Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia.

BACKGROUND: Silene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes. RESULTS: Here we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30% of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome. CONCLUSIONS: Our BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20%.

An angiosperm-wide analysis of the gynodioecy-dioecy pathway.

BACKGROUND AND AIMS: About 6 % of an estimated total of 240 000 species of angiosperms are dioecious. The main precursors of this sexual system are thought to be monoecy and gynodioecy. A previous angiosperm-wide study revealed that many dioecious species have evolved through the monoecy pathway; some case studies and a large body of theoretical research also provide evidence in support of the gynodioecy pathway. If plants have evolved through the gynodioecy pathway, gynodioecious and dioecious species should co-occur in the same genera. However, to date, no large-scale analysis has been conducted to determine the prevalence of the gynodioecy pathway in angiosperms. In this study, this gap in knowledge was addressed by performing an angiosperm-wide survey in order to test for co-occurrence as evidence of the gynodioecy pathway. METHODS: Data from different sources were compiled to obtain (to our knowledge) the largest dataset on gynodioecy available, with 275 genera that include at least one gynodioecious species. This dataset was combined with a dioecy dataset from the literature, and a study was made of how often dioecious and gynodioecious species could be found in the same genera using a contingency table framework. KEY RESULTS: It was found that, overall, angiosperm genera with both gynodioecious and dioecious species occur more frequently than expected, in agreement with the gynodioecy pathway. Importantly, this trend holds when studying different classes separately (or sub-classes, orders and families), suggesting that the gynodioecy pathway is not restricted to a few taxa but may instead be widespread in angiosperms. CONCLUSIONS: This work complements that previously carried out on the monoecy pathway and suggests that gynodioecy is also a common pathway in angiosperms. The results also identify angiosperm families where some (or all) dioecious species may have evolved from gynodioecious precursors. These families could be the targets of future small-scale studies on transitions to dioecy taking phylogeny explicitly into account.

Dioecy is associated with higher diversification rates in flowering plants.

In angiosperms, dioecious clades tend to have fewer species than their nondioecious sister clades. This departure from the expected equal species richness in the standard sister clade test has been interpreted as implying that dioecious clades diversify less and has initiated a series of studies suggesting that dioecy might be an 'evolutionary dead end'. However, two of us recently showed that the 'equal species richness' null hypothesis is not valid in the case of derived char acters, such as dioecy, and proposed a new test for sister clade comparisons; preliminary results, using a data set available in the litterature, indicated that dioecious clades migth diversify more than expected. However, it is crucial for this new test to distinguish between ancestral and derived cases of dioecy, a criterion that was not taken into account in the available data set. Here, we present a new data set that was obtained by searching the phylogenetic literature on more than 600 completely dioecious angiosperm genera and identifying 115 sister clade pairs for which dioecy is likely to be derived (including > 50% of the dioecious species). Applying the new sister clade test to this new dataset, we confirm the preliminary result that dioecy is associated with an increased diversification rate, a result that does not support the idea that dioecy is an evolutionary dead end in angiosperms. The traits usually associated with dioecy, that is, an arborescent growth form, abiotic pollination, fleshy fruits or a tropical distribution, do not influence the diversification rate. Rather than a low diversification rate, the observed species richness patterns of dioecious clades seem to be better explained by a low transition rate to dioecy and frequent losses.

Prophylaxis of infectious complications with colony-stimulating factors in adult cancer patients undergoing chemotherapy-evidence-based guidelines from the Infectious Diseases Working Party AGIHO of the German Society for Haematology and Medical Oncology (DGHO).

BACKGROUND: Current evidence on myelopoietic growth factors is difficult to overview for the practicing haematologist/oncologist. International guidelines are sometimes conflicting, exclude certain patient groups, or cannot directly be applied to the German health system. This guideline by the Infectious Diseases Working Party (AGIHO) of the German Society of Haematology and Medical Oncology (DGHO) gives evidence-based recommendations for the use of G-CSF, pegylated G-CSF, and biosimilars to prevent infectious complications in cancer patients undergoing chemotherapy, including those with haematological malignancies. METHODS: We systematically searched and evaluated current evidence. An expert panel discussed the results and recommendations. We then compared our recommendations to current international guidelines. RESULTS: We summarised the data from eligible studies in evidence tables, developed recommendations for different entities and risk groups. CONCLUSION: Comprehensive literature search and expert panel consensus confirmed many key recommendations given by international guidelines. Evidence for growth factors during acute myeloid leukaemia induction chemotherapy and pegfilgrastim use in haematological malignancies was rated lower compared with other guidelines.

