In silico prediction of polyketide biosynthetic gene clusters in the genomes of Hypericum-borne endophytic fungi.

BACKGROUND: The search for new bioactive natural compounds with anticancer activity is still of great importance. Even though their potential for diagnostics and treatment of cancer has already been proved, the availability is still limited. Hypericin, a naphthodianthrone isolated essentially from plant source Hypericum perforatum L. along with other related anthraquinones and bisanthraquinones belongs to this group of compounds. Although it has been proven that hypericin is synthesized by the polyketide pathway in plants, none of the candidate genes coding for key enzymes has been experimentally validated yet. Despite the rare occurrence of anthraquinones in plants, their presence in microorganisms, including endophytic fungi, is quite common. Unlike plants, several biosynthetic genes grouped into clusters (BGCs) in fungal endophytes have already been characterized. RESULTS: The aim of this work was to predict, identify and characterize the anthraquinone BGCs in de novo assembled and functionally annotated genomes of selected endophytic fungal isolates (Fusarium oxysporum, Plectosphaerella cucumerina, Scedosporium apiospermum, Diaporthe eres, Canariomyces subthermophilus) obtained from different tissues of Hypericum spp. The number of predicted type I polyketide synthase (PKS) BGCs in the studied genomes varied. The non-reducing type I PKS lacking thioesterase domain and adjacent discrete gene encoding protein with product release function were identified only in the genomes of C. subthermophilus and D. eres. A candidate bisanthraquinone BGC was predicted in C. subthermophilus genome and comprised genes coding the enzymes that catalyze formation of the basic anthraquinone skeleton (PKS, metallo-beta-lactamase, decarboxylase, anthrone oxygenase), putative dimerization enzyme (cytochrome P450 monooxygenase), other tailoring enzymes (oxidoreductase, dehydrogenase/reductase), and non-catalytic proteins (fungal transcription factor, transporter protein). CONCLUSIONS: The results provide an insight into genetic background of anthraquinone biosynthesis in Hypericum-borne endophytes. The predicted bisanthraquinone gene cluster represents a basis for functional validation of the candidate biosynthetic genes in a simple eukaryotic system as a prospective biotechnological alternative for production of hypericin and related bioactive anthraquinones.

Heteromorphism of sperm axonemes in a parasitic flatworm, progenetic Diplocotyle olrikii Krabbe, 1874 (Cestoda, Spathebothriidea).

The spermatozoon ultrastructure of the progenetic cestode Diplocotyle olrikii (Spathebothriidea) has been examined using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) for glycogen. The spermatozoon is a filiform cell, tapered at both extremities. Its moderately electron-dense cytoplasm possesses two parallel axonemes of unequal lengths. New for the Cestoda is a finding of three types of the mature spermatozoa with respect to different axonemal structure. The first type has both axonemes with standard 9 + '1' trepaxonematan pattern. The second type is represented by a spermatozoon having one axoneme with 9 + '1' structure and the second one with 9 + 0 pattern. The third type includes the two axonemes with 9 + 0 pattern. Microtubule doublets of the 9 + 0 axonemes contain either inner dynein arms or no dynein arms. In addition to the two axonemes, all three types of the mature sperm cells contain parallel nucleus, parallel cortical microtubules, four electron-dense plaques/attachment zones, and glycogen. The anterior extremity of the gamete exhibits a centriole surrounded by a semiarc of up to five electron-dense tubular structures. The distal end of the first type spermatozoa exhibits two morphological variants, represented either by (i) nucleus or (ii) remnants of the disorganized axoneme. Distal extremity of the spermatozoa of the second and third types contains doublets and singlets of disorganized axoneme. The ultrastructural characters of the spermatozoon of progenetic D. olrikii support the basal position of the Spathebothriidea within the Eucestoda.

Spermiogenesis produces the spermatozoa with 9 + '1' and 9 + 0 axonemal pattern in progenetic cestode Diplocotyle olrikii Krabbe, 1874 (Spathebothriidea: Acrobothriidae).

