Circular PCR as an efficient and precise umbrella of methods for the generation of circular dsDNA with staggered nicks: Mechanism and types.

Here, we introduce the highly versatile circular polymerase chain reaction (CiPCR) technique, propose a mechanism of action, and describe a number of examples demonstrating the versatility of this technique. CiPCR takes place between two fragments of dsDNA with two homologous regions, as long as one of the fragments carries said regions at its 3'- and 5'-ends. Upon hybridization, elongation by a polymerase occurs from all 3'-ends continuously until a 5'-end is reached, leading to stable circular dsDNA with staggered nicks.

Our current understanding of the toxicity of altered mito-ribosomal fidelity during mitochondrial protein synthesis: What can it tell us about human disease?

Altered mito-ribosomal fidelity is an important and insufficiently understood causative agent of mitochondrial dysfunction. Its pathogenic effects are particularly well-known in the case of mitochondrially induced deafness, due to the existence of the, so called, ototoxic variants at positions 847C (m.1494C) and 908A (m.

Heterologous Inferential Analysis (HIA) and Other Emerging Concepts: In Understanding Mitochondrial Variation In Pathogenesis: There is no More Low-Hanging Fruit.

Here we summarize our latest efforts to elucidate the role of mtDNA variants affecting the mitochondrial translation machinery, namely variants mapping to the mt-rRNA and mt-tRNA genes. Evidence is accumulating to suggest that the cellular response to interference with mitochondrial translation is different from that occurring as a result of mutations in genes encoding OXPHOS proteins. As a result, it appears safe to state that a complete view of mitochondrial disease will not be obtained until we understand the effect of mt-rRNA and mt-tRNA variants on mitochondrial protein synthesis.

Covalently and ionically, dually crosslinked chitosan nanoparticles block quorum sensing and affect bacterial cell growth on a cell-density dependent manner.

In our efforts to improve the quality and stability of chitosan nanoparticles (NPs), we describe here a new type of chitosan NPs dually crosslinked with genipin and sodium tripolyphosphate (TPP) that display quorum quenching activity. These NPs were created using a simplified and robust procedure that resulted in improved physicochemical properties and enhanced stability. This procedure involves the covalent crosslinking of chitosan with genipin, followed by the formation of chitosan NPs by ionic gelation with TPP.

Minority Gene Expression Profiling: Probing the Genetic Signatures of Pathogenesis Using Ribosome Profiling.

Minority Gene Expression Profiling (MGEP) refers to a scenario where the expression profiles of specific genes of interest are concentrated in a small cellular pool that is embedded within a larger, non-expressive pool. An example of this is the analysis of disease-related genes within sub-populations of blood or biopsied tissues. These systems are characterized by low signal-to-noise ratios that make it difficult, if not impossible, to uncover the desired signatures of pathogenesis in the absence of lengthy, and often problematic, technical manipulations.

Precision Medicine and Precision Public Health in the Era of Pathogen Next-Generation Sequencing.

This brief report serves as an introduction to a supplement of the Journal of Infectious Diseases entitled "Next-Generation Sequencing (NGS) Technologies to Advance Global Infectious Disease Research." We briefly discuss the history of NGS technologies and describe how the techniques developed during the past 40 years have impacted our understanding of infectious diseases. Our focus is on the application of NGS in the context of pathogen genomics.

Outer membrane protein FrpA, the siderophore piscibactin receptor of Photobacterium damselae subsp. piscicida, as a subunit vaccine against photobacteriosis in sole (Solea senegalensis).

Photobacteriosis caused by Photobacterium damselae subsp. piscicida (Pdp) remains one of the main infectious diseases affecting cultured fish in Mediterranean countries. Diverse vaccine formulations based in the use of inactivated bacterial cells have been used with unsatisfactory results, especially in newly cultured species like sole (Solea senegalensis).

Effect of the ultrastructure of chitosan nanoparticles in colloidal stability, quorum quenching and antibacterial activities.

