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.

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.

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.

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.

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.

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.

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.

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).

First trimester pregnancy loss: Clinical implications of genetic testing.

Around 10-15% of pregnancies result in a spontaneous first trimester miscarriage, which is most frequently caused by chromosomal abnormalities, mainly aneuploidies. Genetic analysis of pregnancy loss includes conventional G-banding karyotyping and various molecular methods. Apart from variable methodological limitations, the effectiveness of genetic analysis depends on the type and quality of the tested sample. To improve the reliability of genetic testing, we present methods of appropriate collection and pre-laboratory preparation of chorionic villi from first trimester miscarriage. We also discuss issues of maternal cell contamination, placental mosaicism and reciprocal and Robertsonian translocations in the context of interpretation of the results and genetic counseling.

New alkaline lipase from Rhizomucor variabilis: Biochemical properties and stability in the presence of microbial EPS.

A new strain of Rhizomucor variabilis producing an active extracellular lipase was identified and characterized in the present studies. The culture conditions were optimized and the highest lipase production amounting to 136 U/mL was achieved after 4 days of cultivation. The optimum pH (5.5) and temperature (28 °C) were determined as the best conditions for R. variabilis lipase production. The isolated enzyme preparation exhibited maximum activity at 40 °C and pH 8.0. Lipase from R. variabilis was stable up to 50 °C during 2 H retaining 80% of its initial activity. The enzyme was highly stable in the pH range of 7.0-9.0. Moreover, the addition of naturally obtained exopolysaccharides (EPS) significantly enhanced lipase activity. The presence of EPS derived from Ganoderma applanatum and Rhizobium leguminosarum enhanced the lipase activity, which was 22% and 31%, respectively, higher than that in the control experiments. Simultaneously, the pH activity profiles remained unchanged. The Michaelis-Menten constant and the turnover number of the enzyme for p-nitrophenyl palmitate in the standard assay conditions were estimated at a level of 0.631 mM and 0.674 Sec(-1) . In conclusion, the results obtained in this work present a newly isolated lipase preparation stabilized with EPS or without modification as a very effective tool for industrial application.

Complex Biochemical Analysis of Fruiting Bodies from Newly Isolated Polish Flammulina velutipes Strains.

The present study examined Polish strains of Flamulina velutipes as a potential source of nutraceuticals and found that their nutritional value is dependent on the fruiting bodies gathering time. To prove the above hypothesis protein, carbohydrate and phenolic substances concentration were determined. Moreover, catalase, superoxide dismutase, cellobiose dehydrogenase activities were assayed. In order to prove the healing properties of Enoki fruiting bodies the obtained extracts were tested for antioxidant and bacteriostatic abilities. We have proved that Polish F. velutipes fruiting bodies may be a rich source of antioxidants and that they are capable of inhibiting Staphylococcus aureus growth.

Cultivation and utility of Piptoporus betulinus fruiting bodies as a source of anticancer agents.

Piptoporus betulinus is a wood-rotting basidiomycete used in medicine and biotechnology. However, to date, no indoor method for cultivation of this mushroom fruiting bodies has been developed. Here we present the first report of successful production of P. betulinus mature fruiting bodies in artificial conditions. Four P. betulinus strains were isolated from natural habitats and their mycelia were inoculated into birch sawdust substrate supplemented with organic additives. All the strains effectively colonized the medium but only one of them produced fruiting bodies. Moisture and organic supplementation of the substrate significantly determined the fruiting process. The biological efficiency of the P. betulinus PB01 strain cultivated on optimal substrate (moisture and organic substance content of 55 and 65 and 25 or 35 %, respectively) ranged from 12 to 16 %. The mature fruiting bodies reached weight in the range from 50 to 120 g. Anticancer properties of water and ethanol extracts isolated from both cultured and nature-derived fruiting bodies of P. betulinus were examined in human colon adenocarcinoma, human lung carcinoma and human breast cancer cell lines. The studies revealed antiproliferative and antimigrative properties of all the investigated extracts. Nevertheless the most pronounced effects demonstrated the ethanol extracts, obtained from fruiting bodies of cultured P. betulinus. Summarizing, our studies proved that P. betulinus can be induced to fruit in indoor artificial culture and the cultured fruiting bodies can be used as a source of potential anticancer agents. In this respect, they are at least as valuable as those sourced from nature.