Central venous catheter-related infections in hematology and oncology: 2012 updated guidelines on diagnosis, management and prevention by the Infectious Diseases Working Party of the German Society of Hematology and Medical Oncology.

BACKGROUND: Cancer patients are at increased risk for central venous catheter-related infections (CRIs). Thus, a comprehensive, practical and evidence-based guideline on CRI in patients with malignancies is warranted. PATIENTS AND METHODS: A panel of experts by the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Medical Oncology (DGHO) has developed a guideline on CRI in cancer patients. Literature searches of the PubMed, Medline and Cochrane databases were carried out and consensus discussions were held. RESULTS: Recommendations on diagnosis, management and prevention of CRI in cancer patients are made, and the strength of the recommendation and the level of evidence are presented. CONCLUSION: This guideline is an evidence-based approach to the diagnosis, management and prevention of CRI in cancer patients.

PCR survey of 50 introns in animals: cross-amplification of homologous EPIC loci in eight non-bilaterian, protostome and deuterostome phyla.

Exon Primed Intron Crossing (EPIC) markers provide molecular tools that are susceptible to be variable within species while remaining amplifiable by PCR using potentially universal primers. In this study we tested the possibility of obtaining PCR products from 50 EPIC markers on 23 species belonging to seven different phyla (Porifera, Cnidaria, Arthropoda, Nematoda, Mollusca, Annelida, Echinodermata) using 70 new primer pairs. A previous study had identified and tested those loci in a dozen species, including another phylum, Urochordata (Chenuil et al., 2010). Results were contrasted among species. The best results were achieved with the oyster (Mollusca) where 28 loci provided amplicons susceptible to contain an intron according to their size. This was however not the case with the other mollusk Crepidula fornicata, which seems to have undergone a reduction in intron number or intron size. In the Porifera, 13 loci appeared susceptible to contain an intron, a surprisingly high number for this phylum considering its phylogenetic distance with genomic data used to design the primers. For two cnidarian species, numerous loci (24) were obtained. Ecdysozoan phyla (arthropods and nematodes) proved less successful than others as expected considering reports of their rapid rate of genome evolution and the worst results were obtained for several arthropods. Some general patterns among phyla arose, and we discuss how the results of this EPIC survey may give new insights into genome evolution of the study species. This work confirms that this set of EPIC loci provides an easy-to-use toolbox to identify genetic markers potentially useful for population genetics, phylogeography or phylogenetic studies for a large panel of metazoan species. We then argue that obtaining diploid sequence genotypes for these loci became simple and affordable owing to Next-Generation Sequencing development. Species surveyed in this study belong to several genera (Acanthaster, Alvinocaris, Aplysina, Aurelia, Crepidula, Eunicella, Hediste, Hemimysis, Litoditis, Lophelia, Mesopodopsis, Mya, Ophiocten, Ophioderma, Ostrea, Pelagia, Platynereis, Rhizostoma, Rimicaris), two of them, belonging to the family Vesicomydae and Eunicidae, could not be determined at the genus level.

Successful systemic high-dose ribavirin treatment of respiratory syncytial virus-induced infections occurring pre-engraftment in allogeneic hematopoietic stem cell transplant recipients.

INTRODUCTION: Respiratory syncytial virus (RSV) is a frequent cause of respiratory tract infectious disease (RTID) in allogeneic hematopoietic stem cell transplant (HSCT) recipients associated with a high mortality once infection has progressed from upper RTID (URTID) to lower RTID (URTID). Aerosolized ribavirin (RBV) is considered a cornerstone of treatment, but is expensive and has toxic side effects on patients and staff. In this study, RSV infection was detected by polymerase chain reaction (PCR) from routinely collected throat swabs in HSCT patients. Infected individuals were treated according to an institutional protocol using intravenous (IV) RBV for patients with LRTID and oral ribavirin for URTID. RESULTS: RSV infection was diagnosed in 10 patients (median age 60 years) a median of 15 days after allogeneic HSCT for high-risk acute myeloid leukemia. Five patients with LRTID received IV RBV within 7 days after HSCT, and 5 with URTID were treated with oral RBV 12-40 days after HSCT. One patient died of septic shock associated with Pseudomonas aeruginosa-induced pneumonia 28 days after HSCT in prolonged neutropenia. All patients became RSV PCR negative on throat swabs within a median of 22 days from start of RBV. Despite severe lymphopenia, no patient treated for URTID progressed to LRTID. Neutrophil recovery was delayed in 3 patients. CONCLUSIONS: We show that IV and oral RBV were efficacious in preventing progression and reducing mortality of RSV infection in this small series of allogeneic HSCT recipients. Randomized studies are not to be expected for this condition and therefore reporting case series could help in determining optimal RSV treatment.

