Metagenomic Analysis of the Composition of Microbial Consortia Involved in Spruce Degradation over Time in Bialowieza Natural Forest.

Deadwood plays an important role in forest ecology; its degradation and, therefore, carbon assimilation is carried out by fungi and bacteria. To quantify the abundance and distribution of microbial taxa inhabiting dead spruce logs fallen over a span of 50 years and the soil beneath, we used taxonomic profiling with NGS sequencing of hypervariable DNA fragments of ITS1 and 16S V3-V4, respectively. The analysis of sequencing data revealed a high level of diversity in microbial communities participating in the degradation of spruce logs. Differences in the relative abundance of microbial taxa between the samples of the wood that died in 1974 and 2014, and of the soil in its immediate vicinity, were visible, especially at the genus level. Based on the Lefse analysis significantly higher numbers of classified bacterial taxa were observed in the wood and soil samples from 2014 (wood: 1974-18 and 2014-28 taxa; soil: 1974-8 and 2014-41 taxa) while the number of classified fungal taxa was significantly higher in the wood and soil samples from 1974 (wood: 1974-17 and 2014-9 taxa; soil: 1974-57 and 2014-28 taxa). Most of the bacterial and fungal amplicon sequence variants (ASVs) unique to wood were found in the samples from 1974, while those unique to soil were detected in the samples from 2014. The ATR-FTIR method supported by CHN analysis revealed physicochemical changes in deadwood induced by the activity of fungal and bacterial organisms.

Properties, Physiological Functions and Involvement of Basidiomycetous Alcohol Oxidase in Wood Degradation.

Extensive research efforts have been devoted to describing yeast alcohol oxidase (AO) and its promoter region, which is vastly applied in studies of heterologous gene expression. However, little is known about basidiomycetous AO and its physiological role in wood degradation. This review describes several alcohol oxidases from both white and brown rot fungi, highlighting their physicochemical and kinetic properties. Moreover, the review presents a detailed analysis of available AO-encoding gene promoter regions in basidiomycetous fungi with a discussion of the manipulations of culture conditions in relation to the modification of alcohol oxidase gene expression and changes in enzyme production. The analysis of reactions catalyzed by lignin-modifying enzymes (LME) and certain lignin auxiliary enzymes (LDA) elucidated the possible involvement of alcohol oxidase in the degradation of derivatives of this polymer. Combined data on lignin degradation pathways suggest that basidiomycetous AO is important in secondary reactions during lignin decomposition by wood degrading fungi. With numerous alcoholic substrates, the enzyme is probably engaged in a variety of catalytic reactions leading to the detoxification of compounds produced in lignin degradation processes and their utilization as a carbon source by fungal mycelium.

Triploid pregnancy-Clinical implications.

Triploidy is a life-limiting genetic aberration resulting from an extra haploid set of chromosomes of paternal (diandric triploidy) or maternal origin (digynic triploidy). Triploidy affects around 1%-2% of all conceptions. The majority of cases is miscarried at early developmental stages. In consequence of genomic imprinting, parental origin affects the phenotype of triploid pregnancies as well as the prevalence and spectrum of related maternal complications. Distinctive ultrasound features of both triploid phenotypes as well as characteristic patterns of biochemical markers may be useful in diagnosis. Molecular confirmation of the parental origin allows to predict the risk of complications, such as gestational trophoblastic neoplasia, hyperthyroidism, hypertension, or preeclampsia associated with the paternal origin of triploidy. Diagnosis of partial hydatidiform mole associated with diandric triploidy is challenging especially in the first trimester pregnancy loss due to the limitations of both histopathology and ultrasound. We present important clinical aspects of triploid pregnancies and indicate unresolved issues demanding further studies.

Distribution of diandric and digynic triploidy depending on gestational age.

