Novel sol–gel preparation of (PO)–(CaO)–(NaO)–(TiO) bioresorbable glasses (X = 0.05, 0.1, and 0.15)

Quaternary phosphate-based glasses in the PO–CaO–NaO–TiO system with a fixed PO and CaO content of 40 and 25 mol% respectively have been successfully synthesised via sol–gel method and bulk, transparent samples were obtained. The structure, elemental proportion, and thermal properties of stabilised sol–gel glasses have been characterised using X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDX), P nuclear magnetic resonance (P NMR), titanium K-edge X-ray absorption near-edge structure (XANES), fourier transform infrared (FTIR) spectroscopy, and differential thermal analysis (DTA). The XRD results confirmed the amorphous nature for all stabilized sol–gel derived glasses. The EDX result shows the relatively low loss of phosphorus during the sol–gel process and Ti K-edge XANES confirmed titanium in the glass structure is in mainly six-fold coordination environment. The P NMR and FTIR results revealed that the glass structure consist of mainly Q and Q phosphate units and the Ti cation was acting as a cross-linking between phosphate units. In addition DTA results confirmed a decrease in the glass transition and crystallisation temperature with increasing NaO content. Ion release studies also demonstrated a decrease in degradation rates with increasing TiO content therefore supporting the use of these glasses for biomedical applications that require a degree of control over glass degradation. These sol–gel glasses also offer the potential to incorporate proactive molecules for drug delivery application due to the low synthesis temperature employed

Structural, mechanistic and functional insight into gliotoxin bis-thiomethylation in Aspergillus fumigatus.

Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus Self-resistance against gliotoxin is effected by the gliotoxin oxidase GliT, and attenuation of gliotoxin biosynthesis is catalysed by gliotoxin S-methyltransferase GtmA. Here we describe the X-ray crystal structures of GtmA-apo (1.66 Å), GtmA complexed to S-adenosylhomocysteine (1.33 Å) and GtmA complexed to S-adenosylmethionine (2.28 Å), providing mechanistic insights into this important biotransformation. We further reveal that simultaneous elimination of the ability of A. fumigatus to dissipate highly reactive dithiol gliotoxin, via deletion of GliT and GtmA, results in the most significant hypersensitivity to exogenous gliotoxin observed to date. Indeed, quantitative proteomic analysis of ΔgliT::ΔgtmA reveals an uncontrolled over-activation of the gli-cluster upon gliotoxin exposure. The data presented herein reveal, for the first time, the extreme risk associated with intracellular dithiol gliotoxin biosynthesis-in the absence of an efficient dismutation capacity. Significantly, a previously concealed protective role for GtmA and functionality of ETP bis-thiomethylation as an ancestral protection strategy against dithiol compounds is now evident

RNA-seq reveals the pan-transcriptomic impact of attenuating the gliotoxin self-protection mechanism in Aspergillus fumigatus.

BACKGROUND: Aspergillus fumigatus produces a number of secondary metabolites, one of which, gliotoxin, has been shown to exhibit anti-fungal activity. Thus, A. fumigatus must be able to protect itself against gliotoxin. Indeed one of the genes in the gliotoxin biosynthetic gene cluster in A. fumigatus, gliT, is required for self-protection against the toxin- however the global self-protection mechanism deployed is unclear. RNA-seq was employed to identify genes differentially regulated upon exposure to gliotoxin in A. fumigatus wild-type and A. fumigatus ∆gliT, a strain that is hypersensitive to gliotoxin. RESULTS: Deletion of A. fumigatus gliT resulted in altered expression of 208 genes (log2 fold change of 1.5) when compared to A. fumigatus wild-type, of which 175 genes were up-regulated and 33 genes were down-regulated. Expression of 164 genes was differentially regulated (log2 fold change of 1.5) in A. fumigatus wild-type when exposed to gliotoxin, consisting of 101 genes with up-regulated expression and 63 genes with down-regulated expression. Interestingly, a much larger number of genes, 1700, were found to be differentially regulated (log2 fold change of 1.5) in A. fumigatus ∆gliT when challenged with gliotoxin. These consisted of 508 genes with up-regulated expression, and 1192 genes with down-regulated expression. Functional Catalogue (FunCat) classification of differentially regulated genes revealed an enrichment of genes involved in both primary metabolic functions and secondary metabolism. Specifically, genes involved in gliotoxin biosynthesis, helvolic acid biosynthesis, siderophore-iron transport genes and also nitrogen metabolism genes and ribosome biogenesis genes underwent altered expression. It was confirmed that gliotoxin biosynthesis is induced upon exposure to exogenous gliotoxin, production of unrelated secondary metabolites is attenuated in A. fumigatus ∆gliT, while quantitative proteomic analysis confirmed disrupted translation in A. fumigatus ∆gliT challenged with exogenous gliotoxin. CONCLUSIONS: This study presents the first global investigation of the transcriptional response to exogenous gliotoxin in A. fumigatus wild-type and the hyper-sensitive strain, ∆gliT. Our data highlight the global and extensive affects of exogenous gliotoxin on a sensitive strain devoid of a self-protection mechanism and infer that GliT functionality is required for the optimal biosynthesis of selected secondary metabolites in A. fumigatus

