Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Comparative genomic sequencing analysis of a region in human chromosome 11p15.3, mouse chromosome 7 and analysis of the novel STK33, Stk33 gene

The comparative genomic sequence analysis of a region in human chromosome 11p15.3 and its homologous segment in mouse chromosome 7 between ST5 and LMO1 genes has been performed. 158,201 bases were sequenced in the mouse and compared with the syntenic region in human, partially available in the public databases. The analysed region exhibits the typical eukaryotic genomic structure and compared with the close neighbouring regions, strikingly reflexes the mosaic pattern distribution of (G+C) and repeats content despites its relative short size. Within this region the novel gene STK33 was discovered (Stk33 in the mouse), that codes for a serine/threonine kinase. The finding of this gene constitutes an excellent example of the strength of the comparative sequencing approach. Poor gene-predictions in the mouse genomic sequence were corrected and improved by the comparison with the unordered data from the human genomic sequence publicly available. Phylogenetical analysis suggests that STK33 belongs to the calcium/calmodulin-dependent protein kinases group and seems to be a novelty in the chordate lineage. The gene, as a whole, seems to evolve under purifying selection whereas some regions appear to be under strong positive selection. Both human and mouse versions of serine/threonine kinase 33, consists of seventeen exons highly conserved in the coding regions, particularly in those coding for the core protein kinase domain. Also the exon/intron structure in the coding regions of the gene is conserved between human and mouse. The existence and functionality of the gene is supported by the presence of entries in the EST databases and was in vivo fully confirmed by isolating specific transcripts from human uterus total RNA and from several mouse tissues. Strong evidence for alternative splicing was found, which may result in tissue-specific starting points of transcription and in some extent, different protein N-termini. RT-PCR and hybridisation experiments suggest that STK33/Stk33 is differentially expressed in a few tissues and in relative low levels. STK33 has been shown to be reproducibly down-regulated in tumor tissues, particularly in ovarian tumors. RNA in-situ hybridisation experiments using mouse Stk33-specific probes showed expression in dividing cells from lung and germinal epithelium and possibly also in macrophages from kidney and lungs. Preliminary experimentation with antibodies designed in this work, performed in parallel to the preparation of this manuscript, seems to confirm this expression pattern. The fact that the chromosomal region 11p15 in which STK33 is located may be associated with several human diseases including tumor development, suggest further investigation is necessary to establish the role of STK33 in human health.

Metodologie per l'analisi statistica di dati di sequencing relativi a un esperimento di rna interference

La RNA interference è un processo attraverso il quale alcuni piccoli frammenti di RNA (19-25 nucleotidi) sono in grado di silenziare l'espressione genica. La sua scoperta, nel 1998, ha rivoluzionato le concezioni della biologia molecolare, minando le basi del cosiddetto Dogma Centrale. Si è visto che la RNAi riveste ruoli fondamentali in meccanismi di regolazione genica, nello spegnimento dell'espressione e funziona come meccanismo di difesa innata contro varie tipologie di virus. Proprio a causa di queste implicazioni richiama interesse non solo dal punto di vista scientifico, ma anche da quello medico, in quanto potrebbe essere impiegata per lo sviluppo di nuove cure. Nonostante la scoperta di tale azione desti la curiosità e l'interesse di molti, i vari processi coinvolti, soprattutto a livello molecolare, non sono ancora chiari. In questo lavoro si propongono i metodi di analisi di dati di un esperimento prodotto dall'Istituto di Biologia molecolare e cellulare di Strasburgo. Nell'esperimento in questione vengono studiate le funzioni che l'enzima Dicer-2 ha nel pathway - cioè la catena di reazioni biomolecolari - della RNA interference durante un'infezione virale nel moscerino della frutta Drosophila Melanogaster. Per comprendere in che modo Dicer-2 intervenga nel silenziamento bisogna capire in quali casi e quali parti di RNA vengono silenziate, a seconda del diverso tipo di mutazione dell'enzima stesso. Dunque è necessario sequenziare l'RNA nelle diverse condizioni sperimentali, ottenendo così i dati da analizzare. Parte dei metodi statistici che verranno proposti risultano poco convenzionali, come conseguenza della peculiarità e della difficoltà dei quesiti che l'esperimento mette in luce. Siccome le tematiche affrontate richiedono un approccio sempre più interdisciplinare, è aumentata considerevolmente la richiesta di esperti di altri settori scientifici come matematici, informatici, fisici, statistici e ingegneri. Questa collaborazione, grazie a una diversità di approccio ai problemi, può fornire nuovi strumenti di comprensione in ambiti che, fino a poco tempo fa, rientravano unicamente nella sfera di competenza dei biologi.

