Phylogenetic placement of Iodosphaeriaceae (Xylariales, Ascomycota), designation of an epitype for the type species of Iodosphaeria, I. phyllophila, and description of I. foliicola sp. nov.

The Iodosphaeriaceae is represented by the single genus, Iodosphaeria, which is composed of nine species with superficial, black, globose ascomata covered with long, flexuous, brown hairs projecting from the ascomata in a stellate fashion, unitunicate asci with an amyloid apical ring or ring lacking and ellipsoidal, ellipsoidal-fusiform or allantoid, hyaline, aseptate ascospores. Members of Iodosphaeria are infrequently found worldwide as saprobes on various hosts and a wide range of substrates. Only three species have been sequenced and included in phylogenetic analyses, but the type species, I. phyllophila, lacks sequence data. In order to stabilize the placement of the genus and family, an epitype for the type species was designated after obtaining ITS sequence data and conducting maximum likelihood and Bayesian phylogenetic analyses. Iodosphaeria foliicola occurring on overwintered Alnus sp. leaves is described as new. Five species in the genus form a well-supported monophyletic group, sister to the Pseudosporidesmiaceae in the Xylariales. Selenosporella-like and/or ceratosporium-like synasexual morphs were experimentally verified or found associated with ascomata of seven of the nine accepted species in the genus. Taxa included and excluded from Iodosphaeria are discussed.

Genotyping-by-Sequencing for Analysis of the Genetic Variation of Podosphaera xanthii, Incitant of Cucurbit Powdery Mildew.

This research was conducted to identify species causing powdery mildew on cucurbits and to determine genetic variations among isolates of the pathogen. We collected 109 isolates from six cucurbit species hosts (Cucumis melo, Cucumis sativus, Cucurbita maxima, Cucurbita moschata, Cucurbita pepo, and Lagenaria siceraria) in California, Illinois, Indiana, Michigan, New York, Texas, Washington, and Wisconsin in the United States and in Italy. By sequencing the internal transcribed spacer region of the nuclear rDNA of these 109 isolates, Podosphaera xanthii was found as the only species causing powdery mildew on cucurbits in the United States. Genotyping-by-sequencing was applied to these 109 isolates to investigate their genetic diversity, which showed a trend of isolates clustering from New York and Italy. In addition, the virulence of 36 isolates was compared and a significant difference (P < 0.0001) was found among them. Furthermore, results of the virulence tests of 28 isolates from Illinois showed significant effects of collection years, hosts, and locations on the virulence of the isolates.

Phylogenetic classification and generic delineation of Calyptosphaeria gen. nov., Lentomitella, Spadicoides and Torrentispora (Sordariomycetes).

The genus Ceratostomella has a long history of taxonomic confusion. While species with evanescent asci have been transferred to the Microascales and Ophiostomatales, the taxonomic status of species with persistent asci has not been completely resolved. In previous studies using DNA sequence data, cultures and morphology, several Ceratostomella spp. were allocated in 13 genera in the Eurotiomycetes and Sordariomycetes. In our study, the systematics of the remaining Ceratostomella spp. with persistent asci is revisited with new collection data, cultures and phylogeny based on novel DNA sequences from six nuclear loci. Bayesian inference and Maximum Likelihood analyses support the monophyly of several wood-inhabiting species formerly classified in Ceratostomella and other unknown morphologically similar taxa and their division into four genera, i.e. Lentomitella, Spadicoides, Torrentispora and the newly described Calyptosphaeria. This robust clade represents the order Xenospadicoidales in the Sordariomycetidae. Comparative analysis of the ITS2 secondary structure revealed a genetic variation among Lentomitella isolates; 11 species were recognised, of which five are newly introduced and two are new combinations. Other taxonomic novelties include four new species and eight new combinations in Calyptosphaeria, Spadicoides, and Torrentispora. Molecular data suggest that Spadicoides is polyphyletic. The core of the genus is positioned in the Xenospadicoidales; Spadicoides s. str. is experimentally linked with sexual morphs for the first time. Based on DNA sequence data, the monotypic genera Xenospadicoides and Pseudodiplococcium are reduced to synonymy under Spadicoides, while Fusoidispora and Pseudoannulatascus are synonymised with Torrentispora. Members of the Xenospadicoidales inhabit decaying wood in terrestrial and freshwater environments and share a few morphological characters such as the absence of stromatic tissue, ascomata with a cylindrical or rostrate neck, similar anatomies of the ascomatal walls, thin-walled unitunicate asci with a non-amyloid apical annulus, disintegrating paraphyses, usually ellipsoidal to fusiform ascospores and holoblastic-denticulate or tretic conidiogenesis. Revised Ceratostomella spp. with persistent asci are listed and the taxonomic status of each species is re-evaluated based on revision of the holotype and other representative material, published details and available phylogenetic data.