Systems impact of zinc chelation by the epipolythiodioxopiperazine dithiol gliotoxin in Aspergillus fumigatus: a new direction in natural product functionality.

The non-ribosomal peptide gliotoxin, which autoinduces its own biosynthesis, has potent anti-fungal activity, especially in the combined absence of the gliotoxin oxidoreductase GliT and bis-thiomethyltransferase GtmA. Dithiol gliotoxin (DTG) is a substrate for both of these enzymes. Herein we demonstrate that DTG chelates Zn2+ (m/z 424.94), rapidly chelates Zn2+ from Zn(4-(2-pyridylazo)-resorcinol) (Zn(PAR)2) and also inhibits a Zn2+-dependent alkaline phosphatase (AP). Zn2+ addition rescues AP function following DTG-associated inhibition, and pre-incubation of DTG with Zn2+ completely protects AP activity. Zn2+ (1-50 μM) also significantly relieves the potent gliotoxin-mediated inhibition of Aspergillus fumigatus ΔgliT::ΔgtmA (p < 0.05), which infers in vivo dithiol gliotoxin-mediated sequestration of free Zn2+ or chelation from intracellular metalloenzymes as inhibitory mechanisms. Quantitative proteomic analysis revealed that excess Zn2+ alters the effect of gliotoxin on A. fumigatus ΔgliT, with differential abundance of secondary metabolism-associated proteins in the combinatorial condition. GtmA abundance increased 18.8 fold upon co-addition of gliotoxin and Zn2+ compared to gliotoxin alone, possibly to compensate for disruption to GtmA activity, as seen in in vitro assays. Furthermore, DTG effected significant in vitro aggregation of a number of protein classes, including Zn2+-dependent enzymes, while proteins were protected from aggregation by pre-incubating DTG with Zn2+. We conclude that DTG can act in vivo as a Zn2+ chelator, which can significantly impede A. fumigatus growth in the absence of GliT and GtmA

Gliotoxin and related metabolites as zinc chelators: implications and exploitation to overcome antimicrobial resistance

Antimicrobial resistance (AMR) is a major global problem and threat to humanity. The search for new antibiotics is directed towards targeting of novel microbial systems and enzymes, as well as augmenting the activity of pre-existing antimicrobials. Sulphur-containing metabolites (e.g., auranofin and bacterial dithiolopyrrolones [e.g., holomycin]) and Zn2+-chelating ionophores (PBT2) have emerged as important antimicrobial classes. The sulphur-containing, non-ribosomal peptide gliotoxin, biosynthesised by Aspergillus fumigatus and other fungi exhibits potent antimicrobial activity, especially in the dithiol form (dithiol gliotoxin; DTG). Specifically, it has been revealed that deletion of the enzymes gliotoxin oxidoreductase GliT, bis-thiomethyltransferase GtmA or the transporter GliA dramatically sensitise A. fumigatus to gliotoxin presence. Indeed, the double deletion strain A. fumigatus ΔgliTΔgtmA is especially sensitive to gliotoxin-mediated growth inhibition, which can be reversed by Zn2+ presence. Moreover, DTG is a Zn2+ chelator which can eject zinc from enzymes and inhibit activity. Although multiple studies have demonstrated the potent antibacterial effect of gliotoxin, no mechanistic details are available. Interestingly, reduced holomycin can inhibit metallo-β-lactamases. Since holomycin and gliotoxin can chelate Zn2+, resulting in metalloenzyme inhibition, we propose that this metal-chelating characteristic of these metabolites requires immediate investigation to identify new antibacterial drug targets or to augment the activity of existing antimicrobials. Given that (i) gliotoxin has been shown in vitro to significantly enhance vancomycin activity against Staphylococcus aureus, and (ii) that it has been independently proposed as an ideal probe to dissect the central 'Integrator' role of Zn2+ in bacteria - we contend such studies are immediately undertaken to help address AMR