Laccase production and metabolic diversity among Flammulina velutipes strains.

Twelve Flammulina velutipes strains originating from Poland were identified using internal transcribed spacer (ITS) region sequencing. Based on the sequences obtained, the genomic relationship of the analyzed strains was determined. All F. velutipes strains were also characterized using Biolog FF MicroPlates to obtain data on C-substrate utilization and mitochondrial activity. The ability to decompose various substrates differed among the F. velutipes strains up to five times. The highest catabolic activities were characteristic for only two strains with capabilities to decompose up to 22 carbon sources. The correlation between carbon repression and laccase production by F. velutipes was analyzed based on glucose assimilation by these strains. Moreover, the influence of metal ions (Cu(2+), Cd(2+)), veratric and ferulic acids, and temperature on laccase activities in the analyzed strains was determined. The results obtained proved that all the inducers influenced laccase expression in almost all the analyzed strains. However, the degree of induction depended not only on the strain used but also on the day of the induction.

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.

Genetic diversity of the edible mushroom Pleurotus sp. by amplified fragment length polymorphism.

Pleurotus strains are the most important fungi used in the agricultural industry. The exact characterization and identification of Pleurotus species is fundamental for correct identification of the individuals and exploiting their full potential in food industry. The amplified fragment length polymorphism (AFLP) method was applied for genomic fingerprinting of 21 Pleurotus isolates of Asian and European origin. Using one PstI restriction endonuclease and four selective primers in an AFLP assay, 371 DNA fragments were generated, including 308 polymorphic bands. The AFLP profiles were found to be highly specific for each strain and they unambiguously distinguished 21 Pleurotus sp. fungi. The coefficient of Jaccard's genome profile similarity between the analyzed strains ranged from 0.0 (Pleurotus sp. I vs. P. sajor-caju 237 and P. eryngii 238) to 0.750 (P. ostreatus 246 vs. P. ostreatus 248), and the average was 0.378. The AFLP-based dendrogram generated by the UPGMA method grouped all the Pleurotus fungi studied into two major clusters and one independent lineage located on the outskirt of the tree occupied by naturally growing Pleurotus species strain I. The results of the present study suggest the possible applicability of the AFLP-PstI method in effective identification and molecular characterization of Pleurotus sp. strains.

Comparative studies on the induction of Trichoderma harzianum mutanase by α-(1→3)-glucan-rich fruiting bodies and mycelia of Laetiporus sulphureus.

Mutanase (α-(1→3)-glucanase) is a little-known inductive enzyme that is potentially useful in dentistry. Here, it was shown that the cell wall preparation (CWP) obtained from the fruiting body or vegetative mycelium of polypore fungus Laetiporus sulphureus is rich in α-(1→3)-glucan and can be successfully used for mutanase induction in Trichoderma harzianum. The content of this biopolymer in the CWP depended on the age of fruiting bodies and increased along with their maturation. In the case of CWP prepared from vegetative mycelia, the amount of α-(1→3)-glucan depended on the mycelium age and also on the kind of medium used for its cultivation. All CWPs prepared from the individually harvested fruiting body specimens induced high mutanase activity (0.53-0.82 U/mL) in T. harzianum after 3 days of cultivation. As for the CWPs obtained from the hyphal mycelia of L. sulpureus, the maximal enzyme productivity (0.34 U/mL after 3 days of incubation) was recorded for CWP prepared from the 3 week-old mycelium cultivated in Sabouraud medium. Statistically, a high positive correlation was found between the total percentage content of α-(1→3)-glucan in the CWP and the mutanase activity.

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.