PURPOSE: To establish the distribution of diandric and digynic triploidy depending on gestational age. METHODS: 107 triploid samples tested prospectively in a single genetic department during a four-year period were analyzed for parental origin of triploidy by Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) (n=95) with the use of matching parental samples or by MS-MLPA (n=12), when parental samples were unavailable. Tested pregnancies were divided into three subgroups with regard to the gestational age at spontaneous pregnancy loss: <11 gestational weeks, 11-14 gestational weeks, and >14 gestational weeks. RESULTS: Diandric triploidy constituted overall 44.9% (46.5% in samples miscarried <11 gestational weeks, 64.3% in samples miscarried between 11 and 14 gestational weeks, and 27.8% in pregnancies which survived >14 gestational weeks). CONCLUSIONS: The distribution of diandric and digynic triploidy depends on gestational age. The majority of diandric triploid pregnancies is lost in the first trimester of pregnancy. In the second trimester, diandric cases are at least twice less frequent than digynic ones.

Cerrena unicolor Laccases, Genes Expression and Regulation of Activity.

A white rot fungus Cerrena unicolor has been identified as an important source of laccase, unfortunately regulation of this enzyme genes expression is poorly understood. Using 1D and 2D PAGE and LC-MS/MS, laccase isoenzymes were investigated in the liquid filtrate of C. unicolor culture. The level of expression of laccase genes was measured using qPCR. The elevated concentrations of copper and manganese in the medium caused greatest change in genes expression and three laccase transcripts were significantly affected after culture temperature was decreased from 28 to 4 °C or increased to 40 °C. The small differences in the PAGE band intensities of individual laccase proteins were also observed, indicating that given compound affect particular laccase's transcript. Analyses of laccase-specific activity, at all tested conditions, showed the increased activities as compared to the control, suggesting that enzyme is regulated at the post-translational stage. We observed that the aspartic protease purified from C. unicolor, significantly stimulate laccase activity. Moreover, electrochemical analysis of protease-treated laccase sample had 5 times higher redox peaks. The obtained results indicate that laccases released by C. unicolor are regulated at transcriptional, translational, and at the post-translational steps of gene expression helping fungus adapt to the environmental changes.

Twin pregnancies discordant for digynic triploidy - A case series.

OBJECTIVE: To analyse natural course and perinatal management in twin pregnancies discordant for digynic triploidy. CASE REPORT: We present five cases of twins discordant for digynic triploidy. Pregnancy outcome was known for three of them. In one case, premature rupture of membranes occurred at 20 gestational weeks and both fetuses were miscarried. In two other pregnancies healthy co-twins were born at term after the triploid fetuses demise at 28 and 37 weeks. No maternal complications were observed. CONCLUSION: Twin pregnancies discordant for triploidy poses a challenge for perinatal management. Expectant management should be considered in digynic triploid cases.

Phytopathogenic Cercosporoid Fungi-From Taxonomy to Modern Biochemistry and Molecular Biology.

Phytopathogenic cercosporoid fungi have been investigated comprehensively due to their important role in causing plant diseases. A significant amount of research has been focused on the biology, morphology, systematics, and taxonomy of this group, with less of a focus on molecular or biochemical issues. Early and extensive research on these fungi focused on taxonomy and their classification based on in vivo features. Lately, investigations have mainly addressed a combination of characteristics such as morphological traits, host specificity, and molecular analyses initiated at the end of the 20th century. Some species that are important from an economic point of view have been more intensively investigated by means of genetic and biochemical methods to better understand the pathogenesis processes. Cercosporin, a photoactivated toxin playing an important role in Cercospora diseases, has been extensively studied. Understanding cercosporin toxicity in relation to reactive oxygen species (ROS) production facilitated the discovery and regulation of the cercosporin biosynthesis pathway, including the gene cluster encoding pathway enzymes. Furthermore, these fungi may be a source of other biotechnologically important compounds, e.g., industrially relevant enzymes. This paper reviews methods and important results of investigations of this group of fungi addressed at different levels over the years.

Lighting Conditions Influence the Dynamics of Protease Synthesis and Proteasomal Activity in the White Rot Fungus Cerrena unicolor.