Five ways to enhance the impact of climate science

Policy-making is rarely driven by evidence alone. Thus, climate scientists who adopt an ‘evidence-based’ mindset, expecting more science to lead automatically to better policy, are likely to be disappointed. Instead, embracing an ‘evidence-informed’ attitude to policy-making will be more productive, recognising that evidence must be deployed in such a way as to interact persuasively with other factors. Using the 5th Assessment Report of the IPCC as inspiration, this commentary argues that climate scientists would do well to consider five ideas and ultimately embrace an evidence-informed approach to presenting evidence.This work is taken from a larger PhD project currently being undertaken in the Department of Geography at the University of Cambridge. This work is very kindly funded by the Economic and Social Research Council (grant number ES/I901957/1) and by the Homerton College Charter Scholarship scheme. I would like to thank S. E. Owens, A. Donovan and W. M. Adams for comments, and D. Watson for help with the figures.This is the accepted manuscript. The final version is available in Nature Climate Change 4, 522–524 (2014) doi:10.1038/nclimate2270 . http://www.nature.com/nclimate/journal/v4/n7/full/nclimate2270.htm

Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences

<p>Abstract</p> <p>Background</p> <p>Adeno-associated virus type 2 (AAV2) preferentially integrates its DNA at a ~2 kb region of human chromosome 19, designated <it>AAVS1 </it>(also known as <it>MBS85</it>). Integration at <it>AAVS1 </it>requires the AAV2 replication (Rep) proteins and a DNA sequence within <it>AAVS1 </it>containing a 16 bp Rep recognition sequence (RRS) and closely spaced Rep nicking site (also referred to as a terminal resolution site, or <it>trs</it>). The AAV2 genome is flanked by inverted terminal repeats (ITRs). Each ITR contains an RRS and closely spaced <it>trs</it>, but the sequences differ from those in <it>AAVS1</it>. These ITR sequences are required for replication and packaging.</p> <p>Results</p> <p>In this study we demonstrate that the <it>AAVS1 </it>RRS and <it>trs </it>can function in AAV2 replication, packaging and integration by replacing a 61 bp region of the AAV2 ITR with a 49 bp segment of <it>AAVS1 </it>DNA. Modifying one or both ITRs did not have a large effect on the overall virus titers. These modifications did not detectably affect integration at <it>AAVS1</it>, as measured by semi-quantitative nested PCR assays. Sequencing of integration junctions shows the joining of the modified ITRs to <it>AAVS1 </it>sequences.</p> <p>Conclusions</p> <p>The ability of these <it>AAVS1 </it>sequences to substitute for the AAV2 RRS and <it>trs </it>provides indirect evidence that the stable secondary structure encompassing the <it>trs </it>is part of the AAV2 packaging signal.</p

Quantum interferometry with three-dimensional geometry

Quantum interferometry uses quantum resources to improve phase estimation with respect to classical methods. Here we propose and theoretically investigate a new quantum interferometric scheme based on three-dimensional waveguide devices. These can be implemented by femtosecond laser waveguide writing, recently adopted for quantum applications. In particular, multiarm interferometers include "tritter" and "quarter" as basic elements, corresponding to the generalization of a beam splitter to a 3- and 4-port splitter, respectively. By injecting Fock states in the input ports of such interferometers, fringe patterns characterized by nonclassical visibilities are expected. This enables outperforming the quantum Fisher information obtained with classical fields in phase estimation. We also discuss the possibility of achieving the simultaneous estimation of more than one optical phase. This approach is expected to open new perspectives to quantum enhanced sensing and metrology performed in integrated photonic.Comment: 7 pages (+4 Supplementary Information), 5 figure