Fungal Planet description sheets: 625-715.

Novel species of fungi described in this study include those from various countries as follows: Antarctica: Cadophora antarctica from soil. Australia: Alfaria dandenongensis on Cyperaceae, Amphosoma persooniae on Persoonia sp., Anungitea nullicana on Eucalyptus sp., Bagadiella eucalypti on Eucalyptus globulus, Castanediella eucalyptigena on Eucalyptus sp., Cercospora dianellicola on Dianella sp., Cladoriella kinglakensis on Eucalyptus regnans, Cladoriella xanthorrhoeae (incl. Cladoriellaceae fam. nov. and Cladoriellales ord. nov.) on Xanthorrhoea sp., Cochlearomyces eucalypti (incl. Cochlearomyces gen. nov. and Cochlearomycetaceae fam. nov.) on Eucalyptus obliqua, Codinaea lambertiae on Lambertia formosa, Diaporthe obtusifoliae on Acacia obtusifolia, Didymella acaciae on Acacia melanoxylon, Dothidea eucalypti on Eucalyptus dalrympleana, Fitzroyomyces cyperi (incl. Fitzroyomyces gen. nov.) on Cyperaceae, Murramarangomyces corymbiae (incl. Murramarangomyces gen. nov., Murramarangomycetaceae fam. nov. and Murramarangomycetales ord. nov.) on Corymbia maculata, Neoanungitea eucalypti (incl. Neoanungitea gen. nov.) on Eucalyptus obliqua, Neoconiothyrium persooniae (incl. Neoconiothyrium gen. nov.) on Persoonia laurina subsp. laurina, Neocrinula lambertiae (incl. Neocrinulaceae fam. nov.) on Lambertia sp., Ochroconis podocarpi on Podocarpus grayae, Paraphysalospora eucalypti (incl. Paraphysalospora gen. nov.) on Eucalyptus sieberi, Pararamichloridium livistonae (incl. Pararamichloridium gen. nov., Pararamichloridiaceae fam. nov. and Pararamichloridiales ord. nov.) on Livistona sp., Pestalotiopsis dianellae on Dianella sp., Phaeosphaeria gahniae on Gahnia aspera, Phlogicylindrium tereticornis on Eucalyptus tereticornis, Pleopassalora acaciae on Acacia obliquinervia, Pseudodactylaria xanthorrhoeae (incl. Pseudodactylaria gen. nov., Pseudodactylariaceae fam. nov. and Pseudodactylariales ord. nov.) on Xanthorrhoea sp., Pseudosporidesmium lambertiae (incl. Pseudosporidesmiaceae fam. nov.) on Lambertia formosa, Saccharata acaciae on Acacia sp., Saccharata epacridis on Epacris sp., Saccharata hakeigena on Hakea sericea, Seiridium persooniae on Persoonia sp., Semifissispora tooloomensis on Eucalyptus dunnii, Stagonospora lomandrae on Lomandra longifolia, Stagonospora victoriana on Poaceae, Subramaniomyces podocarpi on Podocarpus elatus, Sympoventuria melaleucae on Melaleuca sp., Sympoventuria regnans on Eucalyptus regnans, Trichomerium eucalypti on Eucalyptus tereticornis, Vermiculariopsiella eucalypticola on Eucalyptus dalrympleana, Verrucoconiothyrium acaciae on Acacia falciformis, Xenopassalora petrophiles (incl. Xenopassalora gen. nov.) on Petrophile sp., Zasmidium dasypogonis on Dasypogon sp., Zasmidium gahniicola on Gahnia sieberiana.Brazil: Achaetomium lippiae on Lippia gracilis, Cyathus isometricus on decaying wood, Geastrum caririense on soil, Lycoperdon demoulinii (incl. Lycoperdon subg. Arenicola) on soil, Megatomentella cristata (incl. Megatomentella gen. nov.) on unidentified plant, Mutinus verrucosus on soil, Paraopeba schefflerae (incl. Paraopeba gen. nov.) on Schefflera morototoni, Phyllosticta catimbauensis on Mandevilla catimbauensis, Pseudocercospora angularis on Prunus persica, Pseudophialophora sorghi