Spermiogenesis in the progenetic spathebothriidean cestode Diplocotyle olrikii has been examined using transmission electron microscopy (TEM) for the first time. Along with the typical features of spermatozoon cytodifferentiation (e.g., the electron-dense material in the apical region of the differentiation zone in the early stage of spermiogenesis, the intercentriolar body which is composed of three electron-dense plates and two electron-lucent zones, the orthogonal development of the two flagella, a flagellar rotation, proximo-distal fusion, the presence of two pairs of electron-dense attachment zones), new for the Eucestoda is detection of the formation of two types of free flagella during spermiogenesis in progenetic D. olrikii, exhibiting either standard 9 + '1' trepaxonematan pattern, or atypical 9 + 0 structure. Various combinations of these two types of flagella resulted in the production of three types of male gametes during spermiogenesis in this spathebothriidean cestode. The first type is represented with the two axonemes of the 9 + '1' structure; the second type exhibits two different axonemes, i.e., one with 9 + '1' and the other of 9 + 0 pattern; and the third type has two axonemes with atypical 9 + 0 structure. The occurrence of three sperm types in progenetic D. olrikii is associated with typical spermiogenesis and has never been described previously in the Platyhelminthes. We suppose that heteromorphism of male gametes in D. olrikii might be linked to progenesis, i.e., the programmed sexual maturation detected during the larval/developmental stage of an organism.

First ultrastructural and cytochemical data on the spermatozoon and its differentiation in progenetic and adult Archigetes sieboldi Leuckart, 1878 (Cestoda, Caryophyllidea, Caryophyllaeidae).

Spermiogenesis in progenetic and adult stages of Archigetes sieboldi Leuckart, 1878, a tapeworm parasitic in oligochaetes and fish respectively, has been examined using transmission electron microscopy and cytochemical staining for glycogen. General pattern of spermiogenesis is essentially like that of other caryophyllideans, i.e., apical dense material in the zone of differentiation in the early stages of spermiogenesis, rotation of free flagellum and a flagellar bud, and proximo-distal fusion. Interestingly, rotation of a free flagellum and flagellar bud to the median cytoplasmic process (MCP) has been observed unconventionally at > 90° only in progenetic stages. Typical striated roots associated with the centrioles occur rarely in A. sieboldi, and only in form of faint structures in advanced stages of spermiogenesis. In contrast to most caryophyllideans studied to date, penetration of the nucleus into the spermatid body has started before the fusion of the free flagellum with the MCP. This feature has been reported rarely but exclusively in the family Caryophyllaeidae. The unipartite mature spermatozoon of A. sieboldi is composed of one axoneme of the 9 + '1' trepaxonematan pattern with its centriole, parallel nucleus, and parallel cortical microtubules which are situated in a moderately electron-dense cytoplasm with glycogen particles. An unusual arrangement of cortical microtubules in the two parallel rows in region I of the spermatozoon is described here for the first time in the Caryophyllidea. Ultrastructural data on spermiogenesis and the spermatozoon in A. sieboldi from tubuficids and carp are compared and discussed with those in other caryophyllideans and/or Neodermata.

Phytotoxic Effect of Invasive Heracleum mantegazzianum Essential Oil on Dicot and Monocot Species.

Spreading of the plant species in new areas is supported by the hypothesis in which chemicals produced by alien species are allopathic to native plants. A novel weapon hypothesis was tested by using essential oil of dangerous alien species Heracleum mantegazzianum in laboratory conditions. Aboveground plant material was collected in south-east part of Slovakia, dried and hydrodistilled for essential oil isolation. Dominant compounds as octyl acetate (62.6%), hexyl 2-metylbutyrate (10.7%), hexyl isobutyrate (7.5%) and hexyl butyrate (6.5%) were identified by GC-MS. Potential phytotoxic activity was tested on three dicot plant species garden cress (Lepidium sativum L.), radish (Raphanus sativus L.) and lettuce (Lactuca sativa L.) and on one monocot plant species wheat Triticum aestivum L. Germination of the seeds of model plant species after influencing by different doses of essential oil of H. mantegazzianum as well as the roots length was evaluated. Lepidium sativum L. and Raphanus sativus L. were generally not sensitive to applied doses of essential oil although a little stimulation effect at some concentrations prevailed over inhibition effect. Similarly, in monocot species Triticum aestivum L., stimulation was visible in both root length and root number at two or one highest doses, respectively.

Ultrastructural, cytochemistry and electron tomography analysis of Caryophyllaeides fennica (Schneider, 1902) (Cestoda: Lytocestidae) reveals novel spermatology characteristics in the Eucestoda.

