Pilot study on circulating miRNA signature in children with obesity born small for gestational age and appropriate for gestational age.

BACKGROUND: Children born small for gestational age (SGA) are at increased risk of metabolic dysfunction. Dysregulation of specific microRNAs (miRNAs) contributes to aberrant gene expression patterns underlying metabolic dysfunction. OBJECTIVE: We aimed to determine and compare circulating miRNA (c-miRNA) profile of SGA and appropriate for gestational age (AGA) children with obesity and with normal weight, in order to identify biomarkers for early detection of increased risk of developing metabolic dysfunction in SGA and AGA children with obesity. METHODS: Small non-coding RNAs from serum of 15 SGA children with obesity (OB-SGA), 10 SGA children with normal weight (NW-SGA), 17 AGA children with obesity (OB-AGA) and 12 AGA children with normal weight (NW-AGA) (mean age 11.2 ± 2.6) have been extracted and sequenced in order to detect and quantify miRNA expression profiles. RESULTS: RNA-seq analyses showed 28 miRNAs dysregulated in OB-SGA vs. NW-SGA and 19 miRNAs dysregulated in OB-AGA vs. NW-AGA. Among these, miR-92a-3p, miR-122-5p, miR-423-5p, miR-484, miR-486-3p and miR-532-5p were up regulated, and miR-181b-5p was down regulated in both OB-SGA and OB-AGA compared with normal weight counterparts. Pathway analysis and miRNA target prediction suggested that these miRNAs were particularly involved in insulin signalling, glucose transport, insulin resistance, cholesterol and lipid metabolism. CONCLUSION: We identified a specific profile of c-miRNAs in SGA and AGA children with obesity compared with SGA and AGA children with normal weight. These c-miRNAs could represent specific biomarkers for early detection of increased risk of developing metabolic dysfunction in SGA and AGA children with obesity.

RNA editing signature during myeloid leukemia cell differentiation.

Adenosine deaminases acting on RNA (ADARs) are key proteins for hematopoietic stem cell self-renewal and for survival of differentiating progenitor cells. However, their specific role in myeloid cell maturation has been poorly investigated. Here we show that ADAR1 is present at basal level in the primary myeloid leukemia cells obtained from patients at diagnosis as well as in myeloid U-937 and THP1 cell lines and its expression correlates with the editing levels. Upon phorbol-myristate acetate or Vitamin D3/granulocyte macrophage colony-stimulating factor (GM-CSF)-driven differentiation, both ADAR1 and ADAR2 enzymes are upregulated, with a concomitant global increase of A-to-I RNA editing. ADAR1 silencing caused an editing decrease at specific ADAR1 target genes, without, however, interfering with cell differentiation or with ADAR2 activity. Remarkably, ADAR2 is absent in the undifferentiated cell stage, due to its elimination through the ubiquitin-proteasome pathway, being strongly upregulated at the end of the differentiation process. Of note, peripheral blood monocytes display editing events at the selected targets similar to those found in differentiated cell lines. Taken together, the data indicate that ADAR enzymes play important and distinct roles in myeloid cells.

A novel group of IncQ1 plasmids conferring multidrug resistance.

The IncQ is a group of non-conjugative but mobilisable plasmids that are found and stably maintained in a wide range of bacteria contributing to the spread of antimicrobial resistance genes and to the insurgence of multidrug resistant bacteria. Here we report the identification, in clinical Salmonella Typhimurium strains, of an IncQ1 plasmid (pNUC) which confers resistance to sulfamethoxazole, streptomycin and tetracycline through the presence of sul2, strAB and tetA genes, respectively. pNUC was detected in five multidrug resistant S. Typhimurium strains collected in Southern Italy from various hospitals and years of isolation. Bioinformatics analyses highlighted the presence of pNUC-like plasmids in pathogenic bacteria of various Enterobacteriaceae genera or species. Taken as a whole, these plasmids constitute a novel group of IncQ1 plasmids that might have originated through recombination events between a tetR-tetA gene cluster (possibly derived from a Tn1721) and a recipient IncQ1 plasmid related to RSF1010. Our findings raise concerns regarding the possible contribution of the newly identified group of IncQ1 plasmids to the spread of tetracycline resistance.

Diversity and temporal patterns of planktonic protist assemblages at a Mediterranean Long Term Ecological Research site.