on Sorghum bicolor, Spumula piptadeniae on Piptadenia paniculata.Bulgaria: Yarrowia parophonii from gut of Parophonus hirsutulus. Croatia: Pyrenopeziza velebitica on Lonicera borbasiana.Cyprus: Peziza halophila on coastal dunes. Czech Republic: Aspergillus contaminans from human fingernail. Ecuador: Cuphophyllus yacurensis on forest soil, Ganoderma podocarpense on fallen tree trunk. England: Pilidium anglicum (incl. Chaetomellales ord. nov.) on Eucalyptus sp. France: Planamyces parisiensis (incl. Planamyces gen. nov.) on wood inside a house. French Guiana: Lactifluus ceraceus on soil. Germany: Talaromyces musae on Musa sp. India: Hyalocladosporiella cannae on Canna indica, Nothophoma raii from soil. Italy: Setophaeosphaeria citri on Citrus reticulata, Yuccamyces citri on Citrus limon.Japan: Glutinomyces brunneus (incl. Glutinomyces gen. nov.) from roots of Quercus sp. Netherlands (all from soil): Collariella hilkhuijsenii, Fusarium petersiae, Gamsia kooimaniorum, Paracremonium binnewijzendii, Phaeoisaria annesophieae, Plectosphaerella niemeijerarum, Striaticonidium deklijnearum, Talaromyces annesophieae, Umbelopsis wiegerinckiae, Vandijckella johannae (incl. Vandijckella gen. nov. and Vandijckellaceae fam. nov.), Verhulstia trisororum (incl. Verhulstia gen. nov.). New Zealand: Lasiosphaeria similisorbina on decorticated wood. Papua New Guinea: Pseudosubramaniomyces gen. nov. (based on Pseudosubramaniomyces fusisaprophyticus comb. nov.). Slovakia: Hemileucoglossum pusillum on soil. South Africa: Tygervalleyomyces podocarpi (incl. Tygervalleyomyces gen. nov.) on Podocarpus falcatus.Spain: Coniella heterospora from herbivorous dung, Hymenochaete macrochloae on Macrochloa tenacissima, Ramaria cistophila on shrubland of Cistus ladanifer.Thailand: Polycephalomyces phaothaiensis on Coleoptera larvae, buried in soil. Uruguay: Penicillium uruguayense from soil. Vietnam: Entoloma nigrovelutinum on forest soil, Volvariella morozovae on wood of unknown tree. Morphological and culture characteristics along with DNA barcodes are provided.

Generic circumscriptions in Geoglossomycetes.

The class Geoglossomycetes is a recently created class of Ascomycota, currently comprised of one family (Geoglossaceae) and five genera (Geoglossum, Nothomitra, Sarcoleotia, Thuemenidium and Trichoglossum). These fungi, commonly known as earth tongues, have long been a subject of mycological research. However, the taxonomy within the group has historically been hindered by the lack of reliable morphological characters, uncertain ecological associations, and the inability to grow these fungi in culture. The phylogenetic relationships of Geoglossomycetes were investigated by conducting maximum likelihood and Bayesian analyses using a 4-gene dataset (ITS, LSU, MCM7, RPB1). Five well-supported monophyletic clades were found that did not correspond exactly with the currently recognised genera, necessitating a taxonomic revision of the group. Two new genera are proposed: Glutinoglossum to accommodate G. glutinosum and the newly described species G. heptaseptatum, and Sabuloglossum to accommodate S. arenarium. The type species of Thuemenidium, traditionally included within the Geoglossaceae, is confirmed as belonging to a separate lineage that is only distantly related to Geoglossomycetes.