Recent transcriptomic and biochemical studies have revealed that light influences the global gene expression profile and metabolism of the white-rot fungus Cerrena unicolor. Here, we aimed to reveal the involvement of proteases and ubiquitin-mediated proteolysis by the 26S proteasome in the response of this fungus to white, red, blue and green lighting conditions and darkness. The changes in the expression profile of C. unicolor genes putatively engaged in proteolysis were found to be unique and specific to the applied wavelength of light. It was also demonstrated that the activity of proteases in the culture fluid and mycelium measured using natural and synthetic substrates was regulated by light and was substrate-dependent. A clear influence of light on protein turnover and the qualitative and quantitative changes in the hydrolytic degradation of proteins catalyzed by various types of proteases was shown. The analysis of activity associated with the 26S proteasome showed a key role of ATP-dependent proteolysis in the initial stages of adaptation of fungal cells to the stress factors. It was suggested that the light-sensing pathways in C. unicolor are cross-linked with stress signaling and secretion of proteases presumably serving as regulatory molecules.

Usefulness of methylation-specific multiplex ligation-dependent probe amplification for identification of parental origin of triploidy.

Triploidy is a genetic aberration resulting from an extra haploid set of chromosomes of paternal (diandric) or maternal (digynic) origin. Diandric cases, opposite to digynic ones, may lead to gestational trophoblastic neoplasia (GTN) or generate maternal complications, therefore their identification is crucial, but reproducibility of traditionally used histopathological assessment is poor. The aim of the study was to analyse the usefulness of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) with probes for two differentially methylated regions (DMR) at chromosome 11p.15.5 for identification of the parental origin of triploidy. 84 triploid DNA samples were tested with MS-MLPA: 34 paternal cases (40.5%) and 50 maternal ones (59.5%) according to the reference results of QF-PCR. Methylation ratio (MR) was calculated. Reference values proposed by the MRC-Holland for diploid samples (MR 0.8-1.2) were used. The values outside these ranges were used to diagnose parental origin of triploidy-paternal (MR > 1.2) or maternal (MR < 0.8). The effectiveness of MS-MLPA was 94.0%. The mean MR in paternal triploidy was 1.7 (SD-0.25; n = 34) compared with 0.56 in maternal triploidy (SD-0.12; n = 50). MR values in paternal and maternal triploidy did not overlap. In five samples (6.0%) parental origin of triploidy could not be accurately established by MS-MLPA, probably due to the maternal cell contamination (MCC). MS-MLPA can be used as a convenient method for distinguishing between paternal and maternal triploidy without the necessity for parental samples testing. It enables adequate selection of the paternal triploid cases for follow up in order to exclude post-molar GTN.

Combined Effect of Light and Nutrients on the Micromorphology of the White rot Fungus Cerrena Unicolor.

Light influences developmental pathways in fungi. Recent transcriptomic and biochemical analyses have demonstrated that light influences the metabolism of a white-rot basidiomycete Cerrena unicolor. However, the expression profile of genes involved in the growth and development, or micromorphological observations of the mycelium in response to variable lighting and culturing media, have not performed. We aim to reveal the effect of light and nutrients on C. unicolor growth and a potential relationship between the culture medium and lighting conditions on fungus micromorphological structures. Confocal laser scanning microscopy and scanning electron microscopy were employed for morphological observations of C. unicolor mycelium cultivated in red, blue, green, and white light and darkness on mineral and sawdust media. A comprehensive analysis of C. unicolor differentially expressed genes (DEGs) was employed to find global changes in the expression profiles of genes putatively involved in light-dependent morphogenesis. Both light and nutrients influenced C. unicolor growth and development. Considerable differences in the micromorphology of the mycelia were found, which were partially reflected in the functional groups of DEGs observed in the fungus transcriptomes. A complex cross-interaction of nutritional and environmental signals on C. unicolor growth and morphology was suggested. The results are a promising starting point for further investigations of fungus photobiology.

Laccase Properties, Physiological Functions, and Evolution.

Discovered in 1883, laccase is one of the first enzymes ever described. Now, after almost 140 years of research, it seems that this copper-containing protein with a number of unique catalytic properties is widely distributed across all kingdoms of life. Laccase belongs to the superfamily of multicopper oxidases (MCOs)-a group of enzymes comprising many proteins with different substrate specificities and diverse biological functions. The presence of cupredoxin-like domains allows all MCOs to reduce oxygen to water without producing harmful byproducts. This review describes structural characteristics and plausible evolution of laccase in different taxonomic groups. The remarkable catalytic abilities and broad substrate specificity of laccases are described in relation to other copper-containing MCOs. Through an exhaustive analysis of laccase roles in different taxa, we find that this enzyme evolved to serve an important, common, and protective function in living systems.