Deciphering the aetiology of a mixed fungal infection by broad-range PCR with sequencing and fluorescence in situ hybridisation.

Simultaneous infections with multiple fungi may be misinterpreted as monomicrobial infections by current diagnostics with ramifications for the choice of antimicrobial agents that may impact patient outcomes. The application of molecular methods on tissue samples may be useful to decipher the aetiology of mixed fungal infections. We present a leukaemic patient who died from sepsis due to candidaemia. The postmortem examination documented fungal elements in lung tissue. Fungal DNA was amplified from the lung sample by broad-range PCR assays targeting the 28S ribosomal RNA gene or the internal transcribed spacer 2 (ITS-2). Fluorescence in situ hybridisation (FISH) using differentially labelled fungal probes was applied on the tissue. Sequencing identified the PCR amplicons as Aspergillus fumigatus (28S assay) and Candida tropicalis (ITS-2 assay). As a chromatogram suggested mixed amplicons, the Isentio ripseq(®) tool for in silico analysis was applied and confirmed the presence of both amplicons in the PCR products of both assays. FISH confirmed the presence of Aspergillus and Candida within the infectious process, a prerequisite for inferring a causal relationship with the infection. The combination of broad-range PCR with sequence analysis and FISH applied on tissue samples is a powerful approach to identify the aetiology of invasive fungal infections, including mixed infections.

Geographical delimitation of a partial selective sweep in African Drosophila melanogaster.

Positive selection leaves characteristic footprints on DNA variation but detecting such patterns is challenging as the age, the intensity and the mode of selection as well as demography and evolutionary parameters (mutation and recombination rates) all play roles and these are difficult to disentangle. We recorded nucleotide variation in a sample of isogenic chromosomes from a western African population of Drosophila melanogaster at a locus (Fbp2) for which a partial selective sweep had previously been reported. We compared this locus to four other genes from the same chromosomes and from a European and an East African population. Then, we assessed Fbp2 variation in a sample of 370 chromosomes covering a comprehensive geographic sampling of 16 African localities. The signature of selection was tested while accounting for the demographic history of the populations. We found a significant signal of selection in two West African localities including Ivory Coast. Variation at Fpb2 would thus represent a case of an ongoing selective sweep in the range of this species. A weaker, nonsignificant, signal of selection was, however, apparent in some other populations, thus leaving open several possibilities: (i) the selective sweep originated in Ivory Coast and has spread to the rest of the continent; (ii) several African populations report the signature of a selective event having occurred in an ancestral population; (iii) this genome region is subject to independent selective events in African populations; and (iv) A neutral scenario with population subdivision and local bottleneck cannot be fully excluded to explain the molecular patterns observed in some populations.

Human herpesvirus 6 in biopsies from patients with gastrointestinal symptoms after allogeneic stem cell transplantation.

Gastrointestinal complications are frequent after allogeneic stem cell transplantation (allo-SCT). Main differential diagnoses are graft-versus-host disease (GvHD) and viral infections. In this retrospective analysis, we included 50 patients with severe vomiting or diarrhea in the first year after allo-SCT. One hundred two biopsies obtained by colonoscopy or endoscopy of the upper gastrointestinal tract were analysed by conventional histology for signs of GvHD and by qualitative polymerase chain reaction (PCR) for viral DNA of human herpesvirus 6 (HHV-6) and other virus of the herpes family. DNA of HHV-6 was detected in 38 of 75 initial samples (51%) and in 19 of 27 follow-up biopsies (70%). In the initial samples (n = 75), HHV-6 DNA was detected in 20/37 (54%) biopsies in the presence of GvHD compared to 18/38 (47%) biopsies without signs of GvHD. At the time of the first endoscopic investigation, most patients received antiviral prophylaxis with aciclovir. None of the follow-up biopsies was HHV-6 DNA negative after antiviral treatment with aciclovir, foscarnet or ganciclovir. By univariate analysis, no risk factor for HHV-6 detection could be demonstrated. In this cohort of patients with severe gastrointestinal complications, there was no significant difference in the overall survival between patients with or without HHV-6 DNA detection in the gastrointestinal tract. In summary, the detection of HHV-6 DNA had no impact on overall survival. Moreover, antiviral therapy against HHV-6 was without effect. Thus, positive PCR results in GI tract samples do not necessarily reflect reactivation of HHV-6. Further studies are needed to define the significance of HHV-6 for GI tract symptoms after allo-SCT.