A new species of Acanthostigma (Tubeufiaceae, Dothideomycetes) from the southern hemisphere.

A new species belonging to the Dothideomycete genus Acanthostigma is described from bark of two Nothofagus species from Argentina. Its identity as a new species is based on both morphology and molecular sequence data. Acanthostigma patagonica differs from other species in the genus by having larger ascomata and setae and wider, asymmetrical ascospores. An amended key to Acanthostigma species is provided along with a discussion of other species previously described from South America.

A molecular re-appraisal of taxa in the Sordariomycetidae and a new species of Rimaconus from New Zealand.

Several taxa that share similar ascomatal and ascospore characters occur in monotypic or small genera throughout the Sordariomycetidae with uncertain relationships based on their morphology. Taxa in the genera Duradens, Leptosporella, Linocarpon, and Rimaconus share similar morphologies of conical ascomata, carbonised peridia and elongate ascospores, while taxa in the genera Caudatispora, Erythromada and Lasiosphaeriella possess clusters of superficial, obovoid ascomata with variable ascospores. Phylogenetic analyses of 28S large-subunit nrDNA sequences were used to test the monophyly of these genera and provide estimates of their relationships within the Sordariomycetidae. Rimaconus coronatus is described as a new species from New Zealand; it clusters with the type species, R. jamaicensis. Leptosporella gregaria is illustrated and a description is provided for this previously published taxon that is the type species and only sequenced representative of the genus. Both of these genera occur in separate, well-supported clades among taxa that form unsupported groups near the Chaetosphaeriales and Helminthosphaeriaceae. Lasiosphaeriella and Linocarpon appear to be polyphyletic with species occurring in several clades throughout the subclass. Caudatispora and Erythromada represented by single specimens and two putative Duradens spp. have unclear affinities in the Sordariomycetidae.

Growth factor and patient-adapted use of plerixafor is superior to CY and growth factor for autologous hematopoietic stem cells mobilization.

The ideal method to mobilize autologous hematopoietic stem cells (AHSCs) in patients with lymphoma or multiple myeloma remains to be determined. The use of plerixafor, added to growth factor, may overcome the limitations to the use of growth factor mobilization without chemotherapy. We developed and validated a cost-based decision-making algorithm that uses the CD34+ cell count in the peripheral blood on the fourth day of G-CSF administration and the target CD34+ cell count for the specific patient to decide on the use of plerixafor (MUSC algorithm). We compared this approach (MA cohort) with a historical cohort of patients undergoing mobilization with CY 2000 mg/m(2) followed by G-CSF and GM-CSF (CY cohort). Fifty individuals are included in the MA cohort and 81 in the CY cohort. The mobilization failure rate was 2% in the MA cohort vs 22% in the CY cohort (P=0.01). Fewer patients in the MA cohort than in the CY cohort had infectious complications during mobilization requiring hospitalization (2 vs 30% P<0.01). There was significant shortening in the median number of days between starting mobilization and undergoing transplantation in the MA cohort (14 vs 43 days, P<0.01). In conclusion, growth factor and patient-adapted use of plerixafor provides safer hematopoietic stem cell mobilization and faster access to AHSC transplantation.

DNA copy number aberrations in small-cell lung cancer reveal activation of the focal adhesion pathway.

