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Efflux-mediated macrolide resistance due to mef (E) and mel , carried by the mega element, is common in Streptococcus pneumoniae , for which it was originally characterized, but it is rare in Streptococcus pyogenes . In S. pyogenes , mega was previously found to be enclosed in Tn 2009 , a composite genetic element of the Tn 916 family containing tet (M) and conferring erythromycin and tetracycline resistance. In this study, S. pyogenes isolates containing mef (E), apparently not associated with other resistance determinants, were examined to characterize the genetic context of mega. By whole-genome sequencing of one isolate, MB56Spyo009, we identified a novel composite integrative and conjugative element (ICE) carrying mega, designated ICE Spy 009, belonging to the ICE Sa 2603 family. ICE Spy 009 was 55 kb long, contained 61 putative open reading frames (ORFs), and was found to be integrated into hylA , a novel integration site for the ICE Sa 2603 family. The modular organization of the ICE was similar to that of members of the ICE Sa 2603 family carried by different streptococcal species. In addition, a novel cluster of accessory resistance genes was found inside a region that encloses mega. PCR mapping targeting ICE Spy 009 revealed the presence of a similar ICE in five other isolates under study. While in three isolates the integration site was the same as that of ICE Spy 009, in two isolates the ICE was integrated into rplL , the typical integration site of the ICE Sa 2603 family. ICE Spy 009 was able to transfer macrolide resistance by conjugation to both S. pyogenes and S. pneumoniae , showing the first evidence of the transferability of mega from S. pyogenes .

Multiple myeloma (MM) is a clonal proliferation of bone marrow plasma cells characterized by highly heterogeneous genetic background and clinical course, whose pathogenesis remains largely unknown. Long ncRNAs (lncRNAs) are a large class of non-protein-coding RNA, involved in many physiological cellular and genomic processes as well as in carcinogenesis and tumor evolution. Although still in its infancy, the role of lncRNAs in MM is progressively expanding. Besides studies on selected candidates, lncRNAs expression at genome-wide transcriptome level is confined to microarray technologies, thus investigating a limited collection of transcripts. In the present study investigating a cohort of 30 MM patients, a deep RNA-sequencing analysis overwhelmed previous array studies and allowed the most accurate definition of lncRNA transcripts structure and expression, ultimately providing a comprehensive catalogue of lncRNAs specifically associated with the main MM molecular subgroups and genetic alterations. Despite the small number of analyzed samples, the high accuracy of RNA-sequencing approach for complex transcriptome processing led to the identification of 391 deregulated lncRNAs, 67% of which were also detectable and validated by whole-transcript microarrays. In addition, we identified a list of lncRNAs, with potential relevance in MM, co-expressed and in close proximity to genes that might undergo a cis-regulatory relationship.

Recent advances in genome sequencing technologies provided unexpected opportunities to characterize individual genomic landscape and identify mutations relevant for diagnosis and therapy in clinics. Specifically, whole-exome sequencing for complex disease (such as tumor/normal matched sample) and target resequencing for Mendelian disease, using next-generation sequencing (NGS) technologies, are gaining popularity in the human genetics community due to the moderate costs and the huge quantity of information provided by each experiment. However, NGS data analysis still remains the crucial bottleneck in this approach because of the great amount of data containing millions of potential disease-causing variants and difficulties involving the integration of different sources. Here, we describe the application of a bioinformatics analysis pipeline for NGS data in two case studies about rare cardiac diseases. The case 1 is focused on the target sequencing of 158 candidate genes in 91 patients affected by Brugada Syndrome (BrS). To date the clinical phenotype is associated with mutations in the SCN5A gene but explain only the 30% of the BrS cases. Therefore we selected a panel of genes previously associated to cases of arrhythmogenic disorders in literature and we analysed them in a cohort of BrS patients, which were negative for known SCN5A mutations. We found 98 novel genetic variations and 60 clinical rs belonging to 70 genes. In particular we found 13 genes significantly mutated in our cohort compared to healthy controls in 1000 Genomes data that were not previously associated to BrS phenotype. In case study 2 we performed a whole-exome sequencing experiment of trio family where the child is affected by a severe cardiac disease with unclear diagnosis. We developed a specific bioinformatics pipeline to filter out the germline mutations. We found six genes with novel deleterious mutations that were homozygous in the affected child, and heterozygous in both parents. Among the six mutated genes the TRDN and UNC45A genes were already associated to cardiac dysfunctions in literature. Functional studies will be performed to evaluate the involvement of the mutated genes in the disease onset. In conclusion, we developed an automatic and versatile pipeline to analyse NGS data coming from whole-exome sequencing and target sequencing strategies. In addition, we integrated in the pipeline several public variation databases to evaluate and interpret the candidate mutations. The mutations found were validated by Sanger sequencing to evaluate the strength of the pipeline filters.