We tracked temporal changes in protist diversity at the Long Term Ecological Research (LTER) station MareChiara in the Gulf of Naples (Mediterranean Sea) on eight dates in 2011 using a metabarcoding approach. Illumina analysis of the V4 and V9 fragments of the 18S rDNA produced 869 522 and 1 410 071 sequences resulting in 6517 and 6519 OTUs, respectively. Marked compositional variations were recorded across the year, with less than 2% of OTUs shared among all samples and similar patterns for the two marker tags. Alveolata, Stramenopiles and Rhizaria were the most represented groups. A comparison with light microscopy data indicated an over-representation of Dinophyta in the sequence dataset, whereas Bacillariophyta showed comparable taxonomic patterns between sequence and light microscopy data. Shannon diversity values were stable from February to September, increasing thereafter with a peak in December. Community variance was mainly explained by seasonality (as temperature), trophic status (as chlorophyll a), and influence of coastal waters (as salinity). Overall, the background knowledge of the system provided a sound context for the result interpretation, showing that LTER sites provide an ideal setting for high-throughput sequencing (HTS) metabarcoding characterisation of protist assemblages and their relationships with environmental variations.

ASPicDB: a database resource for alternative splicing analysis.

MOTIVATION: Alternative splicing has recently emerged as a key mechanism responsible for the expansion of transcriptome and proteome complexity in human and other organisms. Although several online resources devoted to alternative splicing analysis are available they may suffer from limitations related both to the computational methodologies adopted and to the extent of the annotations they provide that prevent the full exploitation of the available data. Furthermore, current resources provide limited query and download facilities. RESULTS: ASPicDB is a database designed to provide access to reliable annotations of the alternative splicing pattern of human genes and to the functional annotation of predicted splicing isoforms. Splice-site detection and full-length transcript modeling have been carried out by a genome-wide application of the ASPic algorithm, based on the multiple alignments of gene-related transcripts (typically a Unigene cluster) to the genomic sequence, a strategy that greatly improves prediction accuracy compared to methods based on independent and progressive alignments. Enhanced query and download facilities for annotations and sequences allow users to select and extract specific sets of data related to genes, transcripts and introns fulfilling a combination of user-defined criteria. Several tabular and graphical views of the results are presented, providing a comprehensive assessment of the functional implication of alternative splicing in the gene set under investigation. ASPicDB, which is regularly updated on a monthly basis, also includes information on tissue-specific splicing patterns of normal and cancer cells, based on available EST sequences and their library source annotation. AVAILABILITY: www.caspur.it/ASPicDB

New p53 mutations in hilar cholangiocarcinoma.

BACKGROUND: Inactivation of the tumour suppressor gene, p53, is the commonest genetic abnormality in human cancer. The study of the type of p53 mutation in a given tumour may provide prognostic information, clues to aetiology and become useful for therapeutics. MATERIALS AND METHODS: The molecular characterisation of p53 was performed by restriction analysis, denaturing gradient gel electrophoresis, and gene sequencing for exons 5-9. RESULTS: We report, p53 mutational analysis in exons 5-9 in 29 European patients with hilar cholangiocarcinoma who underwent attempted resection. Four patients (14%) showed somatic single nucleotide substitutions with amino acid changes (146, 163, 175, 158, and 175) with one showing mutations in two different positions involving a loss of two CfoI sites. All the mutations occurred in exon 5. Three patients had a germline polymorphism (10%) with a silent substitution in codon 213 (exon 6). CONCLUSIONS: The systematic screening for p53 mutations in European patients with hilar cholangiocarcinoma has shown that the type of mutation (except 175) is different and its incidence is much lower when compared to the pattern previously reported for intrahepatic cholangiocarcinoma in East Asian patients. A probable explanation is that the presence and type of p53 mutation is dependent on geographic and environmental factors which vary in different populations.

Characterization of p53 mutations in colorectal liver metastases and correlation with clinical parameters.

The presence and type of mutations of the p53 tumor suppressor gene were determined in 40 patients undergoing curative hepatic resection for metastatic colorectal carcinoma. This represents the largest series in the literature on the screening of p53 mutations for liver metastases. The analysis was performed in exons 5-9 by denaturing gradient gel electrophoresis followed by direct sequencing. Forty-five percent of tumors showed mutation in p53, and this was observed only in exons 5-8. Mutations at codon positions 167, 196, 204, 213, 245, 281, 282, 286, and 306; deletion of codon 251 and of the first nucleotide of codon 252; and Leu residue (CTC) insertion downstream codon 252 are reported for the first time in colorectal liver metastasis. Mutations at codon positions 163, 248, and 273 have been reported previously. Correlation of p53 status with clinical parameters showed that patients with mutated p53 had a statistically higher number of lesions when compared with patients with wild-type p53 (P<0.050). In particular, of patients with mutated p53, 41% had three or more metastases compared with 14% of patients with wild-type p53. Synchronous metastases were present in 70% of the patients with p53 mutations and in only 29% of patients with wild-type p53 (P<0.025). In addition, patients with p53 mutations are more likely to develop recurrence (73%) compared with patients with wild-type p53 (33%; P<0.001). Other factors considered, including preoperative carcinoembryonic antigen level, bilobar distribution, and size of the lesion(s), did not show significant correlation with p53 status. These results suggest that p53 status might be an important prognostic indicator to predict the pattern and likelihood of treatment failure after hepatic resection.

