Molecular recognition of oligonucleotides and plasmid DNA by l‐methionine

The present study explores the effect of oligonucleotide composition on the mechanism of retention to l ‐methionine agarose support by chromatography and saturation transfer difference (STD)‐nuclear magnetic resonance (NMR) techniques. All chromatographic experiments were performed using 1.5 M (NH₄)₂SO₄. The binding profiles obtained by chromatography show that oligonucleotides with thymine had the highest retention time. In general, the larger homo‐oligonucleotides are more retained to the l ‐methionine agarose support. Moreover, the study with hetero‐oligonucleotides confirms that the presence of guanine reduces the retention on the l ‐methionine chromatographic support. These results are in accord with STD‐NMR experiments, which show that the strongest signals were observed for the methyl group of thymine, and no STD signals were observed for the guanosine protons. Finally, the retention behaviour of linear plasmid DNA (pDNA) with different sizes and base composition (2.7‐kbp pUC19, 6.05‐kbp pVAX1‐LacZ, 7.4‐kbp pVAX1‐LacZgag and 14‐kbp pcDNA‐based plasmid) was also evaluated by chromatography. The results indicate that the underlying mechanism of retention involves not only hydrophobic interactions but also other elementary interactions responsible for the biorecognition of pDNA molecules by l ‐methionine ligands. Copyright © 2014 John Wiley & Sons, Ltd.     

Intra-articular injection of collagenase in the knee of rats as an alternative model to study nociception associated with osteoarthritis

Introduction

Animal models currently used in osteoarthritis-associated pain research inadequately reproduce the initiating events and structural pathology of human osteoarthritis. Conversely, intra-articular injection of collagenase is a structurally relevant model, as it induces articular degeneration both by digesting collagen from cartilage and by causing articular instability, thereby reproducing some of the main events associated with osteoarthritis onset and development. Here, we evaluated if the intra-articular injection of collagenase can be an alternative model to study nociception associated with osteoarthritis.

Methods

Osteoarthritis was induced by two intra-articular injections of either 250 U or 500 U of collagenase into the left knee joint of adult male Wistar rats. A six weeks time-course assessment of movement- and loading-induced nociception was performed by the Knee-Bend and CatWalk tests. The effect of morphine, lidocaine and diclofenac on nociceptive behaviour was evaluated in animals injected with 500 U of collagenase. Joint histopathology was scored for both doses throughout time. The expression of transient receptor potential vanilloid 1 (TRPV1) in ipsilateral dorsal root ganglia (DRG) was evaluated.

Results

An increase in nociceptive behaviour associated with movement and loading of affected joints was observed after intra-articular collagenase injection. With the 500 U dose of collagenase, there was a significant correlation between the behavioural and the histopathological osteoarthritis-like structural changes developed after six weeks. One week after injection of 500 U collagenase, swelling of the injected knee and inflammation of the synovial membrane were also observed, indicating the occurrence of an early inflammatory reaction. Behavioural changes induced by the 500 U dose of collagenase were overall effectively reversed by morphine and lidocaine. Diclofenac was effective one week after injection. TRPV1 expression increased six weeks after 500 U collagenase injection.

Conclusion

We conclude that the intra-articular injection of 500 U collagenase in the knee of rats can be an alternative model for the study of nociception associated with osteoarthritis, since it induces significant nociceptive alterations associated with relevant osteoarthritis-like joint structural changes.     

The horizontal flow of the plasmid resistome: clues from inter‐generic similarity networks

By integrating sequence similarity data of plasmid‐encoded antibiotic resistance determinants with those coming from a less transferred molecular marker, we constructed a network in which all the sequences that most likely underwent horizontal gene transfer (HGT) were linked together. The analysis of this network revealed that either geographical barriers or taxonomical distance can often be overcome since phylogenetically unrelated bacteria, and/or those inhabiting distinct environments, were found to share common antibiotic resistance determinants, probably as a result of (one or multiple) HGT event(s). Data obtained also revealed that bacteria viable through multiple environments (ubiquitous) are likely to give a crucial contribution to the spreading of bacterial resistance towards antimicrobial compounds. These analyses represent a first attempt to give an almost global picture of the horizontal flow of antibiotic resistance determinants at the whole bacterial community level, also underlining the power of HGT among bacteria and how this ‘horizontal flow’ is poorly affected by both taxonomy and physical distance. Finally, data presented may be useful in the infections control procedures, suggesting which bacterial species are more likely acting as vectors of antibiotic resistance determinants.     

The Evolution of Histidine Biosynthesis in Archaea: Insights into the <Emphasis Type="Italic">his</Emphasis> Genes Structure and Organization in LUCA

The available sequences of genes encoding the enzymes associated with histidine biosynthesis suggest that this is an ancient metabolic pathway that was assembled prior to the diversification of Bacteria, Archaea, and Eucarya. Paralogous duplication, gene elongation, and fusion events of several different his genes have played a major role in shaping this biosynthetic route. We have analyzed the structure and organization of histidine biosynthetic genes from 55 complete archaeal genomes and combined it with phylogenetic inference in order to investigate the mechanisms responsible for the assembly of the his pathway and the origin of his operons. We show that a wide variety of different organizations of his genes exists in Archaea and that some his genes or entire his (sub-)operons have been likely transferred horizontally between Archaea and Bacteria. However, we show that, in most Archaea, his genes are monofunctional (except for hisD) and scattered throughout the genome, suggesting that his operons might have been assembled multiple times during evolution and that in some cases they are the result of recent evolutionary events. An evolutionary model for the structure and organization of his genes in LUCA is proposed.     

