Heterothallism revealed in the root rot fungi Berkeleyomyces basicola and B. rouxiae

Berkeleyomyces basicola and Berkeleyomyces rouxiae, two sister species previously treated collectively as Thielaviopsis basicola, reside in the Ceratocystidaceae (Microascales, Ascomycota). Both species are important root pathogens of many important agricultural crops and ornamental plants. Although T. basicola has been known for more than 150y, a sexual state has never been found and it has been assumed to be an asexual pathogen. The aim of this study was to determine the mating strategy of the two Berkeleyomyces species. Investigation of the genome sequences of two B. basicola isolates allowed for the complete characterization of the MATlocus, revealing that it has a typical heterothallic mating system with the MAT1-1andMAT1-2 idiomorphs occurring in different isolates. PCR amplification using mating type primers developed in this study, showed that the MAT1-1-1andMAT1-2-1 genes were also present in different isolates of B. rouxiae. Pairing of isolates representing the two mating types of both species,using a variety of techniques failed to produce sexual structures. Although we have found no direct evidence that they reproduce sexually, these fungi are clearly heterothallic with both mating types occurring in some countries suggesting that a cryptic sexual cycle could exist for them.     

Unveiling Gymnosporangium corniforme, G. unicorne, and G. niitakayamense sp. nov. in Taiwan

Three rust fungi from high mountains and pear-producing areas in Taiwan were described using morphological and molecular data based on 34 specimens. Gymnosporangium corniforme was demonstrated to produce spermogonia and aecia on Sorbus randaiensis based on molecular analyses and inoculation experiments. The pear rust pathogen G. unicorne was discovered in Taiwan for the first time. Gymnosporangium niitakayamense sp. nov. was observed on the leaves of Photinia niitakayamensis. It was distinct from other species in peridial cell wall structures, i.e., smooth outer wall, rugose side wall, and coralloid projections on the inner wall, and in having echinulate aeciospores.     

Hidden diversity uncovered in Hygrophorus sect. Aurei (Hygrophoraceae), including the Mediterranean H. meridionalis and the North American H. boyeri, spp. nov.

For many years, the binomial Hygrophorus hypothejus was widely applied to collections from various geographical regions in different continents, assuming a circum-boreal and circum-mediterranean distribution for this species. This hypothesis, however, had never been put to the test. To assess the diversity and species-limits within this complex of yellow-coloured waxcaps, a phylogenetic, morphological and taxonomical investigation into Hygrophorus sect. Aurei and similar species in sect. Olivaceoumbrini was carried out, including material of pan-European origin, as well as the east and west coasts of North America. Following sequencing of the ITS rDNA locus, nine lineages are confirmed in sect. Aurei, most of them highly continentalised. Of these, two are new to science, introduced here as Hygrophorus boyeri sp. nov., from Pinus banksiana and P. rigida forests in eastern North America and from P. muricata and P. contorta forests in western North America, and Hygrophorus meridionalis sp. nov., from Pinus brutia and Pinus halepensis forests in the island of Cyprus and mainland Greece. H. hypothejus is lectotypified and epitypified, and here resolved as a strictly European species, with the old forgotten taxon Hygrophorus siccipes revived as its North American vicariant. The placement of Hygrophorus fuligineus in sect. Aurei is phylogenetically confirmed and detailed comparisons between morphologically similar and phylogenetically affiliated taxa in sect. Aurei and sect. Olivaceoumbrini are provided. The chronic confusion associated with Hygrophorus fuscoalbus, a highly controversial taxon described from Germany nearly two centuries ago and variously interpreted since, is discussed, concluding that this name is too ambiguous to be applied to any currently recognized species.     

Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli

Poly(ethylene terephthalate) (PET) is the most commonly used polyester polymer resin in fabrics and storage materials, and its accumulation in the environment is a global problem. The ability of PET hydrolase from Ideonella sakaiensis 201-F6 (IsPETase) to degrade PET at moderate temperatures has been studied extensively. However, due to its low structural stability and solubility, it is difficult to apply standard laboratory-level IsPETase expression and purification procedures in industry. To overcome this difficulty, the expression of IsPETase can be improved by using a secretion system. This is the first report on the production of an extracellular IsPETase, active against PET film, using Sec-dependent translocation signal peptides from E. coli. In this work, we tested the effects of fusions of the Sec-dependent and SRP-dependent signal peptides from E. coli secretory proteins into IsPETase, and successfully produced the extracellular enzyme using pET22b-SP_MalE:IsPETase and pET22b-SP_LamB:IsPETase expression systems. We also confirmed that the secreted IsPETase has PET-degradation activity. The work will be used for development of a new E. coli strain capable of degrading and assimilating PET in its culture medium.     

Multiple β-galactosidase activities in petunia hybrida: Separation and characterization

Using isoelectrofocusing (IEF), multiple forms of Petunia β-galactosidase activity could be detected. The β-galactosidase pattern showed only minor tissue-specific differences. There were, however, species-specific differences. Zea mays, for instance, showed two bands which differed from the zones obtained with Petunia preparations. Petunia and corn leaves were mixed and extracted commonly. The species-specific activity patterns remained unchanged.Petunia preparations were inactivated by 8 Murea. Following dialysis, enzymatic activity and the Petunia-specific pattern were restored. The same holds true for a mixture of Petunia and E. coli β-galactosidase preparations. On refocusing isolated Petunia zones, untreated or inactivated by 8 M urea and reactivated by dialysis, the original mobilities were shown. Therefore, it seems highly improbable that the β-galactosidase pattern was due to artefacts. Using a Petunia line which was ‘pure’, also in respect to its β-galactosidase pattern, the four main bands were preparatively separated by IEF and characterized. They showed the same pH optimum (4.3), the same temperature optimum (55°), the same inactivation kinetics by urea, the same sensitivity against Cl^?, and closely related K_m. values. In sucrose gradient centrifugation they invariably showed S values of 8–10. The multiple activities could not be separated by zone electrophoresis using various carrier systems, or by gel filtration. It seems possible that they represent forms which differ only in isoelectric points, not in MW.     

Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS

High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MSⁿ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MSⁿ detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.     

5′-Monohydroxyphylloquinone from Anacystis and Euglena

The monohydroxy analogue of phylloquinone found in Anacystis nidulans and Euglena gracilis has been characterized as 5′-monohydroxyphylloquinone by MS analysis.     

Hybridization and introgression in Carex aquatilis and C. paleacea

The diversification and distribution of Abies species throughout the Mediterranean region has led to a complex of species which are difficult to classify. An open question is whether these mainly allopatric taxa have exchanged genetic information via secondary contact. We studied the variation and geographic distribution of paternally inherited chloroplast DNA markers in nine Mediterranean Abies taxa. Markers with high and low mutation rates were applied in order to differentiate among a scenario of secondary genetic contact and a scenario of complete isolation after speciation. The observed molecular variation was analysed using statistical parsimony, geostatistics, and measures of population genetic differentiation. Ancient paternal lineages, represented by markers with low mutation rates, were shared among species. The central and widespread A. alba retained all ancient lineages whereas other species exhibited fewer, down to a single lineage. In contrast, modern lineages, depicted by markers with high mutation rates, were largely separated among species. The western Mediterranean A. pinsapo and A. numidica were clearly separated from each other and from the remaining Abies species. This indicates the absence of secondary contact. The same scenario applies to the eastern Mediterranean Abies species. An exception is the parapatric complex of A. alba, A. cephalonica, and their supposed hybrid A. borisii regis, which exhibited evidence of secondary contact.     

