sequencing, transcripts involved in the mechanism of TCDD action in Chinese hamster ovary (CHO) cells, known to not express CYP1A1 enzyme. The CHO cells were treated with TCDD for 3, 12 or 24 h, and total RNA was isolated and sequenced. Thirty six (padjusted < 0.05) or six (padjusted < 0.05, log2FC ≥ 1.0/log2FC≤-1.0) differentially expressed genes (DEGs) were identified in TCDD-treated cells depending on the assumed statistical criteria. The dioxin up- and downregulated the expression of genes associated with ovarian follicle functions, development, cardiovascular system, signal transduction, inflammation and carcinogenesis. TCDD did not affect the expression of any of 522 miRNAs which were identified in the cells. The expression of CYP1A1, CYP1A2 and CYP1B1 was demonstrated neither in control nor in TCDD-treated CHO cells, although the respective genes were found in the cell genome. Twenty two other CYP enzymes were identified in CHO cells, however their expression was also not affected by TCDD.

Sex- and season-dependent differences in the expression of adiponectin and adiponectin receptors (AdipoR1 and AdipoR2) in the hypothalamic-pituitary-adrenal axis of the Eurasian beaver (Castor fiber L.).

Adiponectin, a product of the Adipoq gene, is an adipocyte-derived protein hormone of the cytokine family and the most abundantly expressed adipokine. Adiponectin and its receptors AdipoR1 and AdipoR2 (collectively referred to as the adiponectin system) are widely expressed in the central nervous system and other tissues, which suggests that this hormone has pleiotropic effects. Adiponectin could also play a role in the modulation of the hypothalamic-pituitaryadrenal (HPA) hormonal regulatory axis. There is a general scarcity of data on the adiponectin system in wild animals where annual changes in reproductive activity are linked with fluctuations in the activity of the HPA axis. The Eurasian beaver (Castor fiber L.) could be an interesting and suitable model for investigating the above processes. We hypothesized that the expression of the adiponectin system in the tissues of the beaver HPA axis is sex- and season-dependent. The study was performed on adult animals harvested during three different stages of reproductive activity: April ('breeding'), July ('post-breeding') and November ('pre-breeding'). The expression of the adiponectin system was confirmed in all branches (mediobasal hypothalamus, pituitary, adrenal cortex) of the HPA axis in both sexes and during all periods of reproductive activity. The expression of Adipoq, AdipoR1 and AdipoR2 was generally dependent on sex and the period of the reproductive season. The expression of adiponectin system genes was particularly pronounced in the adrenal cortex. These findings suggest that the adiponectin system in the Eurasian beaver could link reproductive processes with stress responses and energy metabolism.

Diversity of Mobile Genetic Elements in the Mitogenomes of Closely Related Fusarium culmorum and F. graminearum sensu stricto Strains and Its Implication for Diagnostic Purposes.

Much of the mitogenome variation observed in fungal lineages seems driven by mobile genetic elements (MGEs), which have invaded their genomes throughout evolution. The variation in the distribution and nucleotide diversity of these elements appears to be the main distinction between different fungal taxa, making them promising candidates for diagnostic purposes. Fungi of the genus Fusarium display a high variation in MGE content, from MGE-poor (Fusarium oxysporum and Fusarium fujikuroi species complex) to MGE-rich mitogenomes found in the important cereal pathogens F. culmorum and F. graminearum sensu stricto. In this study, we investigated the MGE variation in these latter two species by mitogenome analysis of geographically diverse strains. In addition, a smaller set of F. cerealis and F. pseudograminearum strains was included for comparison. Forty-seven introns harboring from 0 to 3 endonucleases (HEGs) were identified in the standard set of mitochondrial protein-coding genes. Most of them belonged to the group I intron family and harbored either LAGLIDADG or GIY-YIG HEGs. Among a total of 53 HEGs, 27 were shared by all fungal strains. Most of the optional HEGs were irregularly distributed among fungal strains/species indicating ancestral mosaicism in MGEs. However, among optional MGEs, one exhibited species-specific conservation in F. culmorum. While in F. graminearum s.s. MGE patterns in cox3 and in the intergenic spacer between cox2 and nad4L may facilitate the identification of this species. Thus, our results demonstrate distinctive traits of mitogenomes for diagnostic purposes of Fusaria.

Molecular delimitation of European leafy liverworts of the genus Calypogeia based on plastid super-barcodes.

BACKGROUND: Molecular research revealed that some of the European Calypogeia species described on the basis of morphological criteria are genetically heterogeneous and, in fact, are species complexes. DNA barcoding is already commonly used for correct identification of difficult to determine species, to disclose cryptic species, or detecting new taxa. Among liverworts, some DNA fragments, recommend as universal plant DNA barcodes, cause problems in amplification. Super-barcoding based on genomic data, makes new opportunities in a species identification. RESULTS: On the basis of 22 individuals, representing 10 Calypogeia species, plastid genome was tested as a super-barcode. It is not effective in 100%, nonetheless its success of species discrimination (95.45%) is still conspicuous. It is not excluded that the above outcome may have been upset by cryptic speciation in C. suecica, as our results indicate. Having the sequences of entire plastomes of European Calypogeia species, we also discovered that the ndhB and ndhH genes and the trnT-trnL spacer identify species in 100%. CONCLUSIONS: This study shows that even if a super-barcoding is not effective in 100%, this method does not close the door to a traditional single- or multi-locus barcoding. Moreover, it avoids many complication resulting from the need to amplify selected DNA fragments. It seems that a good solution for species discrimination is a development of so-called "specific barcodes" for a given taxonomic group, based on plastome data.