ligation overcomes terminal underrepresentation in multiple displacement amplification of linear dna

The amount of DNA available for informative genomic studies is often limiting. Thus, several methods have been developed to achieve a whole genome amplification (WGA). Particu-larly promising is a variant, called multiple displacement amplification (MDA) (1,2), which exploits the high processivity of Φ29 phage DNA polymerase and suffers an amplifi-cation bias significantly lower than previous, PCR-based WGA (3).Even though MDA-DNA has been successfully used for many applica-tions (1,4–7), some problems have been reported. For example, sequences near the ends of linear chromosomes appear underrepresented after MDA (6,8,9). Both defective priming events and abortive chain terminations could contribute to this problem, which limits the applications of MDA and jeopardizes its use on short linear DNA (e.g., cDNA and degraded genomes from forensic, archeological, and fetal origin) (10). In these cases, the terminal underrepresentation would cause the loss of substantial information.We studied this problem using λ phage DNA (48.5 kb; Promega Biosciences, San Luis Obispo, CA, USA). MDA was performed using the Genomiph

Precocious puberty and microbiota: The role of the sex hormone-gut microbiome axis

Puberty is a critical phase of life associated with physiological changes related to sexual maturation, and represents a complex process regulated by multiple endocrine and genetic controls. Puberty is driven by hormones, and it can impact the gut microbiome (GM). GM differences between sex emerge at puberty onset, confirming a relationship between microbiota and sex hormones. In this narrative review, we present an overview of precocious pubertal development and the changes in the GM in precocious puberty (PP) in order to consider the role of the sex hormone-gut microbiome axis from the perspective of pediatric endocrinology. Bidirectional interactions between the GM and sex hormones have been proposed in different studies. Although the evidence on the interaction between microbiota and sex hormones remains limited in pediatric patients, the evidence that GM alterations may occur in girls with central precocious puberty (CPP) represents an interesting finding for the prediction and prevention of PP. Deepening the understanding of the connection between the sex hormones and the role of microbiota changes can lead to the implementation of microbiota-targeted therapies in pubertal disorders by offering a pediatric endocrinology perspective

MeltingPlot, a user-friendly online tool for epidemiological investigation using High Resolution Melting data

BACKGROUND: The rapid identification of pathogen clones is pivotal for effective epidemiological control strategies in hospital settings. High Resolution Melting (HRM) is a molecular biology technique suitable for fast and inexpensive pathogen typing protocols. Unfortunately, the mathematical/informatics skills required to analyse HRM data for pathogen typing likely limit the application of this promising technique in hospital settings.RESULTS: MeltingPlot is the first tool specifically designed for epidemiological investigations using HRM data, easing the application of HRM typing to large real-time surveillance and rapid outbreak reconstructions. MeltingPlot implements a graph-based algorithm designed to discriminate pathogen clones on the basis of HRM data, producing portable typing results. The tool also merges typing information with isolates and patients metadata to create graphical and tabular outputs useful in epidemiological investigations and it runs in a few seconds even with hundreds of isolates.AVAILABILITY: https://skynet.unimi.it/index.php/tools/meltingplot/ .CONCLUSIONS: The analysis and result interpretation of HRM typing protocols can be not trivial and this likely limited its application in hospital settings. MeltingPlot is a web tool designed to help the user to reconstruct epidemiological events by combining HRM-based clustering methods and the isolate/patient metadata. The tool can be used for the implementation of HRM based real time large scale surveillance programs in hospital settings

Detection of ST1702 Escherichia coli blaNDM-5 and blaCMY-42 genes positive isolates from a Northern Italian hospital

We describe two multi drug-resistant (MDR) carbapenemase-producing Escherichia coli clinical isolates from an acute hospital in Milan. Both strains, isolated from a surgical wound sample and a surveillance rectal swab respectively, were positive for a blaNDM-type gene by Xpert Carba-R test. The whole-genome sequence characterization disclosed several resistance determinants: blaNDM-5, blaCMY-42, blaTEM-198, rmtB, mphA. The two isolates belonged to phylogenetic group A, sequence type (ST) 1702 and serotype O89:H9. PCR-based replicon typing and conjugation assay demonstrated an IncI1 plasmid localization for both blaNDM-5 and blaCMY-42 genes. This is the first report of a ST1702 NDM-5 and CMY-42- producing E. coli clone in Italy

Molecular identification of Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina) haemoparasites in Podarcis muralis lizards from northern Italy and detection of conserved motifs in the 18S rRNA gene

This study applies a non-invasive molecular test on common wall lizards (Podarcis muralis) collected in Northern Italy in order to i) identify protozoan blood parasites using primers targeting a portion of haemogregarine 18S rRNA; ii) perform a detailed bioinformatic and phylogenetic analysis of amplicons in a context where sequence analyses data are very scarce. Indeed the corresponding phylum (Apicomplexa) remains the poorest-studied animal group in spite of its significance for reptile ecology and evolution. A single genus, i.e., Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina) and an identical infecting genotype were identified in all positive hosts. Bioinformatic analyses identified highly conserved sequence patterns, some of which known to be involved in the host-parasite cross-talk. Phylogenetic analyses evidenced a limited host specificity, in accord with existing data. This paper provides the first Hepatozoon sequence from P. muralis and one of the few insights into the molecular parasitology, sequence analysis and phylogenesis of haemogregarine parasites

Probiotics as Therapeutic Tools against Pathogenic Biofilms: Have We Found the Perfect Weapon?

Bacterial populations inhabiting a variety of natural and human-associated niches have the ability to grow in the form of biofilms. A large part of pathological chronic conditions, and essentially all the bacterial infections associated with implanted medical devices or prosthetics, are caused by microorganisms embedded in a matrix made of polysaccharides, proteins, and nucleic acids. Biofilm infections are generally characterized by a slow onset, mild symptoms, tendency to chronicity, and refractory response to antibiotic therapy. Even though the molecular mechanisms responsible for resistance to antimicrobial agents and host defenses have been deeply clarified, effective means to fight biofilms are still required. Lactic acid bacteria (LAB), used as probiotics, are emerging as powerful weapons to prevent adhesion, biofilm formation, and control overgrowth of pathogens. Hence, using probiotics or their metabolites to quench and interrupt bacterial communication and aggregation, and to interfere with biofilm formation and stability, might represent a new frontier in clinical microbiology and a valid alternative to antibiotic therapies. This review summarizes the current knowledge on the experimental and therapeutic applications of LAB to interfere with biofilm formation or disrupt the stability of pathogenic biofilms

Potenzialità della metagenomica per la conservazione dei beni culturali

This paper focuses on the last generation methods for the characterization of biodeteriogens on historical artifacts. New "culture-independent" technologies based on DNA obtained from environmental samples, which have replaced the previous culture-dependent methods, are experiencing a real boom and revolutionized the field of microbial ecology. These methods are based conceptually on metagenomics and, technically, on the so-called NGS (Next-Generation Sequencing) technologies. Metagenomic analysis is defined as the direct genetic analysis of genomes contained in an environmental sample. The NGS technologies are characterized by a power and depth of sequencing totally unthinkable a few years ago and allow the identification also of the rarest species in a community. Even in their simplest declination, that is, the NGS sequencing of amplicons obtained with universal primers, the amount of data generated ranges from hundreds to thousands (for the Roche technology) in the millions-hundreds of millions (for the Illumina platforms)