Small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer in its clinical behavior, with a 5-year overall survival as low as 5%. Despite years of research in the field, molecular determinants of SCLC behavior are still poorly understood, and this deficiency has translated into an absence of specific diagnostics and targeted therapeutics. We hypothesized that tumor DNA copy number alterations would allow the identification of molecular pathways involved in SCLC progression. Array comparative genomic hybridization was performed on DNA extracted from 46 formalin-fixed paraffin-embedded SCLC tissue specimens. Genomic profiling of tumor and sex-matched control DNA allowed the identification of 70 regions of copy number gain and 55 regions of copy number loss. Using molecular pathway analysis, we found a strong enrichment in these regions of copy number alterations for 11 genes associated with the focal adhesion pathway. We verified these findings at the genomic, gene expression and protein level. Focal Adhesion Kinase (FAK), one of the central genes represented in this pathway, was commonly expressed in SCLC tumors and constitutively phosphorylated in SCLC cell lines. Those were poorly adherent to most substrates but not to laminin-322. Inhibition of FAK phosphorylation at Tyr(397) by a small-molecule inhibitor, PF-573,228, induced a dose-dependent decrease of adhesion and an increase of spreading in SCLC cell lines on laminin-322. Cells that tended to spread also showed a decrease in focal adhesions, as demonstrated by a decreased vinculin expression. These results support the concept that pathway analysis of genes in regions of copy number alterations may uncover molecular mechanisms of disease progression and demonstrate a new role of FAK and associated adhesion pathways in SCLC. Further investigations of FAK at the functional level may lead to a better understanding of SCLC progression and may have therapeutic implications.

The radiological evaluation of acetabular fractures in the elderly.

We have examined the accuracy of reduction and the functional outcomes in elderly patients with surgically treated acetabular fractures, based on assessment of plain radiographs and CT scans. There were 45 patients with such a fracture with a mean age of 67 years (59 to 82) at the time of surgery. All patients completed SF-36 questionnaires to determine the functional outcome at a mean follow-up of 72.4 months (24 to 188). All had radiographs and a CT scan within one week of surgery. The reduction was categorised as 'anatomical', 'imperfect', or 'poor'. Radiographs classified 26 patients (58%) as anatomical,13 (29%) as imperfect and six (13%) as poor. The maximum displacement on CT showed none as anatomical, 23 (51%) as imperfect and 22 (49%) as poor, but this was not always at the weight-bearing dome. SF-36 scores showed functional outcomes comparable with those of the general elderly population, with no correlation with the radiological reduction. Perfect anatomical reduction is not necessary to attain a good functional outcome in acetabular fractures in the elderly.

A molecular phylogenetic reappraisal of the Hysteriaceae, Mytilinidiaceae and Gloniaceae (Pleosporomycetidae, Dothideomycetes) with keys to world species.

