Ancient DNA studies: new perspectives on old samples

In spite of past controversies, the field of ancient DNA is now a reliable research area due to recent methodological improvements. A series of recent large-scale studies have revealed the true potential of ancient DNA samples to study the processes of evolution and to test models and assumptions commonly used to reconstruct patterns of evolution and to analyze population genetics and palaeoecological changes. Recent advances in DNA technologies, such as next-generation sequencing make it possible to recover DNA information from archaeological and paleontological remains allowing us to go back in time and study the genetic relationships between extinct organisms and their contemporary relatives. With the next-generation sequencing methodologies, DNA sequences can be retrieved even from samples (for example human remains) for which the technical pitfalls of classical methodologies required stringent criteria to guaranty the reliability of the results. In this paper, we review the methodologies applied to ancient DNA analysis and the perspectives that next-generation sequencing applications provide in this field

In vitro transcription profiling of the σS subunit of bacterial RNA polymerase: re-definition of the σS regulon and identification of σS-specific promoter sequence elements

Specific promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble with RNA polymerase core enzyme (E) during transcription initiation. However, σ70 (the housekeeping σ subunit) and σS (an alternative σ subunit mostly active during slow growth) recognize almost identical promoter sequences, thus raising the question of how promoter selectivity is achieved in the bacterial cell. To identify novel sequence determinants for selective promoter recognition, we performed run-off/microarray (ROMA) experiments with RNA polymerase saturated either with σ70 (Eσ70) or with σS (EσS) using the whole Escherichia coli genome as DNA template. We found that Eσ70, in the absence of any additional transcription factor, preferentially transcribes genes associated with fast growth (e.g. ribosomal operons). In contrast, EσS efficiently transcribes genes involved in stress responses, secondary metabolism as well as RNAs from intergenic regions with yet-unknown function. Promoter sequence comparison suggests that, in addition to different conservation of the −35 sequence and of the UP element, selective promoter recognition by either form of RNA polymerase can be affected by the A/T content in the −10/+1 region. Indeed, site-directed mutagenesis experiments confirmed that an A/T bias in the −10/+1 region could improve promoter recognition by Eσ

Un modello di danno fragile per mezzi porosi: esempi di applicazione

Si presenta un approccio accoppiato per modellare il danneggiamento indotto da sollecitazioni idrauliche e meccaniche in ammassi rocciosi. Il danneggiamento del materiale è legato alla formazione a scala microstrutturale di diverse famiglie di fratture parallele, annidate una nell’altra, ciascuna caratterizzata da una propria orientazione e spaziatura. La semplicità della geometria delle fratture permette di esprimere analiticamente la variazione di porosità e di permeabilità causate dal progressivo danneggiamento del materiale. Si illustrano alcune simulazioni, sia a livello di punto di volume sia come problema al contorno, per mettere in evidenza i potenziali campi di applicazione del modello, tra i quali si individuano la stabilità di perforazioni in roccia e l’ottimizzazione di processi di fratturazione idraulica

High taxonomic level fingerprint of the human intestinal microbiota by Ligase Detection Reaction - Universal Array approach

<p>Abstract</p> <p>Background</p> <p>Affecting the core functional microbiome, peculiar high level taxonomic unbalances of the human intestinal microbiota have been recently associated with specific diseases, such as obesity, inflammatory bowel diseases, and intestinal inflammation.</p> <p>Results</p> <p>In order to specifically monitor microbiota unbalances that impact human physiology, here we develop and validate an original DNA-microarray (HTF-Microbi.Array) for the high taxonomic level fingerprint of the human intestinal microbiota. Based on the Ligase Detection Reaction-Universal Array (LDR-UA) approach, the HTF-Microbi.Array enables specific detection and approximate relative quantification of 16S rRNAs from 30 phylogenetically related groups of the human intestinal microbiota. The HTF-Microbi.Array was used in a pilot study of the faecal microbiota of eight young adults. Cluster analysis revealed the good reproducibility of the high level taxonomic microbiota fingerprint obtained for each of the subject.</p> <p>Conclusion</p> <p>The HTF-Microbi.Array is a fast and sensitive tool for the high taxonomic level fingerprint of the human intestinal microbiota in terms of presence/absence of the principal groups. Moreover, analysis of the relative fluorescence intensity for each probe pair of our LDR-UA platform can provide estimation of the relative abundance of the microbial target groups within each samples. Focusing the phylogenetic resolution at division, order and cluster levels, the HTF-Microbi.Array is blind with respect to the inter-individual variability at the species level.</p

WoPPER: Web server for Position Related data analysis of gene Expression in Prokaryotes

The structural and conformational organization of chromosomes is crucial for gene expression regulation in eukaryotes and prokaryotes as well. Up to date, gene expression data generated using either microarray or RNA-sequencing are available for many bacterial genomes. However, differential gene expression is usually investigated with methods considering each gene independently, thus not taking into account the physical localization of genes along a bacterial chromosome. Here, we present WoPPER, a web tool integrating gene expression and genomic annotations to identify differentially expressed chromosomal regions in bacteria. RNA-sequencing or microarray-based gene expression data are provided as input, along with gene annotations. The user can select genomic annotations from an internal database including 2780 bacterial strains, or provide custom genomic annotations. The analysis produces as output the lists of positionally related genes showing a coordinated trend of differential expression. Graphical representations, including a circular plot of the analyzed chromosome, allow intuitive browsing of the results. The analysis procedure is based on our previously published R-package PREDA. The release of this tool is timely and relevant for the scientific community, as WoPPER will fill an existing gap in prokaryotic gene expression data analysis and visualization tools. WoPPER is open to all users and can be reached at the following URL: https://WoPPER.ba.itb.cnr.it

