What are the current and future requirements for magnetic resonance imaging interpretation skills in radiotherapy? A critical review

AbstractPurposeIncreasing usage of magnetic resonance imaging (MRI) in radiotherapy (RT) and the advent of MRI-based image-guided radiotherapy (IGRT) suggests a need for additional training within the RT profession. This critical review aimed to identify potential gaps in knowledge by evaluating the current skill base in MRI among therapeutic radiographers as evidenced by published research.MethodsPapers related to MRI usage were retrieved. Topic areas included outlining, planning and IGRT; diagnosis, follow-up and staging-related papers were excluded. After selection and further text analysis, papers were grouped by tumour site and year of publication.ResultsThe literature search and filtering resulted in a total of 123 papers, of which 66 were related to ‘outlining’, 37 to ‘planning’ and 20 to ‘IGRT’. The main sites of existing MRI expertise in RT were brain, central nervous system, prostate, and head and neck tumours. Expertise was clearly related to regions where MRI offered improved soft-tissue contrast. MRI studies within RT have been published from 2007 onwards at a steadily increasing rate.ConclusionCurrent use of MRI in RT is mainly restricted to sites where MRI offers a considerable imaging advantage over computed tomography. Given the changing use of MRI for image guidance, emerging therapeutic radiographers will require training in MRI interpretation across a wider range of anatomical regions.</jats:sec

Chemical Rescue of Active Site Mutants of S. pneumoniae Surface Endonuclease EndA and Other Nucleases of the HNH Family by Imidazole

The His-Asn-His (HNH) motif characterizes the active sites of a large number of different nucleases such as homing endonucleases, restriction endonucleases, structure-specific nucleases and, in particular, nonspecific nucleases. Several biochemical studies have revealed an essential catalytic function for the first amino acid of this motif in HNH nucleases. This histidine residue was identified as the general base that activates a water molecule for a nucleophilic attack on the sugar phosphate backbone of nucleic acids. Replacement of histidine by an amino acid such as glycine or alanine, which lack the catalytically active imidazole side chain, leads to decreases of several orders of magnitude in the nucleolytic activities of members of this nuclease family. We were able, however, to restore the activity of HNH nuclease variants (i.e., EndA (Streptococcus pneumoniae), SmaNuc (Serratia marcescens) and NucA (Anabaena sp.)) that had been inactivated by His→Gly or His→Ala substitution by adding excess imidazole to the inactive enzymes in vitro. Imidazole clearly replaces the missing histidine side chain and thereby restores nucleolytic activity. Significantly, this chemical rescue could also be observed in vivo (Escherichia coli). The in vivo assay might be a promising starting point for the development of a high-throughput screening system for functional EndA inhibitors because, unlike the wild-type enzyme, the H160G and H160A variants of EndA can easily be produced in E. coli. A simple viability assay would allow inhibitors of EndA to be identified because these would counteract the toxicities of the chemically rescued EndA variants. Such inhibitors could be used to block the nucleolytic activity of EndA, which as a surface-exposed enzyme in its natural host destroys the DNA scaffolds of neutrophil extracellular traps (NETs) and thereby allows S. pneumoniae to escape the innate immune response. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Mutational and biochemical analysis of the DNA-entry nuclease EndA from Streptococcus pneumoniae

EndA is a membrane-attached surface-exposed DNA-entry nuclease previously known to be required for genetic transformation of Streptococcus pneumoniae. More recent studies have shown that the enzyme also plays an important role during the establishment of invasive infections by degrading extracellular chromatin in the form of neutrophil extracellular traps (NETs), enabling streptococci to overcome the innate immune system in mammals. As a virulence factor, EndA has become an interesting target for future drug design. Here we present the first mutational and biochemical analysis of recombinant forms of EndA produced either in a cell-free expression system or in Escherichia coli. We identify His160 and Asn191 to be essential for catalysis and Asn182 to be required for stability of EndA. The role of His160 as the putative general base in the catalytic mechanism is supported by chemical rescue of the H160A variant of EndA with imidazole added in excess. Our study paves the way for the identification and development of protein or low-molecular-weight inhibitors for EndA in future high-throughput screening assays

Chemical Rescue of Active Site Mutants of S. pneumoniae Surface Endonuclease EndA and Other Nucleases of the HNH Family by Imidazole