Acute toxicity,critical body residues,Michaelis–Menten analysis of bioaccumulation,and ionoregulatory disturbance in response to waterborne nickel in four invertebrates: Chironomus riparius, Lymnaea stagnalis, Lumbriculus variegatus and Daphnia pulex

We investigated the bioaccumulation and acute toxicity (48 h or 96 h) of Ni in four freshwater invertebrate species in two waters with hardness of 40 (soft water) and 140 mg L^− 1 as CaCO₃ (hard water). Sensitivity order (most to least) was Lymnaea stagnalis > Daphnia pulex > Lumbriculus variegatus > Chironomus riparius. In all cases water hardness was protective against acute Ni toxicity with LC50 values 3–3.5 × higher in the hard water vs. soft water. In addition, higher water hardness significantly reduced Ni bioaccumulation in these organisms suggesting that competition by Ca and Mg for uptake at the biotic ligand may contribute to higher metal resistance. CBR50 values (Critical Body Residues) were less dependent on water chemistry (i.e. more consistent) than LC50 values within and across species by ~ 2 fold. These data support one of the main advantages of the Tissue Residue Approach (TRA) where tissue concentrations are generally less variable than exposure concentrations with respect to toxicity. Whole body Ni bioaccumulation followed Michaelis–Menten kinetics in all organisms, with greater hardness tending to decrease B_max with no consistent effect on K_d. Across species, acute Ni LC50 values tended to increase with both K_d and B_max values — i.e. more sensitive species exhibited higher binding affinity and lower binding capacity for Ni, but there was no correlation with body size. With respect to biotic ligand modeling, log K_NiBL values derived from Ni bioaccumulation correlated well with log K_NiBL values derived from toxicity testing. Both whole body Na and Mg levels were disturbed, suggesting that disruption of ionoregulatory homeostasis is a mechanism of acute Ni toxicity. In L. stagnalis, Na depletion was a more sensitive endpoint than mortality, however, the opposite was true for the other organisms. This is the first study to show the relationship between Na and Ni.     

Isoform- and dose-sensitive feedback interactions between paired box 6 gene and δ-catenin in cell differentiation and death

Pax6, a mammalian homolog of the Drosophila paired box gene family member expressed in stem and progenitor cells, resides at the top of the genetic hierarchy in controlling cell fates and morphogenesis. While Pax6 activation can lead to mitotic arrest, premature neurogenesis, and apoptosis, the underlying molecular mechanisms have not been resolved. Here we report that either Pax6(+5a) or Pax6(-5a) was sufficient to promote, whereas their knockdown reduced the expression of δ-catenin (CTNND2), a neural specific member of the armadillo/β-catenin superfamily. Pax6(+5a) elicited stronger effects on δ-catenin than Pax6(-5a). Inducible Pax6(+5a) expression demonstrated a biphasic and dose-dependent regulation of δ-catenin expression and cell fates. A moderate upregulation of Pax6(+5a) promoted δ-catenin expression and induced neurite-like cellular protrusions, but increasing expression of Pax6(+5a) reversed these processes. Furthermore, sustained high expression of Pax6(+5a) triggered apoptosis as determined by the reduction of phospho-Bad, Bcl-2, survivin and procaspases, as well as the increases in Bax and cleaved poly(ADP-ribose) polymerase. Importantly, re-introducing δ-catenin by ectopic expression elicited a feedback suppression on Pax6(+5a) expression and reduced Pax6(+5a) induced apoptosis. Therefore, δ-catenin expression is not only controlled by Pax6, but it also provides a feedback suppression mechanism for their functional interactions with important implications in cellular morphogenesis, apoptosis, and cancer.     