Evolution of lactic acid bacteria in the order Lactobacillales as depicted by analysis of glycolysis and pentose phosphate pathways

Lactic acid bacteria (LAB) represent a functional group of bacteria that are fundamental in human nutrition because of their prominent role in fermented food production and their presence as commensals in the gut. LAB co-evolution and niche-adaptation have been analyzed in several phylogenomic studies due to the availability of complete genome sequences. The aim of this study was to provide novel insights into LAB evolution through the comparative analysis of the metabolic pathways related to carbohydrate metabolism. The analysis was based on 42 LAB genome sequences of representative strains belonging to Enterococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae. A reference phylogenetic tree was inferred from concatenation of 42 ribosomal proteins; then 42 genes belonging to the Embden–Meyerhof–Parnas (or glycolysis; EMPP) and pentose phosphate (PPP) pathways were analyzed in terms of their distribution and organization in the genomes. Phylogenetic analyses confirmed the paraphyly of the Lactobacillaceae family, while the distribution and organization of the EMPP and PPP genes revealed the occurrence of lineage-specific trends of gene loss/gain within the two metabolic pathways examined. In addition, the investigation of the two pathways as structures resulting from different evolutionary processes provided new information concerning the genetic bases of heterofermentative/homofermentative metabolism.     

Integrating docking and molecular dynamics approaches for a series of proline-based 2,5-diketopiperazines as novel αβ-tubulin inhibitors

In this work, docking tools were utilized in order to study the binding properties of more than five hundred of proline-based 2,5-diketopiperazine in the binding site of αβ-tubulin. Results revealed that 20 compounds among them showed lower binding energies in comparison with Tryprostatin-A, a well known tubulin inhibitor and therefore could be potential inhibitors of tubulin. However, the precise evaluation of binding poses represents the similar binding modes for all of these compounds and Tryprostatin-A. Finally, the best docked complex was subjected to a 25 ns molecular dynamics simulation to further validate the proposed binding mode of this compound.     

Primary and Secondary Antifungal Prophylaxis in the Immunocompromised Child: Where do we Stand?

Invasive opportunistic fungal infections are important causes of morbidity and mortality in immunocompromised children undergoing chemotherapy or haematopoietic stem cell transplantation (HSCT). Primary and secondary chemoprophylaxis of invasive fungal infections targets high risk disease-related patients with acute myeloid leukaemia, high risk acute lymphoblastic leukaemias, recurrent leukaemias and those following allogeneic HSCT. The rationale for antifungal prophylaxis in high risk patients comes from two different aspects. On the one hand, is the difficulty of instant diagnosis and, on the other hand, the consequences of morbidity and mortality by invasive infectious diseases. Although we have limited pediatric data concerning antifungal prophylaxis, it has become part of infectious disease supportive care schemes in most of paediatric leukaemia and HSCT centres. This review has insights on the evidence concerning primary and secondary antifungal prophylaxis in immunocompromised children. Although our knowledge comes from large adult studies concerning antifungal agents, there is a great need for evidence of primary or secondary antifungal prophylaxis in large pediatric clinical trials in order to have a consensus in primary and secondary antifungal prophylaxis in immunocompromised children.     

Whole genome sequencing of penicillin-resistant Streptococcus pneumoniae reveals mutations in penicillin-binding proteins and in a putative iron permease

Background

Penicillin resistance in Streptococcus pneumoniae is mediated by a mosaic of genes encoding altered penicillin-binding proteins (PBPs). Nonetheless, S. pneumoniae has also developed non-PBP mechanisms implicated in penicillin resistance. In this study, whole genome sequencing of resistant organisms was used to discover mutations implicated in resistance to penicillin.

Results

We sequenced two S. pneumoniae isolates selected for resistance to penicillin in vitro. The analysis of the genome assemblies revealed that six genes were mutated in both mutants. These included three pbp genes, and three non-pbp genes, including a putative iron permease, spr1178. The nonsense mutation in spr1178 always occurred in the first step of the selection process. Although the mutants had increased resistance to penicillin, the introduction of altered versions of PBPs into a penicillin-susceptible strain by sequential transformation led to strains with a minimal increase in resistance, thus implicating other genes in resistance. The introduction by transformation of the non-PBP recurrent mutations did not increase penicillin resistance, but the introduction of the nonsense mutation in the putative iron permease spr1178 led to a reduced accumulation of reactive oxygen species following exposure to penicillin and to other bactericidal antibiotics as well.

Conclusions

This study indicates that the selection of resistance to penicillin in S. pneumoniae involves the acquisition of mutations conferring tolerance to the antibiotic-induced accumulation of oxidants, which translates into an increased survival that putatively enables the selection of major resistance determinants such as mutations in PBPs.