The His-Asn-His (HNH) motif characterizes the active sites of a large number of different nucleases such as homing endonucleases, restriction endonucleases, structure-specific nucleases and, in particular, nonspecific nucleases. Several biochemical studies have revealed an essential catalytic function for the first amino acid of this motif in HNH nucleases. This histidine residue was identified as the general base that activates a water molecule for a nucleophilic attack on the sugar phosphate backbone of nucleic acids. Replacement of histidine by an amino acid such as glycine or alanine, which lack the catalytically active imidazole side chain, leads to decreases of several orders of magnitude in the nucleolytic activities of members of this nuclease family. We were able, however, to restore the activity of HNH nuclease variants (i.e., EndA (Streptococcus pneumoniae), SmaNuc (Serratia marcescens) and NucA (Anabaena sp.)) that had been inactivated by His→Gly or His→Ala substitution by adding excess imidazole to the inactive enzymes in vitro. Imidazole clearly replaces the missing histidine side chain and thereby restores nucleolytic activity. Significantly, this chemical rescue could also be observed in vivo (Escherichia coli). The in vivo assay might be a promising starting point for the development of a high-throughput screening system for functional EndA inhibitors because, unlike the wild-type enzyme, the H160G and H160A variants of EndA can easily be produced in E. coli. A simple viability assay would allow inhibitors of EndA to be identified because these would counteract the toxicities of the chemically rescued EndA variants. Such inhibitors could be used to block the nucleolytic activity of EndA, which as a surface-exposed enzyme in its natural host destroys the DNA scaffolds of neutrophil extracellular traps (NETs) and thereby allows S. pneumoniae to escape the innate immune response. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Chemical Rescue of Active Site Mutants of S. pneumoniae Surface Endonuclease EndA and Other Nucleases of the HNH Family by Imidazole

The His-Asn-His (HNH) motif characterizes the active sites of a large number of different nucleases such as homing endonucleases, restriction endonucleases, structure-specific nucleases and, in particular, nonspecific nucleases. Several biochemical studies have revealed an essential catalytic function for the first amino acid of this motif in HNH nucleases. This histidine residue was identified as the general base that activates a water molecule for a nucleophilic attack on the sugar phosphate backbone of nucleic acids. Replacement of histidine by an amino acid such as glycine or alanine, which lack the catalytically active imidazole side chain, leads to decreases of several orders of magnitude in the nucleolytic activities of members of this nuclease family. We were able, however, to restore the activity of HNH nuclease variants (i.e., EndA (Streptococcus pneumoniae), SmaNuc (Serratia marcescens) and NucA (Anabaena sp.)) that had been inactivated by His→Gly or His→Ala substitution by adding excess imidazole to the inactive enzymes in vitro. Imidazole clearly replaces the missing histidine side chain and thereby restores nucleolytic activity. Significantly, this chemical rescue could also be observed in vivo (Escherichia coli). The in vivo assay might be a promising starting point for the development of a high-throughput screening system for functional EndA inhibitors because, unlike the wild-type enzyme, the H160G and H160A variants of EndA can easily be produced in E. coli. A simple viability assay would allow inhibitors of EndA to be identified because these would counteract the toxicities of the chemically rescued EndA variants. Such inhibitors could be used to block the nucleolytic activity of EndA, which as a surface-exposed enzyme in its natural host destroys the DNA scaffolds of neutrophil extracellular traps (NETs) and thereby allows S. pneumoniae to escape the innate immune response. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Novel Role of the Antimicrobial Peptide LL-37 in the Protection of Neutrophil Extracellular Traps against Degradation by Bacterial Nucleases

Neutrophil extracellular traps (NETs) have been described as a fundamental innate immune defence mechanism. They consist of a nuclear DNA backbone associated with different antimicrobial peptides (AMPs) which are able to engulf and kill pathogens. The AMP LL-37, a member of the cathelicidin family, is highly present in NETs. However, the function of LL-37 within NETs is still unknown because it loses its antimicrobial activity when bound to DNA in the NETs. Using immunofluorescence microscopy, we demonstrate that NETs treated with LL-37 are distinctly more resistant to S. aureus nuclease degradation than nontreated NETs. Biochemical assays utilising a random LL-37-fragment library indicated that the blocking effect of LL-37 on nuclease activity is based on the cationic character of the AMP, which facilitates the binding to neutrophil DNA, thus protecting it from degradation by the nuclease. In good correlation to these data, the cationic AMPs human beta defensin-3 and human neutrophil peptide-1 showed similar protection of neutrophil-derived DNA against nuclease degradation. In conclusion, this study demonstrates a novel role of AMPs in host immune defence: beside its direct antimicrobial activity against various pathogens, cationic AMPs can stabilise neutrophil-derived DNA or NETs against bacterial nuclease degradation. (C) 2014 S. Karger AG, Base