Oxalate oxidase from Abortiporus biennis - protein localisation and gene sequence analysis.

We have described for the first time the localisation of oxalate oxidase (OXO, EC 1.2.3.4) in Abortiporus biennis cells, using histochemical and immunochemical methods coupled with transmission electron microscopy. Rabbit anti-oxalate oxidase immunoglobulins with anti-rabbit secondary antibody conjugated with 10-nm gold particles were used. Moreover, the formation of electron dense precipitation of reaction of diaminobenzidine (DAB) with horseradish peroxidase (HRP) for histochemical localisation of the enzyme was found. OXO was localised close to the membranous structures of the cell membranes, in membranous vesicles located close to the outer cell membrane, and vacuolar membranes surrounding vacuoles. The positive immunoreaction to OXO was also intense in cell wall areas. Moreover, we proved that gene coding for OXO was expressed in the same cultures. Corresponding mRNA was isolated, full length cDNA was synthesized, cloned and sequenced. Two copies of cupin domains were found in the sequence of amino-acids conserved domain coding for the cupin enzyme. Comparison of the genomic DNA and cDNA sequences has revealed the presence of seventeen introns in the gene. The isoelectric point of the protein was estimated at pH 4.5 and several possible N-glycosylation sites were predicted.

Light-regulated synthesis of extra- and intracellular enzymes related to wood degradation by the white rot fungus Cerrena unicolor during solid-state fermentation on ash sawdust-based medium.

The light-dependent metabolism of the white rot basidiomycete Cerrena unicolor FCL139 has already been demonstrated using transcriptomic and Biolog-based approaches. To further analyze the influence of light on C. unicolor wood degradation, we measured the activity of an array of CAZymes (carbohydrate-active enzymes) and enzymes involved in the redox system of fungal cells associated with lignolysis. Extra- and intracellular enzymatic extracts were obtained from solid-state ash sawdust C. unicolor cultures cultivated for 14 days under red, blue, green, or white light conditions, or in the dark. Light greatly influenced the synthesis of MnP, total cellulases, endo-1,4-ß-glucanase, endo-1,4-ß-xylanase, catalase, and superoxide dismutase. The production of MnP and catalase was evidently stimulated by white light. It is also worth noticing that blue light caused a gradual increase in the activity of total cellulases throughout the entire period of C. unicolor growth. Moreover, endo-1,4-ß-glucanase showed the highest activity on day 13 of fungus cultivation and the production of laccase and ß-glucosidase appeared to be the least influenced by light. However, the strongest activity of the endo-1,4-ß-xylanase was observed in the dark. It seemed that light not only influenced the regulation of the synthesis of the wood-degrading enzymes at different levels, but also acted indirectly by affecting production of enzymes managing harmful lignin by-products causing oxidative stress. The ability of the fungus to decompose woody plant material is clearly influenced by environmental factors.

Phytohormones (Auxin, Gibberellin) and ACC Deaminase In Vitro Synthesized by the Mycoparasitic Trichoderma DEMTkZ3A0 Strain and Changes in the Level of Auxin and Plant Resistance Markers in Wheat Seedlings Inoculated with this Strain Conidia.

Both hormonal balance and plant growth may be shaped by microorganisms synthesizing phytohormones, regulating its synthesis in the plant and inducing plant resistance by releasing elicitors from cell walls (CW) by degrading enzymes (CWDE). It was shown that the Trichoderma DEMTkZ3A0 strain, isolated from a healthy rye rhizosphere, colonized the rhizoplane of wheat seedlings and root border cells (RBC) and caused approximately 40% increase of stem weight. The strain inhibited (in over 90%) the growth of polyphagous Fusarium spp. (F. culmorum, F. oxysporum, F. graminearum) phytopathogens through a mechanism of mycoparasitism. Chitinolytic and glucanolytic activity, strongly stimulated by CW of F. culmorum in the DEMTkZ3A0 liquid culture, is most likely responsible for the lysis of hyphae and macroconidia of phytopathogenic Fusarium spp. as well as the release of plant resistance elicitors. In DEMTkZ3A0 inoculated plants, an increase in the activity of the six tested plant resistance markers and a decrease in the concentration of indoleacetic acid (IAA) auxin were noted. IAA and gibberellic acid (GA) but also the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) enzyme regulating ethylene production by plant were synthesized by DEMTkZ3A0 in the liquid culture. IAA synthesis was dependent on tryptophan and negatively correlated with temperature, whereas GA synthesis was positively correlated with the biomass and temperature.