A reappraisal of the phylogenetic integrity of bitunicate ascomycete fungi belonging to or previously affiliated with the Hysteriaceae, Mytilinidiaceae, Gloniaceae and Patellariaceae is presented, based on an analysis of 121 isolates and four nuclear genes, the ribosomal large and small subunits, transcription elongation factor 1 and the second largest RNA polymerase II subunit. A geographically diverse and high density taxon sampling strategy was employed, including multiple isolates/species from the following genera: Anteaglonium (6/4), Encephalographa (1/1), Farlowiella (3/1), Gloniopsis (8/4), Glonium (4/2), Hysterium (12/5), Hysterobrevium (14/3), Hysterographium (2/1), Hysteropatella (2/2), Lophium (4/2), Mytilinidion (13/10), Oedohysterium (5/3), Ostreichnion (2/2), Patellaria (1/1), Psiloglonium (11/3), Quasiconcha (1/1), Rhytidhysteron (8/3), and 24 outgroup taxa. Sequence data indicate that although the Hysteriales are closely related to the Pleosporales, sufficient branch support exists for their separation into separate orders within the Pleosporomycetidae. The Mytilinidiales are more distantly related within the subclass and show a close association with the Gloniaceae. Although there are examples of concordance between morphological and molecular data, these are few. Molecular data instead support the premise of a large number of convergent evolutionary lineages, which do not correspond to previously held assumptions of synapomorphy relating to spore morphology. Thus, within the Hysteriaceae, the genera Gloniopsis, Glonium, Hysterium and Hysterographium are highly polyphyletic. This necessitated the transfer of two species of Hysterium to Oedohysteriumgen. nov. (Od. insidenscomb. nov. and Od. sinense comb. nov.), the description of a new species, Hysterium barrianumsp. nov., and the transfer of two species of Gloniopsis to Hysterobreviumgen. nov. (Hb. smilaciscomb. nov. and Hb. constrictumcomb. nov.). While Hysterographium, with the type Hg. fraxini, is removed from the Hysteriaceae, some of its species remain within the family, transferred here to Oedohysterium (Od. pulchrumcomb. nov.), Hysterobrevium (Hb. moricomb. nov.) and Gloniopsis (Gp. subrugosacomb. nov.); the latter genus, in addition to the type, Gp. praelonga, with two new species, Gp. arciformissp. nov. and Gp. kenyensis sp. nov. The genus Glonium is now divided into Anteaglonium (Pleosporales), Glonium (Gloniaceae), and Psiloglonium (Hysteriaceae). The hysterothecium has evolved convergently no less than five times within the Pleosporomycetidae (e.g., Anteaglonium, Farlowiella, Glonium, Hysterographium and the Hysteriaceae). Similarly, thin-walled mytilinidioid (e.g., Ostreichnion) and patellarioid (e.g., Rhytidhysteron) genera, previously in the Mytilinidiaceae and Patellariaceae, respectively, transferred here to the Hysteriaceae, have also evolved at least twice within the subclass. As such, character states traditionally considered to represent synapomorphies among these fungi, whether they relate to spore septation or the ascomata, in fact, represent symplesiomorphies, and most likely have arisen multiple times through convergent evolutionary processes in response to common selective pressures.

A class-wide phylogenetic assessment of Dothideomycetes.

We present a comprehensive phylogeny derived from 5 genes, nucSSU, nucLSU rDNA, TEF1, RPB1 and RPB2, for 356 isolates and 41 families (six newly described in this volume) in Dothideomycetes. All currently accepted orders in the class are represented for the first time in addition to numerous previously unplaced lineages. Subclass Pleosporomycetidae is expanded to include the aquatic order Jahnulales. An ancestral reconstruction of basic nutritional modes supports numerous transitions from saprobic life histories to plant associated and lichenised modes and a transition from terrestrial to aquatic habitats are confirmed. Finally, a genomic comparison of 6 dothideomycete genomes with other fungi finds a high level of unique protein associated with the class, supporting its delineation as a separate taxon.

The molecular phylogeny of freshwater Dothideomycetes.

The freshwater Dothideomycetes species are an ecological rather than taxonomic group and comprise approximately 178 meiosporic and mitosporic species. Due to convergent or parallel morphological adaptations to aquatic habitats, it is difficult to determine phylogenetic relationships among freshwater taxa and among freshwater, marine and terrestrial taxa based solely on morphology. We conducted molecular sequence-based phylogenetic analyses using nuclear ribosomal sequences (SSU and/or LSU) for 84 isolates of described and undescribed freshwater Dothideomycetes and 85 additional taxa representative of the major orders and families of Dothideomycetes. Results indicated that this ecological group is not monophyletic and all the freshwater taxa, except three aeroaquatic Tubeufiaceae, occur in Pleosporomycetidae as opposed to Dothideomycetidae. Four clades comprised of only freshwater taxa were recovered. The largest of these is the Jahnulales clade consisting of 13 species, two of which are the anamorphs Brachiosphaera tropicalis and Xylomyces chlamydosporus. The second most speciose clade is the Lindgomycetaceae clade consisting of nine taxa including the anamorph Taeniolella typhoides. The Lindgomycetaceae clade consists of taxa formerly described in Massarina, Lophiostoma, and Massariosphaeriae.g.,Massarina ingoldiana, Lophiostoma breviappendiculatum, and Massariosphaeria typhicola and several newly described and undescribed taxa. The aquatic family Amniculicolaceae, including three species of Amniculicola, Semimassariosphaeria typhicola and the anamorph, Anguillospora longissima, was well supported. A fourth clade of freshwater species consisting of Tingoldiago graminicola,Lentithecium aquaticum,L. arundinaceum and undescribed taxon A-369-2b was not well supported with maximum likelihood bootstrap and Bayesian posterior probability. Eight freshwater taxa occurred along with terrestrial species in the Lophiostoma clades 1 and 2. Two taxa lacking statistical support for their placement with any taxa included in this study are considered singletons within Pleosporomycetidae. These singletons, Ocala scalariformis, and Lepidopterella palustris, are morphologically distinct from other taxa in Pleosporomycetidae. This study suggests that freshwater Dothideomycetes are related to terrestrial taxa and have adapted to freshwater habitats numerous times. In some cases (Jahnulales and Lindgomycetaceae), species radiation appears to have occurred. Additional collections and molecular study are required to further clarify the phylogeny of this interesting ecological group.