We have fabricated two types of crosslinked chitosan-based nanoparticles (NPs), namely (1) ionically crosslinked with tripolyphosphate (TPP), designated as IC-NPs and (2) dually co-crosslinked (ionically and covalently with TPP and genipin, respectively) termed CC-NPs. The two types of NPs were physichochemically characterized by means of DLS-NIBS, synchrotron SAXS and M3-PALS (zeta potential). First, we found that covalent co-crosslinking of ionically pre-crosslinked nanoparticles yielded monodisperse CC-NPs in the size range of ∼200 nm, whereas the parental IC-NPs remained highly polydisperse.

Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness.

Diet may be modified seasonally or by biogeographic, demographic or cultural shifts. It can differentially influence mitochondrial bioenergetics, retrograde signalling to the nuclear genome, and anterograde signalling to mitochondria. All these interactions have the potential to alter the frequencies of mtDNA haplotypes (mitotypes) in nature and may impact human health.

Recruitment of RNA molecules by connexin RNA-binding motifs: Implication in RNA and DNA transport through microvesicles and exosomes.

Connexins (Cxs) are integral membrane proteins that form high-conductance plasma membrane channels, allowing communication from cell to cell (via gap junctions) and from cells to the extracellular environment (via hemichannels). Initially described for their role in joining excitable cells (nerve and muscle), gap junctions (GJs) are found between virtually all cells in solid tissues and are essential for functional coordination by enabling the direct transfer of small signalling molecules, metabolites, ions, and electrical signals from cell to cell. Several studies have revealed diverse channel-independent functions of Cxs, which include the control of cell growth and tumourigenicity.

The presence of highly disruptive 16S rRNA mutations in clinical samples indicates a wider role for mutations of the mitochondrial ribosome in human disease.

Mitochondrial DNA mutations are well recognized as an important cause of disease, with over two hundred variants in the protein encoding and mt-tRNA genes associated with human disorders. In contrast, the two genes encoding the mitochondrial rRNAs (mt-rRNAs) have been studied in far less detail. This is because establishing the pathogenicity of mt-rRNA mutations is a major diagnostic challenge.

Heterologous inferential analysis (HIA) as a method to understand the role of mitochondrial rRNA mutations in pathogenesis.

Despite the identification of a large number of potentially pathogenic variants in the mitochondrially encoded rRNA (mt-rRNA) genes, we lack direct methods to firmly establish their pathogenicity. In the absence of such methods, we have devised an indirect approach named heterologous inferential analysis or HIA that can be used to make predictions on the disruptive potential of a large subset of mt-rRNA variants. First, due to the high evolutionary conservation of the rRNA fold, comparison of phylogenetically derived secondary structures of the human mt-rRNAs and those from model organisms allows the location of structurally equivalent residues.

The role of the mitochondrial ribosome in human disease: searching for mutations in 12S mitochondrial rRNA with high disruptive potential.

Mutations of mitochondrial DNA are linked to many human diseases. Despite the identification of a large number of variants in the mitochondrially encoded rRNA (mt-rRNA) genes, the evidence supporting their pathogenicity is, at best, circumstantial. Establishing the pathogenicity of these variations is of major diagnostic importance.

Tertiary interactions between helices h13 and h44 in 16S RNA contribute to the fidelity of translation.

The A-minor interaction, formed between single-stranded adenosines and the minor groove of a receptor helix, is among the most common motifs found in rRNA. Among the A-minors found in 16S rRNA are a set of interactions between adenosines at positions 1433, 1434 and 1468 in helix 44 (h44) and their receptors in the nucleotide 320-340 region of helix 13 (h13). These interactions have been implicated in the maintenance of translational accuracy, because base substitutions at the adjacent C1469 increase miscoding errors.

Mutations in the intersubunit bridge regions of 16S rRNA affect decoding and subunit-subunit interactions on the 70S ribosome.