Breakthrough zygomycosis on posaconazole prophylaxis after allogeneic stem cell transplantation.

Antifungal prophylaxis with posaconazole (POS) has been shown to decrease the mortality associated with invasive fungal infections in high-risk patients. We report on a patient, with severe graft-versus-host disease after allogeneic stem cell transplantation, who developed proven pneumonia due to Rhizopus microsporus after 40 days of POS prophylaxis (fasting serum levels: 691-904 ng/mL). Despite combination treatment with liposomal amphotericin B and POS for 39 days, the patient died from pulmonary hemorrhage. This case highlights the need for continued awareness of breakthrough zygomycosis in patients receiving POS.

Nonsecretory primary plasma cell leukemia with good response to thalidomide-based treatment.

Primary plasma cell leukemia (PCL) is a rare hematologic disorder with distinct features. The criterion for the diagnosis of PCL is based on the finding of malignant plasma cells in the peripheral blood (more than 2 x 10(9)/L or more than 20% of white blood cells). We report a case of a 74-year-old patient with primary nonsecretory PCL. Examination of blood smears led to the diagnosis of PCL, which was confirmed by bone marrow biopsy. Due to the patient's impaired general condition, intensive chemotherapy could not be administered. After an oral induction chemotherapy consisting of cyclophosphamide and high dose dexamethasone followed by one cycle of high-dose dexamethasone and thalidomide no evidence of the disease in the peripheral blood was detectable. Consequently, the patient was put on a thalidomide maintenance therapy. Six months after first diagnosis, the patient was found to have bone marrow and peripheral blood relapse with anemia and neutropenia in the clinical context of acute on chronic renal failure. After a limited response to further chemotherapy, the patient died 14 months after the first diagnosis while on dexamethasone maintenance. We conclude that monotherapy with thalidomide might be an alternative maintenance strategy with limited response duration for patients with primary PCL in impaired general condition.

An infected esophageal duplication cyst in a patient with non-Hodgkin's lymphoma mimicking persistent disease.

Differentiation of mediastinal cysts appearing as soft-tissue attenuation masses on computed tomography (CT) scans from malignant mediastinal masses is difficult. We report a patient with non-Hodgkin's lymphoma, who was considered to have persistent disease in the posterior mediastinum based on CT scans. However, endoscopic ultrasound (EUS) demonstrated a paraesophageal, fluid-filled cyst with echodens inclusions and no evidence of any solid component. EUS-guided fine-needle aspiration (FNA) revealed mucous, epithelial and inflammatory cells, and additionally candida albicans was cultured. Based on these findings and constant size during follow-up, the diagnosis of an infected esophageal duplication cyst was made. Thus, this report further demonstrated the impact of EUS and EUS-FNA for management of posterior mediastinal cystic lesions in selected cases.

Gene flow in a facultative apomictic poacea, the savanna grass Hyparrhenia diplandra.

The genetics of the poacea Hyparrhenia diplandra was studied in four natural populations from an ecological station in West Africa, where it makes up 80% of grasses from wet savanna and constitutes a dense continuum of randomly distributed individuals. DNA content and cytogenetical observations suggest it is an allotetraploid. Using two highly variable microsatellites (heterozygosity H = 0.615-0.616), we show that this species is an apomict with rare sexual reproduction events that account for approximately 0.5% of seeds pollinated in the wild. Hexaploid individuals were also produced, corroborating the observation of aberrant genotypes in the wild. The spatial extent of asexual clones in the field was low in comparison with the predominance of apomixis, thus indicating a low dispersal of seeds from their parent. Heterozygosity and departure from Hardy-Weinberg predictions were similar in the four populations, revealing a high apparent selfing rate s = 0.599 among sexually produced seeds. This is an overestimate since we could not distinguish true selfing from reciprocal outcrosses between neighboring individuals from the same apomictic clone. Gene flow by pollen could be substantial, possibly explaining the absence of isolation by distance in the studied area.

Selective sweep near the In(2L)t inversion breakpoint in an African population of Drosophila melanogaster.