Freshwater ascomycetes: Aquapoterium pinicola, a new genus and species of Helotiales (leotiomycetes) from Florida.

As part of a survey of freshwater ascomycetes in Florida an unusual discomycete fungus belonging in the Helotiales was found on submerged Pinus needles. This fungus is described and illustrated as a new genus and species, Aquapoterium pinicola, based on morphological data. Aquapoterium pinicola is characterized by minute, hyaline apothecia with an excipulum one cell layer thick of parallel hyphae composed of chains of cells narrow at the basal end and enlarged at the apical end and aseptate ascospores that are surrounded by a gelatinous sheath. Analyses of the nuclear ribosomal large subunit DNA sequence data confirmed its placement within the Helotiales but failed to resolve its familial placement.

The effects of Arcanobacterium pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo.

Uterine bacterial infection after parturition causes endometritis, perturbs ovarian function and leads to infertility in cattle. Although endometritis is caused by mixed infections, endometrial pathology is associated with the presence of Arcanobacterium pyogenes. The aims of the present study were to determine the effects of A. pyogenes on endometrial function in vitro, and on uterine and ovarian function in vivo. Heat-killed A. pyogenes did not affect the production of prostaglandin F2alpha (PGF) or prostaglandin E(2) (PGE) from endometrial explants, or purified populations of endometrial epithelial or stromal cells. However, the explants produced more PGF and PGE than controls when treated with a bacteria-free filtrate (BFF) cultured from A. pyogenes. Similarly, BFF stimulated PGF and PGE production by epithelial and stromal cells, respectively. So, BFF or control PBS was infused into the uterus of heifers (n=7 per group) for 8 days, starting the day after estrus. Emergence of the follicle wave, dominant follicle or corpus luteum diameter, and peripheral plasma FSH, LH, estradiol, progesterone, PGFM, or acute phase protein concentrations were unaffected by the BFF infusion. In the live animal it is likely that the intact uterine mucosa limits the exposure of the endometrial cells to the exotoxin of A. pyogenes, whereas the cells are readily exposed to the toxin in vitro.

Differential gene expression in haemocytes of the snail Biomphalaria glabrata: effects of Schistosoma mansoni infection.

Parasite encapsulation and destruction in Biomphalaria glabrata has been shown to involve the cellular component of the snail's internal defence system, the haemocytes. To identify genes involved in the immunobiology of these cells, we used the method of differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) to investigate differential gene regulation in haemocytes isolated from Schistosoma mansoni exposed and unexposed snails. RNA isolated from circulating haemocytes from resistant snails (BS-90 stock), previously exposed to S. mansoni, was analysed using 12 different arbitrary primers in conjunction with an anchored Oligo d(T(11)CG) primer. Transcription profiles between haemocytes of parasite exposed and unexposed snails were compared and a total of 87 differentially regulated bands were identified and isolated. Of these, 65 bands were cloned and used as probes in Southern blots to show the presence of corresponding sequences in the snail genome. RT-PCR was performed to verify the regulation of these transcripts. DNA sequence analysis showed that the majority of the cloned sequences were novel, although a few showed a high degree of sequence similarity to other sequences in the DNA and protein databases. One of these included a differentially expressed transcript that showed a significant degree of sequence identity to E. coli transposase Tn5, an enzyme whose activity is normally associated with generating mobility and instability in the genome.