Ecological modelling for next generation sequencing data

Le tecniche di next generation sequencing costituiscono un potente strumento per diverse applicazioni, soprattutto da quando i loro costi sono iniziati a calare e la qualità dei loro dati a migliorare. Una delle applicazioni del sequencing è certamente la metagenomica, ovvero l'analisi di microorganismi entro un dato ambiente, come per esempio quello dell'intestino. In quest'ambito il sequencing ha permesso di campionare specie batteriche a cui non si riusciva ad accedere con le tradizionali tecniche di coltura. Lo studio delle popolazioni batteriche intestinali è molto importante in quanto queste risultano alterate come effetto ma anche causa di numerose malattie, come quelle metaboliche (obesità, diabete di tipo 2, etc.). In questo lavoro siamo partiti da dati di next generation sequencing del microbiota intestinale di 5 animali (16S rRNA sequencing) [Jeraldo et al.]. Abbiamo applicato algoritmi ottimizzati (UCLUST) per clusterizzare le sequenze generate in OTU (Operational Taxonomic Units), che corrispondono a cluster di specie batteriche ad un determinato livello tassonomico. Abbiamo poi applicato la teoria ecologica a master equation sviluppata da [Volkov et al.] per descrivere la distribuzione dell'abbondanza relativa delle specie (RSA) per i nostri campioni. La RSA è uno strumento ormai validato per lo studio della biodiversità dei sistemi ecologici e mostra una transizione da un andamento a logserie ad uno a lognormale passando da piccole comunità locali isolate a più grandi metacomunità costituite da più comunità locali che possono in qualche modo interagire. Abbiamo mostrato come le OTU di popolazioni batteriche intestinali costituiscono un sistema ecologico che segue queste stesse regole se ottenuto usando diverse soglie di similarità nella procedura di clustering. Ci aspettiamo quindi che questo risultato possa essere sfruttato per la comprensione della dinamica delle popolazioni batteriche e quindi di come queste variano in presenza di particolari malattie.

On the car sequencing problem: analysis and solution methods

This work deals with the car sequencing (CS) problem, a combinatorial optimization problem for sequencing mixed-model assembly lines. The aim is to find a production sequence for different variants of a common base product, such that work overload of the respective line operators is avoided or minimized. The variants are distinguished by certain options (e.g., sun roof yes/no) and, therefore, require different processing times at the stations of the line. CS introduces a so-called sequencing rule H:N for each option, which restricts the occurrence of this option to at most H in any N consecutive variants. It seeks for a sequence that leads to no or a minimum number of sequencing rule violations. In this work, CS’ suitability for workload-oriented sequencing is analyzed. Therefore, its solution quality is compared in experiments to the related mixed-model sequencing problem. A new sequencing rule generation approach as well as a new lower bound for the problem are presented. Different exact and heuristic solution methods for CS are developed and their efficiency is shown in experiments. Furthermore, CS is adjusted and applied to a resequencing problem with pull-off tables.

Next-Generation Sequencing-Based Mutations Scanning Strategy of the BCR-ABL Kinase Domain in Patients with PhiladelPhia-Chromosome Positive Leukemias Treated with Tyrosine Kinase Inhibitors

In chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL kinase domain mutation status is an essential component of the therapeutic decision algorithm. The recent development of Ultra-Deep Sequencing approach (UDS) has opened the way to a more accurate characterization of the mutant clones surviving TKIs conjugating assay sensitivity and throughput. We decided to set-up and validated an UDS-based for BCR-ABL KD mutation screening in order to i) resolve qualitatively and quantitatively the complexity and the clonal structure of mutated populations surviving TKIs, ii) study the dynamic of expansion of mutated clones in relation to TKIs therapy, iii) assess whether UDS may allow more sensitive detection of emerging clones, harboring critical 2GTKIs-resistant mutations predicting for an impending relapse, earlier than SS. UDS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II (Interlaboratory Robustness of Next-Generation Sequencing) International consortium.Samples from CML and Ph+ ALL patients who had developed resistance to one or multiple TKIs and collected at regular time-points during treatment were selected for this study. Our results indicate the technical feasibility, accuracy and robustness of our UDS-based BCR-ABL KD mutation screening approach. UDS was found to provide a more accurate picture of BCR-ABL KD mutation status, both in terms of presence/absence of mutations and in terms of clonal complexity and showed that BCR-ABL KD mutations detected by SS are only the “tip of iceberg”. In addition UDS may reliably pick 2GTKIs-resistant mutations earlier than SS in a significantly greater proportion of patients.The enhanced sensitivity as well as the possibility to identify low level mutations point the UDS-based approach as an ideal alternative to conventional sequencing for BCR-ABL KD mutation screening in TKIs-resistant Ph+ leukemia patients

Identification of novel genetic alterations in pediatric cytogenetically normal acute myeloid leukemia by next-generation sequencing

