Nitroheterocyclic drug resistance mechanisms in <i>Trypanosoma brucei</i>

OBJECTIVES: The objective of this study was to identify the mechanisms of resistance to nifurtimox and fexinidazole in African trypanosomes. METHODS: Bloodstream-form Trypanosoma brucei were selected for resistance to nifurtimox and fexinidazole by stepwise exposure to increasing drug concentrations. Clones were subjected to WGS to identify putative resistance genes. Transgenic parasites modulating expression of genes of interest were generated and drug susceptibility phenotypes determined. RESULTS: Nifurtimox-resistant (NfxR) and fexinidazole-resistant (FxR) parasites shared reciprocal cross-resistance suggestive of a common mechanism of action. Previously, a type I nitroreductase (NTR) has been implicated in nitro drug activation. WGS of resistant clones revealed that NfxR parasites had lost >100 kb from one copy of chromosome 7, rendering them hemizygous for NTR as well as over 30 other genes. FxR parasites retained both copies of NTR, but lost >70 kb downstream of one NTR allele, decreasing NTR transcription by half. A single knockout line of NTR displayed 1.6- and 1.9-fold resistance to nifurtimox and fexinidazole, respectively. Since NfxR and FxR parasites are ∼6- and 20-fold resistant to nifurtimox and fexinidazole, respectively, additional factors must be involved. Overexpression and knockout studies ruled out a role for a putative oxidoreductase (Tb927.7.7410) and a hypothetical gene (Tb927.1.1050), previously identified in a genome-scale RNAi screen. CONCLUSIONS: NTR was confirmed as a key resistance determinant, either by loss of one gene copy or loss of gene expression. Further work is required to identify which of the many dozens of SNPs identified in the drug-resistant cell lines contribute to the overall resistance phenotype

Catalyst Efficacy of Homogeneous and Heterogeneous Palladium Catalysts in the Direct Arylation of Common Heterocycles

The direct arylation of several common heterocycles, using homogeneous and heterogeneous palladium (pre)catalysts, has been examined by initial rate analysis. The study reveals that apparently distinct palladium catalysts can display similar activities in such transformations, implying formation of a comparable active palladium catalyst phase. A substrate dependence was noted for the palladium catalysts examined

Isolation and characterization of kinetoplast DNA from bloodstream form of Trypanosoma brucei

We have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African Trypanosomiasis Research Organization [EATRO] 427). Endonuclease PstI and S1 nuclease cut the maxi-circle at a single site, allowing its isolation in a linear form with a mol wt of 12.2 x 10(6), determined by electron microscopy. The other enzymes give multiple maxi-circle fragments, whose added mol wt is 12-13 x 10(6), determined by gel electrophoresis. The maxi-circle in another T. brucei isolate (EATRO 1125) yields similar fragments but appears to contain a deletion of about 0.7 x 10(6) daltons. Electron microscopy of kDNA shows the presence of DNA considerably longer than the mini-circle contour length (0.3 micron) either in the network or as loops extending from the edge. This long DNA never exceeds the maxi-circle length (6.3 microns) and is completely removed by digestion with endonuclease PstI. 5-10% of the networks are doublets with up to 40 loops of DNA clustered between the two halves of the mini-circle network and probably represent a division stage of the kDNA. Digestion with PstI selectively removes these loops without markedly altering the mini-circle network. We conclude that the long DNA in both single and double networks represents maxi-circles and that long tandemly repeated oligomers of mini-circles are (virtually) absent. kDNA from Trypanosoma equiperdum, a trypanosome species incapable of synthesizing a fully functional mitochondrion, contains single and double networks of dimensions similar to those from T. brucei but without any DNA longer than mini-circle contour length. We conclude that the maxi-circle of trypanosomes is the genetic equivalent of the mitochondrial DNA (mtDNA) of other organisms

Theoretical rationalisation for the mechanism of N-heterocyclic carbene-halide reductive elimination at CuIII, AgIII and AuIII

Reductive elimination of imidazolium salts from CuIII is extremely sensitive to the anionic ligand (X or Y) type on Cu (e.g. ΔG‡ ranges from 4.7 kcal mol-1 to 31.8 kcal mol-1, from chloride to benzyl). Weakly σ-donating ligands dramatically accelerate reductive elimination. Comparison with Ag/Au shows that the HOMO energy, strength of M-NHC and M-Y bonds and inherent stability of MIII with respect to MI are critical to governing reaction feasibility

Chemotherapy versus supportive care in advanced non-small cell lung cancer: improved survival without detriment to quality of life

BACKGROUND: In 1995 a meta-analysis of randomised trials investigating the value of adding chemotherapy to primary treatment for non-small cell lung cancer (NSCLC) suggested a small survival benefit for cisplatin-based chemotherapy in each of the primary treatment settings. However, the metaanalysis included many small trials and trials with differing eligibility criteria and chemotherapy regimens. METHODS: The aim of the Big Lung Trial was to confirm the survival benefits seen in the meta-analysis and to assess quality of life and cost in the supportive care setting. A total of 725 patients were randomised to receive supportive care alone (n = 361) or supportive care plus cisplatin-based chemotherapy (n = 364). RESULTS: 65% of patients allocated chemotherapy (C) received all three cycles of treatment and a further 27% received one or two cycles. 74% of patients allocated no chemotherapy (NoC) received thoracic radiotherapy compared with 47% of the C group. Patients allocated C had a significantly better survival than those allocated NoC: HR 0.77 (95% CI 0.66 to 0.89, p = 0.0006), median survival 8.0 months for the C group v 5.7 months for the NoC group, a difference of 9 weeks. There were 19 (5%) treatment related deaths in the C group. There was no evidence that any subgroup benefited more or less fromchemotherapy. No significant differences were observed between the two groups in terms of the pre-defined primary and secondary quality of life end points, although large negative effects of chemotherapy were ruled out. The regimens used proved to be cost effective, the extra cost of chemotherapy being offset by longer survival. CONCLUSIONS: The survival benefit seen in this trial was entirely consistent with the NSCLC meta-analysis and subsequent similarly designed large trials. The information on quality of life and cost should enablepatients and their clinicians to make more informed treatment choices