The identification of markers segregating with resistance to Schistosoma mansoni infection in the snail Biomphalaria glabrata.

Both snail and parasite genes determine the susceptibility of the snail Biomphalaria glabrata to infection with the trematode Schistosoma mansoni. To identify molecular markers associated with resistance to the parasite in the snail host, we performed genetic crosses between parasite-resistant and -susceptible isogenic snails. Because resistance to infection in adult snails is controlled by a single locus, DNA samples from individual F2 and F1 backcross progeny, segregating for either the resistant or susceptible phenotypes, were pooled (bulked segregant). Genotypes for both parents were determined with 205 arbitrary decamer primers by random amplified polymorphic DNA-PCR. Of the 205 primers, 144 were informative, and the relative allele frequencies between the pools for these primers were determined. Two primers, OPM-04 and OPZ-11, produced fragments in the resistant parent of one cross that were inherited in a dominant fashion in the resistant F2 and backcross-bulked segregant progeny. Subsequent typing of DNA samples of individual progeny snails showed that the 1.2-kb marker amplified by primer OPM-04 and the 1.0-kb marker produced by primer OPZ-11 segregated in the same dominant fashion with the resistant phenotype. Sequence analysis of the 1.2-kb marker showed that it corresponds to a repetitive sequence in the snail genome with no homology to existing DNA sequences in the public databases. Analysis of the 1. 0-kb marker showed that it also corresponds to a repetitive sequence in the B. glabrata genome that contains an imperfect ORF, with homology to retrovirus-related group-specific antigens (gag) polyprotein.

Characterization of the myoglobin and its coding gene of the mollusc Biomphalaria glabrata.

A cDNA clone isolated from a Biomphalaria glabrata (Mollusca, Gastropoda) neural cDNA library was identified as encoding a myoglobin-like protein of 148 amino acids with a single domain and a calculated mass of 16,049.29. Alignment with globin sequences with known tertiary structure confirms its overall globin nature. The expressed myoglobin was identified in the radular muscle and isolated. Oxygen equilibrium measurements on the protein reveal a high oxygen affinity. Val-B10 and Gln-E7, important residues for the determination of the oxygen affinity, are strikingly different from the standard molluscan pattern (Conti, E., Moser, C., Rizzi, M., Mattevi, A., Lionetti, C., Coda, A., Ascenzi, P., Brunori, M., Bolognesi, M. (1993) J. Mol. Biol. 233, 498-508). The single gene encoding the globin chain is interrupted by three introns at positions A3.2, B12.2, and G7.0. Comparison with other nonvertebrate globin genes reveals on the one hand conservation (B12.2 and G7.0) and on the other hand variability of the insertion positions (A3.2). The Biomphalaria myoglobin sequence was used together with all other molluscan globin sequences available to assess the origin and phylogeny of the phylum. Our results confirm the doubts raised about monophyletic origin of the Mollusca, which was first observed using SSU rRNA as a molecular marker.

Schistosoma mansoni: use of a subtractive cloning strategy to search for RFLPs in parasite-resistant Biomphalaria glabrata.

A subtractive cloning strategy has been applied for the identification of two cDNA clones whose corresponding transcripts were elevated in Schistosoma mansoni-resistant (BS-90) compared to susceptible (M-line) snails. Clone pBS11 encoded a 1.9-kb transcript that was more elevated compared to a 500-bp transcript encoded by clone pBS12. Consequently, more attention was focused on the molecular characterization of clone pBS11. Results showed that the transcript encoded by this clone was expressed in the albumen gland and was developmentally regulated. Sequence analysis of pBS11 demonstrated the presence of an open reading frame that corresponded to a novel Biomphalaria glabrata albumen gland gene product. Comparative Southern analysis of the resistant and susceptible snail lines using pBS11 as probe indicated the presence of a BamHI and EcoRI RFLP between the two strains.