Chromosomal inversions largely inhibit recombination and may be associated with selective forces, such as hitch-hiking effects: the effect of positive selection on linked loci. A West African population of Drosophila melanogaster showed a high frequency (0.61) of the In(2L)t inversion. Departure from neutrality statistically associated with the inversion polymorphism was previously recorded at Su(H), a locus distant from the proximal breakpoint of the inversion. These results were consistent with hitch-hiking effects with recombination. The present sequence polymorphism survey involves a 1 kb fragment of the Vha68-1 locus located closer to the proximal breakpoint of the inversion. It shows a significant deficit of polymorphism with respect to divergence when compared with other loci studied in the same population, thus suggesting selective effects. Only 11 polymorphic sites are present in a sample of 20 chromosomes and these sites present a significant excess of rare-frequency variants. The major haplotype shows an unexpectedly high frequency. Our estimate of the background selection effect is not sufficient to account for the observed reduction of polymorphism. Intraspecific variation is structured between inverted and standard chromosomes; there are no shared polymorphisms but also no fixed differences between them. This pattern, together with that found on other loci previously studied near this inversion breakpoint, suggests hitch-hiking effects enhanced by the inversion.

p53-independent apoptosis and p53-dependent block of DNA rereplication following mitotic spindle inhibition in human cells.

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.

Cysteine control over glutathione homeostasis in Chinese hamster fibroblasts overexpressing a gamma-glutamylcysteine synthetase activity.

Gamma-glutamylcysteine synthetase (GCS) catalyses the first step of glutathione (GSH) biosynthesis and is considered to be the rate-limiting step of this pathway. In several experimental systems, GCS overexpression has been associated with GSH pool expansion and drug resistance. In this report, we describe a mutant line of Chinese hamster fibroblasts that overexpress this activity by 4-5 times, due to the amplification of the gene encoding the catalytic subunit of GCS. These mutant cells contained a wild-type steady-state level of GSH and, after depletion, synthesized GSH at the same rate as wild-type cells because their rate of endogenous production of cysteine was limiting. An exogenous supply of cysteine expanded the pool of GSH in mutant cells by 80% but did not increase that of wild-type cells, and, in GSH-depleted cells, increased the rate of GSH biosynthesis by eight and 35-times in wild-type and mutant cells, respectively. These experiments indicated that GCS overexpression had no consequence on the metabolism of GSH, unless a supply of cysteine was provided. Mutant cells were not resistant to cisplatin or nitrogen mustard.

The cytotoxicity of the parvovirus minute virus of mice nonstructural protein NS1 is related to changes in the synthesis and phosphorylation of cell proteins.

Autonomous parvoviruses exert lytic and cytostatic effects believed to contribute to their antineoplastic activity. Studies with inducible clones have demonstrated a direct involvement of parvovirus nonstructural proteins (NS) in oncolysis. Human and rat fibroblasts have been stably transfected with MVM(p) (minute virus of mice prototype strain) NS genes cloned under the control of a hormone-inducible promoter. Dexamethasone-induced synthesis of the NS proteins in sensitive transformed cells results in cell killing within a few days. From these sensitive cell lines have been isolated some NS-resistant clones that also prove resistant to MVM(p) infection, suggesting that cell factors modulate NS cytotoxicity. We have previously reported that factors involved in cell cycle regulation may contribute to this modulation, since NS toxicity requires cell proliferation and correlates with a cell cycle perturbation leading to an arrest in phase S/G2. In addition to its role in cytotoxicity, NS1 can regulate transcription driven by parvovirus and nonparvovirus promoters. Since phosphorylation is a critical event in controlling the activity of many proteins, notably transcription factors and cell cycle-regulated proteins, we have examined the effect of NS1 on the synthesis and phosphorylation of cell proteins. Our results indicate that NS1 interferes, within 7 h of induction, with phosphorylation of a protein of about 14 kDa (p14). Cell synchronization has enabled us to show that phosphorylation of this protein occurs in early S phase and is prevented when NS1 is induced. This early effect of NS1 on p14 phosphorylation may be directly linked to cytotoxicity and is probably related to the previously reported inhibition of cell DNA synthesis. Late in the induction period (24 h), NS1 also alters the synthesis of a 50-kDa protein and a 35-kDa protein (p50 and p35, respectively). Microsequencing of p35 reveals sequence homology with beta-tubulin. These effects of NS1, observed only in NS1-sensitive cell lines, may be related to the protein's cytotoxicity.