Nonalcoholic fatty liver disease (NAFLD) is a rising cause of hepatocellular carcinoma (HCC). We examined whether inherited pathogenic variants in candidate genes (n = 181) were enriched in patients with NAFLD-HCC. To this end, we resequenced peripheral blood DNA of 142 NAFLD-HCC, 59 NAFLD with advanced fibrosis, and 50 controls, and considered 404 healthy individuals from 1000 G. Pathogenic variants were defined according to ClinVar, likely pathogenic as rare variants predicted to alter protein activity. In NAFLD-HCC patients, we detected an enrichment in pathogenic (p = 0.024), and likely pathogenic variants (p = 1.9*10−6), particularly in APOB (p = 0.047). APOB variants were associated with lower circulating triglycerides and higher HDL cholesterol (p −16), outperforming the diagnostic accuracy of common genetic risk variants, and of clinical risk factors (p

The 1.8 Å resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using β-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography.

Rifamycins are mainstay agents in treatment of many widespread diseases, but how an improved rifamycin producer can be created is still incompletely understood. Here, we describe a comparative genomic approach to investigate the mutational patterns introduced by the classical mutate-and-screen method in the genome of an improved rifamycin producer. Comparing the genome of the rifamycin B overproducer Amycolatopsis mediterranei HP-130 with those of the reference strains A. mediterranei S699 and U32, we identified 250 variations, affecting 227 coding sequences (CDS), 109 of which were HP-130-specific since they were absent in both S699 and U32. Mutational and transcriptional patterns indicated a series of genomic manipulations that not only proved the causative effect of mutB2 (coding for methylmalonyl-CoA mutase large subunit) and argS2 (coding for arginyl tRNA synthetase) mutations on the overproduction of rifamycin, but also constituted a rational strategy to genetically engineer a reference strain into an overproducer. © 2014 International Metabolic Engineering Society.

// Ingrid Cifola 1, * , Marta Lionetti 2, 3, * , Eva Pinatel 1 , Katia Todoerti 4 , Eleonora Mangano 1 , Alessandro Pietrelli 1 , Sonia Fabris 2, 3 , Laura Mosca 2, 3 , Vittorio Simeon 4 , Maria Teresa Petrucci 5 , Fortunato Morabito 6 , Massimo Offidani 7 , Francesco Di Raimondo 8 , Antonietta Falcone 9 , Tommaso Caravita 10 , Cristina Battaglia 1, 11 , Gianluca De Bellis 1 , Antonio Palumbo 12 , Pellegrino Musto 13 , Antonino Neri 2, 3 1 Institute for Biomedical Technologies, National Research Council, Milan, Italy 2 Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy 3 Hematology, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy 4 Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture (PZ), Italy 5 Hematology, Department of Cellular Biotechnologies and Hematology, La Sapienza University, Rome, Italy 6 Hematology Unit, Azienda Ospedaliera di Cosenza, Cosenza, Italy 7 Hematologic Clinic, Azienda Ospedaliero-Universitaria Ospedali Riuniti di Ancona, Ancona, Italy 8 Department of Biomedical Sciences, Division of Hematology, Ospedale Ferrarotto, University of Catania, Catania, Italy 9 Hematology Unit, IRCCS “Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo, Italy 10 Department of Hematology, Ospedale S. Eugenio, Tor Vergata University, Rome, Italy 11 Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy 12 Division of Hematology, University of Torino, A.O.U. San Giovanni Battista, Torino, Italy 13 Scientific Direction, IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture (PZ), Italy * These authors have contributed equally to this work Correspondence to: Antonino Neri, e-mail: antonino.neri@unimi.it Ingrid Cifola, e-mail: ingrid.cifola@itb.cnr.it Keywords: whole-exome sequencing, plasma cell leukemia, multiple myeloma, mutations Received: April 29, 2015 Accepted: May 11, 2015 Published: May 25, 2015 ABSTRACT Primary plasma cell leukemia (pPCL) is a rare and aggressive form of plasma cell dyscrasia and may represent a valid model for high-risk multiple myeloma (MM). To provide novel information concerning the mutational profile of this disease, we performed the whole-exome sequencing of a prospective series of 12 pPCL cases included in a Phase II multicenter clinical trial and previously characterized at clinical and molecular levels. We identified 1, 928 coding somatic non-silent variants on 1, 643 genes, with a mean of 166 variants per sample, and only few variants and genes recurrent in two or more samples. An excess of C > T transitions and the presence of two main mutational signatures (related to APOBEC over-activity and aging) occurring in different translocation groups were observed. We identified 14 candidate cancer driver genes, mainly involved in cell-matrix adhesion, cell cycle, genome stability, RNA metabolism and protein folding. Furthermore, integration of mutation data with copy number alteration profiles evidenced biallelically disrupted genes with potential tumor suppressor functions. Globally, cadherin/Wnt signaling, extracellular matrix and cell cycle checkpoint resulted the most affected functional pathways. Sequencing results were finally combined with gene expression data to better elucidate the biological relevance of mutated genes. This study represents the first whole-exome sequencing screen of pPCL and evidenced a remarkable genetic heterogeneity of mutational patterns. This may provide a contribution to the comprehension of the pathogenetic mechanisms associated with this aggressive form of PC dyscrasia and potentially with high-risk MM.