Pediatric acute myeloid leukemia (AML) is a molecularly heterogeneous disease that arises from genetic alterations in pathways that regulate self-renewal and myeloid differentiation. While the majority of patients carry recurrent chromosomal translocations, almost 20% of childhood AML do not show any recognizable cytogenetic alteration and are defined as cytogenetically normal (CN)-AML. CN-AML patients have always showed a great variability in response to therapy and overall outcome, underlining the presence of unknown genetic changes, not detectable by conventional analyses, but relevant for pathogenesis, and outcome of AML. The development of novel genome-wide techniques such as next-generation sequencing, have tremendously improved our ability to interrogate the cancer genome. Based on this background, the aim of this research study was to investigate the mutational landscape of pediatric CN-AML patients negative for all the currently known somatic mutations reported in AML through whole-transcriptome sequencing (RNA-seq). RNA-seq performed on diagnostic leukemic blasts from 19 pediatric CN-AML cases revealed a considerable incidence of cryptic chromosomal rearrangements, with the identification of 21 putative fusion genes. Several of the fusion genes that were identified in this study are recurrent and might have a prognostic and/or therapeutic relevance. A paradigm of that is the CBFA2T3-GLIS2 fusion, which has been demonstrated to be a common alteration in pediatric CN-AML, predicting poor outcome. Important findings have been also obtained in the identification of novel therapeutic targets. On one side, the identification of NUP98-JARID1A fusion suggests the use of disulfiram; on the other, here we describe alteration-activating tyrosine kinases, providing functional data supporting the use of tyrosine kinase inhibitors to specifically inhibit leukemia cells. This study provides new insights in the knowledge of genetic alterations underlying pediatric AML, defines novel prognostic markers and putative therapeutic targets, and prospectively ensures a correct risk stratification and risk-adapted therapy also for the “all-neg” AML subgroup.

Caratterizzazione genomica di virus influenzali suini in Italia mediante next generation sequencing

I sottotipi H1N1, H1N2 e H3N2 di influenza A virus sono largamente diffusi nella popolazione suina di tutto il mondo. Nel presente lavoro è stato sviluppato un protocollo di sequenziamento di c.d. nuova generazione, su piattaforma Ion Torrent PGM, idoneo per l’analisi di tutti i virus influenzali suini (SIV). Per valutare l’evoluzione molecolare dei SIV italiani, sono stati sequenziati ed analizzati mediante analisi genomica e filogenetica un totale di sessantadue ceppi di SIV appartenenti ai sottotipi H1N1, H1N2 e H3N2, isolati in Italia dal 1998 al 2014. Sono stati evidenziati in sei campioni due fenomeni di riassortimento: tutti i SIV H1N2 esaminati presentavano una neuraminidasi di derivazione umana, diversa da quella dei SIV H1N2 circolanti in Europa, inoltre l’emoagglutinina (HA) di due isolati H1N2 era originata dal riassortimento con un SIV H1N1 avian-like. L’analisi molecolare dell’HA ha permesso di rivelare un’inserzione di due amminoacidi in quattro SIV H1N1 pandemici e una delezione di due aminoacidi in quattro SIV H1N2, entrambe a livello del sito di legame con il recettore cellulare. E’ stata inoltre evidenziata un’elevata omologia di un SIV H1N1 con ceppi europei isolati negli anni ’80, suggerendo la possibile origine vaccinale di questo virus. E’ stato possibile, in aggiunta, applicare il nuovo protocollo sviluppato per sequenziare un virus influenzale aviare altamente patogeno trasmesso all’uomo, direttamente da campione biologico. La diversità genetica nei SIV esaminati in questo studio conferma l’importanza di un continuo monitoraggio della costellazione genomica dei virus influenzali nella popolazione suina.

Deep-sequencing identification of the genomic targets of the cytidine deaminase AID and its cofactor RPA in B lymphocytes

The cytidine deaminase AID hypermutates immunoglobulin genes but can also target oncogenes, leading to tumorigenesis. The extent of AID's promiscuity and its predilection for immunoglobulin genes are unknown. We report here that AID interacted broadly with promoter-proximal sequences associated with stalled polymerases and chromatin-activating marks. In contrast, genomic occupancy of replication protein A (RPA), an AID cofactor, was restricted to immunoglobulin genes. The recruitment of RPA to the immunoglobulin loci was facilitated by phosphorylation of AID at Ser38 and Thr140. We propose that stalled polymerases recruit AID, thereby resulting in low frequencies of hypermutation across the B cell genome. Efficient hypermutation and switch recombination required AID phosphorylation and correlated with recruitment of RPA. Our findings provide a rationale for the oncogenic role of AID in B cell malignancy.

Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus (sensu lato) strains

Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536(T), M. massiliense CIP 108297(T), and M. bolletii CIP 108541(T)) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.

Ambiguous nucleotide calls from population-based sequencing of HIV-1 are a marker for viral diversity and the age of infection

The time passed since the infection of a human immunodeficiency virus (HIV)-infected individual (the age of infection) is an important but often only poorly known quantity. We assessed whether the fraction of ambiguous nucleotides obtained from bulk sequencing as done for genotypic resistance testing can serve as a proxy of this parameter.

Targeted next generation sequencing as a diagnostic tool in epileptic disorders

Epilepsies have a highly heterogeneous background with a strong genetic contribution. The variety of unspecific and overlapping syndromic and nonsyndromic phenotypes often hampers a clear clinical diagnosis and prevents straightforward genetic testing. Knowing the genetic basis of a patient's epilepsy can be valuable not only for diagnosis but also for guiding treatment and estimating recurrence risks.