Spermatozoon formation in Caryophyllaeides fennica (Schneider, 1902) is characterised by the following: (1) apical electron-dense material in the zone of differentiation, (2) typical striated roots situated unconventionally in opposite directions in early stages of spermiogenesis, (3) intercentriolar body composed of three electron-dense and two electron-lucent plates, (4) free flagellum and a flagellar bud that correspond to a greatly reduced flagellum and (5) rotation of free flagellum and a flagellar bud to the median cytoplasmic process at 90°. The development of two flagella of significantly unequal length clearly supports a derived form of spermiogenesis in the Caryophyllidea. New for cestodes is a finding of two additional striated roots situated opposite each other, in conjunction with both the flagellar bud and free flagellum. Mutual position of additional striated roots and typical striated roots is parallel in early stages and perpendicular in advanced stages of spermiogenesis. A complete proximodistal fusion gives rise to a mature spermatozoon consisting of one axoneme, parallel cortical microtubules, a nucleus and a moderately electron-dense cytoplasm with glycogen particles, detected by a technique of Thiéry (J Microsc 6:987-1018, 1967), in the principal regions (II, III, IV). Electron tomography analysis of the free flagellum and one axoneme of a mature spermatozoon of C. fennica provides clear evidence, for the first time, that two tubular structures are present in the central axonemal electron-dense core. Phylogenetically important aspects of spermiogenesis of the Caryophyllidea with one axoneme, and other cestodes with one or two axonemes, are briefly reviewed and discussed.

UV-Induced Nanoparticles-Formation, Properties and Their Potential Role in Origin of Life.

Inorganic nanoparticles might have played a vital role in the transition from inorganic chemistry to self-sustaining living systems. Such transition may have been triggered or controlled by processes requiring not only versatile catalysts but also suitable reaction surfaces. Here, experimental results showing that multicolor quantum dots might have been able to participate as catalysts in several specific and nonspecific reactions, relevant to the prebiotic chemistry are demonstrated. A very fast and easy UV-induced formation of ZnCd quantum dots (QDs) with a quantum yield of up to 47% was shown to occur 5 min after UV exposure of the solution containing Zn(II) and Cd(II) in the presence of a thiol capping agent. In addition to QDs formation, xanthine activity was observed in the solution. The role of solar radiation to induce ZnCd QDs formation was replicated during a stratospheric balloon flight.

New data on spermiogenesis and trepaxonematan axoneme in basal tapeworms (Cestoda, Caryophyllidea, Lytocestidae) parasitizing cyprinid fishes.

Monozoic caryophyllidean cestodes, intestinal parasites of cyprinid fishes, represent a group of tapeworms with an unclear evolutionary history. As spermatology may provide phylogenetically important data, the spermiogenesis and ultrastructure of the mature spermatozoon have been investigated using an integrative approach combining transmission electron microscopy, cytochemistry and electron tomography in Khawia rossittensis (Szidat, 1937). The process of spermatid formation is accompanied by the presence of ultrastructural characters not described in traditional models of spermiogenesis, e.g., apical electron-dense material, the two striated roots situated unusually opposite each other, branching of typical striated roots, an intercentriolar body comprising five electron-dense and four electron-lucent layers, rotation of both free flagella and flagellar buds to the median cytoplasmic process at 90°, and a complete proximodistal fusion. The synchronous rotation of both flagellar buds and growing free flagella is an evolutionarily linked pattern favouring the hypothesis that the Caryophyllidea are not ancestral but are secondarily derived from polyzoic forms. Electron tomography analysis has revealed a unique feature of two helicoidal tubular structures in the central electron-dense core of the axoneme of mature spermatozoon. These data provide new insights into the architecture of the 9 + '1' axoneme, which is shared by male gametes of all trepaxonematan Platyhelminthes.

Potential Phytotoxic Effect of Essential Oil of Non-Native Species Impatiens parviflora DC.

Impatiens parviflora is non-native invasive plant species occupying large areas all over the Europe and threatens native communities by altering their species composition and reducing native biodiversity. The factor responsible for its spreading could be explained by releasing biochemical to the environment. On the other hands, high demand on secondary metabolites as potential source of new ecofriendly biocides could be beneficial. The analysis of I. parviflora essential oil (EO) led us to identify more than 60 volatiles. The main compound was hexahydrofarnesyl acetone, other dominant components were phytol, carvacrol, germacra-4(15),5,10(14)-trien-1-α-ol, and pentacosane. The potential phytotoxic effect of I. parviflora EO collected in two vegetation periods (summer and autumn) was evaluated on seed germination and root elongation of three dicot species (Raphanus sativus, Lepidum sativum, and Lactuca sativa) and on one monocot species (Triticum aestivum). The seed germination of only one dicot species, L. sativa, was affected by both EOs. In contrast, seed germination of monocot species T. aestivum was influenced only by the highest doses of EOs isolated from I. parviflora in autumn. The root elongation of tested plant species was less influenced by I. parviflora EOs. L. sativum showed sensitivity to one dose of EOs hydrodistilled in summer, while the monocot species was influenced by both EOs samples in highest doses. Our findings revealed that I. parviflora contained phenolics that were phytotoxic to the germination of some plant species, mainly at higher EOs doses, while root elongation of tested plants was not suppressed by essential oils.