The wood decay fungus Cerrena unicolor adjusts its metabolism to grow on various types of wood and light conditions.

Cerrena unicolor is a wood-degrading basidiomycete with ecological and biotechnological importance. Comprehensive Biolog-based analysis was performed to assess the metabolic capabilities and sensitivity to chemicals of C. unicolor FCL139 growing in various sawdust substrates and light conditions. The metabolic preferences of the fungus towards utilization of specific substrates were shown to be correlated with the sawdust medium applied for fungus growth and the light conditions. The highest catabolic activity of C. unicolor was observed after fungus precultivation on birch and ash sawdust media. The fungus growing in the dark showed the highest metabolic activity which was indicated by capacity to utilize a broad spectrum of compounds and the decomposition of 74/95 of the carbon sources. In all the culture light conditions, p-hydroxyphenylacetic acid was the most readily metabolized compound. The greatest tolerance to chemicals was also observed during C. unicolor growth in darkness. The fungus was the most sensitive to nitrogen compounds and antibiotics, but more resistant to chelators. Comparative analysis of C. unicolor and selected wood-decay fungi from different taxonomic and ecological groups revealed average catabolic activity of the fungus. However, C. unicolor showed outstanding capabilities to catabolize salicin and arbutin. The obtained picture of C. unicolor metabolism showed that the fungus abilities to decompose woody plant material are influenced by various environmental factors.

RNA Sequencing Reveals Differential Gene Expression of Cerrena Unicolor in Response to Variable Lighting Conditions.

To elucidate the light-dependent gene expression in Cerrena unicolor FCL139, the transcriptomes of the fungus growing in white, blue, green, and red lighting conditions and darkness were analysed. Among 10,413 all-unigenes detected in C. unicolor, 7762 were found to be expressed in all tested conditions. Transcripts encoding putative fungal photoreceptors in the C. unicolor transcriptome were identified. The number of transcripts uniquely produced by fungus ranged from 20 during its growth in darkness to 112 in the green lighting conditions. We identified numerous genes whose expression differed substantially between the darkness (control) and each of the light variants tested, with the greatest number of differentially expressed genes (DEGs) (454 up- and 457 down-regulated) observed for the white lighting conditions. The DEGs comprised those involved in primary carbohydrate metabolism, amino acid metabolism, autophagy, nucleotide repair systems, signalling pathways, and carotenoid metabolism as defined using Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The analysis of the expression profile of genes coding for lignocellulose-degrading enzymes suggests that the wood-degradation properties of C. unicolor may be independent of the lighting conditions and may result from the overall stimulation of fungal metabolism by daylight.

Comparative transcriptomic analysis of Cerrena unicolor revealed differential expression of genes engaged in degradation of various kinds of wood.

To explore the number of enzymes engaged by Cerrena unicolor FCL139 for wood degradation, the transcriptomes of the fungus growing on birch, ash, maple sawdust and the control liquid medium were analyzed. Among 12,966 gene models predicted for the C. unicolor genome, 10,396 all-unigenes were detected, of which 9567 were found to be expressed in each of the tested growth media. The highest number (107) of unique transcripts was detected during fungus growth in the control liquid medium, while the lowest number (11) - in the fungal culture comprising maple saw dust. Analysis of C. unicolor transcriptomes identified numerous genes whose expression differed substantially between the mycelia growing in control medium and each of the sawdust media used, with the highest number (828) of upregulated transcripts observed during the fungus growth on the ash medium. Among the 294 genes that were potentially engaged in wood degradation, the expression of 59 was significantly (p < .01) changed in the tested conditions. The transcripts of 37 of those genes were at least four times more abundant in the cells grown in all sawdust media when compared to the control medium. Upregulated genes coding for cellulases and, to a lower extent, hemicellulases predominated during fungus growth on sawdust. Transcripts encoding cellulolytic enzymes were the most abundant in mycelia grown on birch and maple while lower number of such transcripts was detected in fungus growing on ash. The expression pattern of lignolytic activities-coding genes was strongly dependent on the type of sawdust applied for fungus growth medium.