Comparative genomics and transcriptional profiles of Saccharopolyspora erythraea NRRL 2338 and a classically improved erythromycin over-producing strain

<p>Abstract</p> <p>Background</p> <p>The molecular mechanisms altered by the traditional mutation and screening approach during the improvement of antibiotic-producing microorganisms are still poorly understood although this information is essential to design rational strategies for industrial strain improvement. In this study, we applied comparative genomics to identify all genetic changes occurring during the development of an erythromycin overproducer obtained using the traditional mutate-and- screen method.</p> <p>Results</p> <p>Compared with the parental <it>Saccharopolyspora erythraea </it>NRRL 2338, the genome of the overproducing strain presents 117 deletion, 78 insertion and 12 transposition sites, with 71 insertion/deletion sites mapping within coding sequences (CDSs) and generating frame-shift mutations. Single nucleotide variations are present in 144 CDSs. Overall, the genomic variations affect 227 proteins of the overproducing strain and a considerable number of mutations alter genes of key enzymes in the central carbon and nitrogen metabolism and in the biosynthesis of secondary metabolites, resulting in the redirection of common precursors toward erythromycin biosynthesis. Interestingly, several mutations inactivate genes coding for proteins that play fundamental roles in basic transcription and translation machineries including the transcription anti-termination factor NusB and the transcription elongation factor Efp. These mutations, along with those affecting genes coding for pleiotropic or pathway-specific regulators, affect global expression profile as demonstrated by a comparative analysis of the parental and overproducer expression profiles. Genomic data, finally, suggest that the mutate-and-screen process might have been accelerated by mutations in DNA repair genes.</p> <p>Conclusions</p> <p>This study helps to clarify the mechanisms underlying antibiotic overproduction providing valuable information about new possible molecular targets for rationale strain improvement.</p

Unbalance of intestinal microbiota in atopic children

BACKGROUND: Playing a strategic role in the host immune function, the intestinal microbiota has been recently hypothesized to be involved in the etiology of atopy. In order to investigate the gastrointestinal microbial ecology of atopic disease, here we performed a pilot comparative molecular analysis of the faecal microbiota in atopic children and healthy controls. RESULTS: Nineteen atopic children and 12 healthy controls aged 4–14 years were enrolled. Stools were collected and the faecal microbiota was characterized by means of the already developed phylogenetic microarray platform, HTF-Microbi.Array, and quantitative PCR. The intestinal microbiota of atopic children showed a significant depletion in members of the Clostridium cluster IV, Faecalibacterium prausnitzii, Akkermansia muciniphila and a corresponding increase of the relative abundance of Enterobacteriaceae. CONCLUSION: Depleted in key immunomodulatory symbionts, the atopy-associated microbiota can represent an inflammogenic microbial consortium which can contribute to the severity of the disease. Our data open the way to the therapeutic manipulation of the intestinal microbiota in the treatment of atopy by means of pharmaceutical probiotics

Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

BACKGROUND: PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. RESULTS: Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. CONCLUSIONS: The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria

The germline of the malaria mosquito produces abundant miRNAs, endo-siRNAs, piRNAs and 29-nt small RNA

BACKGROUND Small RNAs include different classes essential for endogenous gene regulation and cellular defence against genomic parasites. However, a comprehensive analysis of the small RNA pathways in the germline of the mosquito Anopheles gambiae has never been performed despite their potential relevance to reproductive capacity in this malaria vector. RESULTS We performed small RNA deep sequencing during larval and adult gonadogenesis and find that they predominantly express four classes of regulatory small RNAs. We identified 45 novel miRNA precursors some of which were sex-biased and gonad-enriched , nearly doubling the number of previously known miRNA loci. We also determine multiple genomic clusters of 24-30 nt Piwi-interacting RNAs (piRNAs) that map to transposable elements (TEs) and 3'UTR of protein coding genes. Unusually, many TEs and the 3'UTR of some endogenous genes produce an abundant peak of 29-nt small RNAs with piRNA-like characteristics. Moreover, both sense and antisense piRNAs from TEs in both Anopheles gambiae and Drosophila melanogaster reveal novel features of piRNA sequence bias. We also discovered endogenous small interfering RNAs (endo-siRNAs) that map to overlapping transcripts and TEs. CONCLUSIONS This is the first description of the germline miRNome in a mosquito species and should prove a valuable resource for understanding gene regulation that underlies gametogenesis and reproductive capacity. We also provide the first evidence of a piRNA pathway that is active against transposons in the germline and our findings suggest novel piRNA sequence bias. The contribution of small RNA pathways to germline TE regulation and genome defence in general is an important finding for approaches aimed at manipulating mosquito populations through the use of selfish genetic elements