The small and large subunits of the ribosome are held together by a series of bridges, involving RNA-RNA, RNA-protein and protein-protein interactions. Some 12 bridges have been described for the Escherichia coli 70S ribosome. In this work, we have targeted for mutagenesis, some of the 16S rRNA residues involved in the formation of intersubunit bridges B3, B5, B6, B7b and B8.

Phylogenetic and multivariate analyses to determine the effects of different tillage and residue management practices on soil bacterial communities.

Bacterial communities are important not only in the cycling of organic compounds but also in maintaining ecosystems. Specific bacterial groups can be affected as a result of changes in environmental conditions caused by human activities, such as agricultural practices. The aim of this study was to analyze the effects of different forms of tillage and residue management on soil bacterial communities by using phylogenetic and multivariate analyses.

Mutagenesis of the modified bases, m(5)U1939 and psi2504, in Escherichia coli 23S rRNA.

Ribosomal RNAs (rRNAs) from all kingdoms contain a variety of post-transcriptional modifications and these are typically clustered in the functional centers of the ribosome. The functions of two bases in the 23S rRNA of Escherichia coli that are post-transcriptionally modified, m(5)U1939 and psi2504, were examined by mutagenesis of the rRNA bases and by inactivation of the RumA methylase that methylates U1939. Base substitutions at U1939 had little effect on growth or the fidelity of translation, but altered the sensitivity of the ribosomes to the antibiotics fusidic acid and capreomycin.

Modification of the Ribosome and the Translational Machinery during Reduced Growth Due to Environmental Stress.

Escherichia coli strains normally used under laboratory conditions have been selected for maximum growth rates and require maximum translation efficiency. Recent studies have shed light on the structural and functional changes undergone by the translational machinery in E. coli during heat and cold shock and upon entry into stationary phase.

Modulation of 16S rRNA function by ribosomal protein S12.

Ribosomal protein S12 is a critical component of the decoding center of the 30S ribosomal subunit and is involved in both tRNA selection and the response to streptomycin. We have investigated the interplay between S12 and some of the surrounding 16S rRNA residues by examining the phenotypes of double-mutant ribosomes in strains of Escherichia coli carrying deletions in all chromosomal rrn operons and expressing total rRNA from a single plasmid-borne rrn operon. We show that the combination of S12 and otherwise benign mutations at positions C1409-G1491 in 16S rRNA severely compromises cell growth while the level and range of aminoglycoside resistances conferred by the G1491U/C substitutions is markedly increased by a mutant S12 protein.

Structural analysis of kasugamycin inhibition of translation.

The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-A resolution.

Structures of the bacterial ribosome at 3.5 A resolution.

We describe two structures of the intact bacterial ribosome from Escherichia coli determined to a resolution of 3.5 angstroms by x-ray crystallography. These structures provide a detailed view of the interface between the small and large ribosomal subunits and the conformation of the peptidyl transferase center in the context of the intact ribosome.

Structural basis for the control of translation initiation during stress.

During environmental stress, organisms limit protein synthesis by storing inactive ribosomes that are rapidly reactivated when conditions improve. Here we present structural and biochemical data showing that protein Y, an Escherichia coli stress protein, fills the tRNA- and mRNA-binding channel of the small ribosomal subunit to stabilize intact ribosomes. Protein Y inhibits translation initiation during cold shock but not at normal temperatures.

X-ray crystal structures of the WT and a hyper-accurate ribosome from Escherichia coli.

Protein biosynthesis on the ribosome requires accurate reading of the genetic code in mRNA. Two conformational rearrangements in the small ribosomal subunit, a closing of the head and body around the incoming tRNA and an RNA helical switch near the mRNA decoding site, have been proposed to select for complementary base-pairing between mRNA codons and tRNA anticodons. We determined x-ray crystal structures of the WT and a hyper-accurate variant of the Escherichia coli ribosome at resolutions of 10 and 9 A, respectively, revealing that formation of the intact 70S ribosome from its two subunits closes the conformation of the head of the small subunit independent of mRNA decoding.