Investigating the inner discs of Herbig Ae/Be stars with CO bandhead and Brγ emission

articleHerbig Ae/Be stars lie in the mass range between low- and high-mass young stars, and therefore offer a unique opportunity to observe any changes in the formation processes that may occur across this boundary. This paper presents medium-resolution Very Large Telescope (VLT)/X-shooter spectra of six Herbig Ae/Be stars, drawn from a sample of 91 targets, and high-resolution VLT/Cryogenic Infrared Echelle Spectrograph (CRIRES) spectra of five Herbig Ae/Be stars, chosen based on the presence of CO first overtone bandhead emission in their spectra. The X-shooter survey reveals a low detection rate of CO first overtone emission (7 per cent), consisting of objects mainly of spectral type B. A positive correlation is found between the strength of the CO v = 2–0 and Brγ emission lines, despite their intrinsic linewidths suggesting a separate kinematic origin. The high-resolution CRIRES spectra are modelled, and are well fitted under the assumption that the emission originates from small scale Keplerian discs, interior to the dust sublimation radius, but outside the corotation radius of the central stars. In addition, our findings are in very good agreement for the one object where spatially resolved near-infrared interferometric studies have also been performed. These results suggest that the Herbig Ae/Be stars in question are in the process of gaining mass via disc accretion, and that modelling of high spectral resolution spectra is able to provide a reliable probe into the process of stellar accretion in young stars of intermediate to high masses.European Union FP7-2011Science and Technology Facilities Counci

Activation of bicyclic nitro-drugs by a novel nitroreductase (NTR2) in <i>Leishmania</i>

Drug discovery pipelines for the "neglected diseases" are now heavily populated with nitroheterocyclic compounds. Recently, the bicyclic nitro-compounds (R)-PA-824, DNDI-VL-2098 and delamanid have been identified as potential candidates for the treatment of visceral leishmaniasis. Using a combination of quantitative proteomics and whole genome sequencing of susceptible and drug-resistant parasites we identified a putative NAD(P)H oxidase as the activating nitroreductase (NTR2). Whole genome sequencing revealed that deletion of a single cytosine in the gene for NTR2 that is likely to result in the expression of a non-functional truncated protein. Susceptibility of leishmania was restored by reintroduction of the wild-type gene into the resistant line, which was accompanied by the ability to metabolise these compounds. Overexpression of NTR2 in wild-type parasites rendered cells hyper-sensitive to bicyclic nitro-compounds, but only marginally to the monocyclic nitro-drugs, nifurtimox and fexinidazole sulfone, known to be activated by a mitochondrial oxygen-insensitive nitroreductase (NTR1). Conversely, a double knockout NTR2 null cell line was completely resistant to bicyclic nitro-compounds and only marginally resistant to nifurtimox. Sensitivity was fully restored on expression of NTR2 in the null background. Thus, NTR2 is necessary and sufficient for activation of these bicyclic nitro-drugs. Recombinant NTR2 was capable of reducing bicyclic nitro-compounds in the same rank order as drug sensitivity in vitro. These findings may aid the future development of better, novel anti-leishmanial drugs. Moreover, the discovery of anti-leishmanial nitro-drugs with independent modes of activation and independent mechanisms of resistance alleviates many of the concerns over the continued development of these compound series

On the origin of the correlations between the accretion luminosity and emission line luminosities in pre-main-sequence stars

Correlations between the accretion luminosity and emission line luminosities (Lacc and Lline) of pre-main-sequence (PMS) stars have been published for many different spectral lines, which are used to estimate accretion rates. Despite the origin of those correlations is unknown, this could be attributed to direct or indirect physical relations between the emission line formation and the accretion mechanism. This work shows that all (near-UV/optical/near-IR) Lacc-Lline correlations are the result of the fact that the accretion luminosity and the stellar luminosity (L*) are correlated, and are not necessarily related with the physical origin of the line. Synthetic and observational data are used to illustrate how the Lacc-Lline correlations depend on the Lacc-L* relationship. We conclude that because PMS stars show the Lacc-L* correlation immediately implies that Lacc also correlates with the luminosity of all emission lines, for which the Lacc-Lline correlations alone do not prove any physical connection with accretion but can only be used with practical purposes to roughly estimate accretion rates. When looking for correlations with possible physical meaning, we suggest that Lacc/L* and Lline/L* should be used instead of Lacc and Lline. Finally, the finding that Lacc has a steeper dependence on L* for T Tauri stars than for intermediate-mass Herbig Ae/Be stars is also discussed. That is explained from the magnetospheric accretion scenario and the different photospheric properties in the near-U