RIPCAL: a tool for alignment-based analysis of repeat-induced point mutations in fungal genomic sequences

Background Repeat-induced point mutation (RIP) is a fungal-specific genome defence mechanism that alters the sequences of repetitive DNA, thereby inactivating coding genes. Repeated DNA sequences align between mating and meiosis and both sequences undergo C:G to T:A transitions. In most fungi these transitions preferentially affect CpA di-nucleotides thus altering the frequency of certain di-nucleotides in the affected sequences. The majority of previously published in silico analyses were limited to the comparison of ratios of pre- and post-RIP di-nucleotides in putatively RIP-affected sequences – so-called RIP indices. The analysis of RIP is significantly more informative when comparing sequence alignments of repeated sequences. There is, however, a dearth of bioinformatics tools available to the fungal research community for alignment-based RIP analysis of repeat families. Results We present RIPCAL http://www.sourceforge.net/projects/ripcal, a software tool for the automated analysis of RIP in fungal genomic DNA repeats, which performs both RIP index and alignment-based analyses. We demonstrate the ability of RIPCAL to detect RIP within known RIP-affected sequences of Neurospora crassa and other fungi. We also predict and delineate the presence of RIP in the genome of Stagonospora nodorum – a Dothideomycete pathogen of wheat. We show that RIP has affected different members of the S. nodorum rDNA tandem repeat to different extents depending on their genomic contexts. Conclusion The RIPCAL alignment-based method has considerable advantages over RIP indices for the analysis of whole genomes. We demonstrate its application to the recently published genome assembly of S. nodorum

A Cytosine Methyltransferase Homologue Is Essential for Sexual Development in Aspergillus nidulans

Background: The genome defense processes RIP (repeat-induced point mutation) in the filamentous fungus Neurospora crassa, and MIP (methylation induced premeiotically) in the fungus Ascobolus immersus depend on proteins with DNA methyltransferase (DMT) domains. Nevertheless, these proteins, RID and Masc1, respectively, have not been demonstrated to have DMT activity. We discovered a close homologue in Aspergillus nidulans, a fungus thought to have no methylation and no genome defense system comparable to RIP or MIP. Principal Findings: We report the cloning and characterization of the DNA methyltransferase homologue A (dmtA) gene from Aspergillus nidulans. We found that the dmtA locus encodes both a sense (dmtA) and an anti-sense transcript (tmdA). Both transcripts are expressed in vegetative, conidial and sexual tissues. We determined that dmtA, but not tmdA, is required for early sexual development and formation of viable ascospores. We also tested if DNA methylation accumulated in any of the dmtA/tmdA mutants we constructed, and found that in both asexual and sexual tissues, these mutants, just like wild-type strains, appear devoid of DNA methylation. Conclusions/Significance: Our results demonstrate that a DMT homologue closely related to proteins implicated in RIP and MIP has an essential developmental function in a fungus that appears to lack both DNA methylation and RIP or MIP. It remains formally possible that DmtA is a bona fide DMT, responsible for trace, undetected DNA methylation that i

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology

Mesotelioma maligno: analisi di una casistica ospedaliera

Nel 2000-2010 sono stati ricoverati presso il nostro Istituto 77 casi di mesotelioma maligno (MM) [67 pleurici (40 maschi, 27 femmine; età media 63,9 anni), 9 peritoneali (7 maschi, 2 femmine; 67,9 anni) e uno testicolare (uomo di 38 anni)], spesso associati, con vario grado di probabilità, a pregresse esposizioni (occupazionali o ambientali) ad amianto. Ventiquattro pazienti con MM pleurico risultavano operati (12 decorticazioni pleuriche e 12 pneumectomie), con mediana di sopravvivenza, rispettivamente, di 14 ± 4,33 (errore standard) e 38 ± 4,27 mesi, superiore a quella dei pazienti non operati (8 ± 0.94 mesi). Quattro casi peritoneali sono stati trattati con peritonectomia e chemioterapia ipertermica intraoperatoria: uno è ancora in vita a 20 mesi dalla diagnosi, gli altri sono deceduti a 8, 18 e 36 mesi. Il caso testicolare, sottoposto a orchiectomia radicale, è tuttora vivente a 6 anni. La casistica conferma che, in seguito al pregresso uso d’amianto, il MM è di osservazione relativamente frequente nell’attuale pratica clinica. I pazienti trattati chirurgicamente presentano sopravvivenze più lunghe

Protocollo interdisciplinare per il reinserimento lavorativo dopo infortunio osteoarticolare sul lavoro: casistica clinica

Sono presentati i risultati ottenuti con un protocollo finalizzato alla ripresa lavorativa dopo infortunio osteoarticolare sul lavoro e successiva riabilitazione: 159 pazienti (102 maschi e 57 femmine; età media 43,7 anni) sono stati valutati dal fisiatra e dal medico del lavoro e forniti d’indicazioni in base alle loro capacità funzionali e alle caratteristiche della mansione. Dopo 6 mesi, 105 dei 130 soggetti raggiungibili telefonicamente (80,7%) erano tornati al lavoro (8 dopo cambio di mansione, 7 parzialmente), in un tempo medio di 9,4 giorni per coloro che avevano ripreso integralmente le mansioni precedenti, di 21,4 giorni per gli altri. A 12 mesi, la percentuale di ripresa lavorativa è risultata sovrapponibile (76 su 93 pazienti: 81,7%), a indicare che il beneficio della riabilitazione è massimo nel breve-medio periodo. Nel complesso, i dati comprovano l’efficacia dell’approccio riabilitativo multidisciplinare utilizzato