Nucleotide diversity and minisatellite in chloroplast Asp(GUC)–Thr(GGU) region in Elymus trachycaulus complex, Elymus alaskanus and Elymus caninus

Nucleotide variation in chloroplast Asp(GUC)–Thr(GGU) intergenic region and genetic relationships among this group were examined among Elymus trachycaulus complex, Elymus alaskanus and Elymus caninus. The estimates of nucleotide diversity (π) ranged from 0.00111 for Elymus virescens to 0.03086 for E. caninus. Highest nucleotide diversity was found for E. caninus among the taxa analyzed here and followed by Elymus hyperarcticus. E. virescens accessions are genetically very uniform. Phylogenetic analysis suggested that E. caninus is paraphyletic. Elymus violaceus is genetically distinct from both E. alaskanus and E. trachycaulus. Our result indicates that Asp (GUC)–Thr (GGU) intergenic region has a high rate of evolutionary in Elymus species. Large indels detected in this region appear to have a highly rate of evolution and are thus more prone to homoplasy. We also first reported a minisatellite discovered in Asp (GUC)–Thr (GGU) region in Elymus species. The minisatellite identified here is an excellent candidate marker for studying population structures of Elymus species.     

Importance of spores and δ-endotoxin protein crystals of Bacillus thuringiensis in Galleria mellonella

Larvae of Galleria mellonella were fed on a honey-rich artificial food containing live spores or toxic crystals of Bacillus thuringiensis serotype V or various combinations of both. In this food; 1:1 combinations were 10 times more potent than live spores alone and about 10⁴ times more potent than crystals alone. Reduction in the proportion of spores, but not in that of crystals, decreased the slope of probit lines from 3.4 to 0.6. One or more factors in the spore are at least partly responsible for the potency of serotype V in G. mellonella. The results suggest than an observed gross loss of potency of this serotype in beehives is more likely to be due to death of spores than to deterioration of crystals. The reaction of G. mellonella to serotype V is nearest to that of a type 3 host species. Spores of serotype I are almost inactive in this host.     

Relationship between GSTM1 and GSTT1 polymorphisms and schizophrenia: A case–control study in a Tunisian population

There is substantial evidence found in the literature that supports the fact that the presence of oxidative stress may play an important role in the pathophysiology of schizophrenia. The glutathione S-transferases (GSTs) forms one of the major detoxifying groups of enzymes responsible for eliminating products of oxidative stress. Interindividual differences observed in the metabolism of xenobiotics have been attributed to the genetic polymorphism of genes coding for enzymes involved in detoxification. Thus, in this study we investigated the association of glutathione S-transferase Mu-1 (GSTM1) and glutathione S-transferase theta-1 (GSTT1) gene deletion polymorphisms and schizophrenia in a Tunisian population. A case–control study including 138 schizophrenic patients and 123 healthy controls was enrolled. The GSTM1 and GSTT1 polymorphisms were analyzed by multiplex polymerase chain reaction (PCR). No association was found between the GSTM1 genotype and schizophrenia, whereas the prevalence of the GSTT1 active genotype was significantly higher in the schizophrenic patients (57.2%) than in the controls (45.5%) with (OR = 0.6, IC 0.37–0.99, p = 0.039). Thus, we noted a significant association between schizophrenia and GSTT1 active genotype. Furthermore, the combination of the GSTM1 and GSTT1 null genotypes showed a non-significant trend to an increased risk of schizophrenia. The present finding indicated that GSTT1 seems to be a candidate gene for susceptibility to schizophrenia in at least Tunisian population.     

Hormonal aspects of oögenesis in the flies Phormia regina and Sarcophaga bullata

The corpora allata (CA) and median neurosecretory cells (MNC) of Phormia regina and Sarcophaga bullata become active with increasing age of the fly, on a diet of sugar alone. To prevent or retard oögenesis the CA or MNCs must be removed shortly after emergence, with subsequent protein meals. Topical JH application partially compensates for CA or MNC removal. This shows that the MNC activate the CA, and not vice versa. The trauma of either operation slightly depresses egg development.Injection of ecdysone into both species in the stage of initial yolk deposition causes the primary oöcytes to degenerate. This leads to development of the penultimate oöcytes. Older and younger egg stages are not sensitive to ecdysone. In P. regina the application of JH to females with developing primary oöcytes stimulates yolk deposition in the penultimate oöcytes.     