Antimelanomic Effects of High- and Low-Molecular Weight Bioactive Subfractions Isolated from the Mossy Maze Mushroom, Cerrena unicolor (Agaricomycetes).

Three bioactive fractions isolated from Cerrena unicolor cultures-crude endopolysaccharide (c-EPS), laccase, and a subfraction of low-molecular weight secondary metabolites-were used to determine potential cytotoxic effects on the mouse melanoma B16-F10 cell line (American Type Culture Collection CRL-6475). The results obtained prove that all examined fractions exhibited activity against the investigated tumor cells. In addition, an evident immunomodulatory effect of the c-EPS fraction was observed. Our results show that the levels of 2 cytokines (tumor necrosis factor-a and chemokine ligand 2) in mouse inner medullary collecting duct mIMCD-3 cells (American Type Culture Collection CRL-2123) stimulated by c-EPS were significantly higher. A lipopolysaccharide model was used at the same concentration (10 µg/mL) as a positive control.

Lignin degradation: microorganisms, enzymes involved, genomes analysis and evolution.

Extensive research efforts have been dedicated to describing degradation of wood, which is a complex process; hence, microorganisms have evolved different enzymatic and non-enzymatic strategies to utilize this plentiful plant material. This review describes a number of fungal and bacterial organisms which have developed both competitive and mutualistic strategies for the decomposition of wood and to thrive in different ecological niches. Through the analysis of the enzymatic machinery engaged in wood degradation, it was possible to elucidate different strategies of wood decomposition which often depend on ecological niches inhabited by given organism. Moreover, a detailed description of low molecular weight compounds is presented, which gives these organisms not only an advantage in wood degradation processes, but seems rather to be a new evolutionatory alternative to enzymatic combustion. Through analysis of genomics and secretomic data, it was possible to underline the probable importance of certain wood-degrading enzymes produced by different fungal organisms, potentially giving them advantage in their ecological niches. The paper highlights different fungal strategies of wood degradation, which possibly correlates to the number of genes coding for secretory enzymes. Furthermore, investigation of the evolution of wood-degrading organisms has been described.

Prenatal diagnosis of Duchenne and Becker muscular dystrophies: Underestimated problem of the secondary prevention of monogenetic disorders.

AIM: The aim of this study was to analyze the influence of effective preconceptional testing for carrier status in women at risk for Duchenne and Becker muscular dystrophies (D/BMD) on the prenatal diagnosis. METHODS: A retrospective analysis of 201 prenatal tests was performed in 169 Polish women at risk, in regard to time of testing for carrier status (prior to conception or during pregnancy) and carrier status of tested women, including confirmed D/BMD carriers (n = 78; 46.2%), D/BMD non-carriers - tested for germline mosaicism risk (n = 23; 13.6%), and women at risk with uncertain carrier status (n = 68; 40.2%). RESULTS: Only 52.7% of women were tested for D/BMD carrier status prior to conception and in these women prenatal diagnosis was carried out more frequently in the first trimester of pregnancy (64.7% vs 47.8%; P = 0.035). The results of prenatal testing in male fetuses in pregnancies of confirmed D/BMD carriers and D/BMD non-carriers - tested for germline mosaicism risk were conclusive in all cases, whereas in women with uncertain carrier status, only 60.0% of results were conclusive. Eighty-five of 103 female fetuses (82.5%) were tested prenatally and in 31.8% of them fetal carrier status was confirmed. CONCLUSION: Carrier status testing in women prior to conception has a positive impact on the frequency of first-trimester prenatal diagnosis and known D/BMD carrier status on the effectiveness of prenatal diagnosis. Due to the low percentage of women tested effectively prior to conception, carrier status testing in the families at risk should be propagated (including possibility of prenatal diagnosis of female fetuses).