Guinea pig p53 mRNA: identification of new elements in coding and untranslated regions and their functional and evolutionary implications.

We report the sequence of the guinea pig p53 cDNA. The comparative analysis of the coding and noncoding regions of p53 cDNAs of all available complete vertebrate sequences has allowed us to single out new conserved signals possibly involved in p53 functional activity. We have focused our attention on the most variable region of the protein, the proline (P)-rich domain, suggested to play a fundamental role in antiproliferative pathways. In this domain we have identified the PXXXXP repeated motif and singled out a common consensus sequence that can be considered a signature for mammalian p53: PXXXXPX{0,4}PX{0,9}PA(T,P,I,)(S,P)WPL. We have demonstrated the significance of the PXXXXP motif in SH3-binding protein and suggested its structure to be a loop. Also, the 5' and 3' untranslated regions (UTRs) of the guinea pig were sequenced, and this study represents the first detailed structural analysis of the UTRs of the p53 mRNAs available in literature. The 5' UTR of guinea pig (233 nt) can be folded into a stable secondary structure resembling that predicted in mouse. The 3' UTR of guinea pig is 771 nt long and shows higher similarity with human than with rodent sequences, having a region of about 350 nt that is deleted in rat and mouse. In the 3' UTR we have identified the presence of a mammalian-wide interspersed repeat sequence and of a cytoplasmic polyadenylation element, which could be involved in translational activation by promoting polyadenylation of mRNA, providing information about a possible mechanism of regulation of p53 expression mediated by the 3' UTR of the mRNA. The observations presented here could open new avenues to targeted mutations and experimental approaches useful in investigating new regulation mechanisms of p53 translation, activity, and stability.

The guinea-pig is not a rodent.

In 1991 Graur et al. raised the question of whether the guinea-pig, Cavia porcellus, is a rodent. They suggested that the guinea-pig and myomorph rodents diverged before the separation between myomorph rodents and a lineage leading to primates and artiodactyls. Several findings have since been reported, both for and against this phylogeny, thereby highlighting the issue of the validity of molecular analysis in mammalian phylogeny. Here we present findings based on the sequence of the complete mitochondrial genome of the guinea-pig, which strongly contradict rodent monophyly. The conclusions are based on cumulative evidence provided by orthologically inherited genes and the use of three different analytical methods, none of which joins the guinea-pig with myomorph rodents. In addition to the phylogenetic conclusions, we also draw attention to several factors that are important for the validity of phylogenetic analysis based on molecular data.

Transcription of rat mitochondrial NADH-dehydrogenase subunits. Presence of antisense and precursor RNA species.

We have characterized the transcriptional pattern of the rat mitochondrial ND6-containing region in vivo. We have identified a stable polyadenylated RNA species complementary for the full length of the ND6 mRNA. The analysis of the ND5 region has revealed the presence of an antisense RNA only at its 3' end. The presence of these stable antisense species complementary to structural genes is intriguing and suggests a possible regulatory function. The quantitative analyses have demonstrated that the H transcripts, both codogenic and non-codogenic, are more stable than the L transcripts. We have defined the 5' end of the ND6 mRNA at the level of the ATG downstream of the tRNA(Glu). The mapping of the ND1 5' end has demonstrated that GTG is the first codon of the mRNA. Our findings suggest that the post-transcriptional mechanisms involved in the expression of the mt genome are much more numerous and complex than those already described in the literature.

Detection of novel transcripts in the human mitochondrial DNA region coding for ATPase8-ATPase6 subunits.

We have analyzed the tRNA(Lys), ATPase8, ATPase6, COIII region of mitochondrial DNA in several human tissues. Beside the mature tRNA(Lys), ATPase8 and ATPase6 common mRNA, and COIII mRNA, we have characterized two new transcripts, called RNA 20 and RNA 21. The RNA 20 is a precursor species which contains the tRNA(Lys) plus the ATPase8 and ATPase6 common mRNA; the RNA 21 is an RNA species shorter than the ATPase8 and ATPase6 common mRNA. The relative concentration of the mature with respect to that of the new species proved different in the various tissues. These findings provide new insights into the mitochondrial transcription mechanism opening the question of a possibly regulatory role of the processing on the expression of the mitochondrial genome.