Candida species in tchapalo and bangui, two traditional alcoholic beverages from Côte d'Ivoire

The increase of infections due to non-Candida albicans species made it very necessary to conduct adequate characterization to be able to identify the species of Candida isolated from traditional fermented foods. In this study, based on their hue on Candida Chromogenic Agar medium, a total of 136 yeast strains were isolated from tchapalo and bangui. Molecular identification based on PCR-RFLP of internal transcribed spacers of rDNA (ITS) and sequencing of the ITS and the D1/D2 regions allowed us to assign these isolates to seven species: Candida tropicalis, Candida inconspicua, Candida rugosa, Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Trichosporon asahii. With the respect to each beverage, six species were found among with four species are regarded as opportunistic pathogens. From these, C. tropicalis, C. inconspicua and K. marxianus were the most commonly encountered. The enzyme activities of the potential pathogens assessed using API ZYM system showed that almost strains had esterase, esterase lipase, valine and cystine arylamidase, alpha chymotrypsin, alkaline phosphatase and naphthol phosphohydrolase activities. The activity of α-glucosidase was found only in C. tropicalis and C. inconspicua strains isolated from tchapalo while β-glucosidase activity was found in all strains from tchapalo and only in C. inconspicua isolated from bangui.     

The Developmental Changes and Effect on IMF Content of H-FABP and PPARγ mRNA Expression in Sheep Muscle

Male Kazak sheep and Xinjiang fine wool sheep of different ages were selected to investigate the developmental changes and effect on intramuscular fat (IMF) content of heart fatty acid-binding protein (H-FABP) and peroxisome proliferator-activated receptor γ (PPARγ) mRNA expression in muscle. Longissimus dorsal muscle was sampled to measure IMF content; and total RNA was extracted to determine H-FABP and PPARγ mRNA expression levels by real-time PCR. The results showed that: (1) The IMF content increased continuously with growing and showed significant differences (P<0.05) between ages in male Kazak sheep, but no such differences (P>0.05) existed in Xinjiang fine wool sheep. Furthermore, the IMF content in Kazak sheep was very much higher (P<0.01) than that of the other breed from day 30 to 90; (2) H-FABP mRNA expression level was the highest on day 2 and showed significant differences (P<0.05) between ages in male Kazak sheep as well as in Xinjiang fine wool sheep. In the former breed, the expression reached the lowest point at day 30, and then rose continuously. But in the latter breed, it declined continuously from day 2 to 90, and then increased; (3) Significant differences (P<0.05) of PPARγ mRNA expression between ages occurred in both breeds. In male Kazak sheep, PPARγ mRNA expression declined from day 2 to 90, while in the other breed it increased continuously from day 2 to 60, but reached the lowest level at day 90, then increased; (4) In male Kazak sheep, the mRNA expression level of H-FABP was highly positively correlated (r=0.737, P<0.01) with IMF content from day 30 to 90, but that of PPARγ was highly negatively correlated (r=−0.835, P<0.01) with IMF content from day 2 to 90.     

In vitro, ex vivo and in vivo models: A comparative analysis of Paracoccidioides spp. proteomic studies

Members of the Paracoccidioides complex are human pathogens that infect different anatomic sites in the host. The ability of Paracoccidioides spp. to infect host niches is putatively supported by a wide range of virulence factors, as well as fitness attributes that may comprise the transition from mycelia/conidia to yeast cells, response to deprivation of micronutrients in the host, expression of adhesins on the cell surface, response to oxidative and nitrosative stresses, as well as the secretion of hydrolytic enzymes in the host tissue. Our understanding of how those molecules can contribute to the infection establishment has been increasing significantly, through the utilization of several models, including in vitro, ex vivo and in vivo infection in animal models. In this review we present an update of our understanding on the strategies used by the pathogen to establish infection. Our results were obtained through a comparative proteomic analysis of Paracoccidioides spp. in models of infection.