Modelling and Visualization of the Geometry of Fibre Materials

A geometry model for three dimensional fibre networks is proposed. The geometry model is intended to be a part of a system, where data from a computer tomography of a fibre material can be fitted to the geometry model and from there, be evaluated according to key parameters such as fibre length, fibre orientation angles and radii of curvature. The model is also intended to give the geometry foundation for a FE-model. The model uses several linked circle arcs to describe a fibre, which accommodates for modelling varying degrees of out of plane curl and kinks. A program has been developed that, from distributions of the aforementioned key parameters, generates a periodic network according to the model which can be visualised by the output of two different file formats, vrml and a specialised format for the fibre network viewing program, FibreScope. Comparisons have been made between microscope photographs of several different fibre materials, including paper and cellulose fibre fluff, and visualisations of generated networks showing versatility in the model to describe a wide variety of fibre material types

Site-directed mutagenesis of Saccharomyces cerevisiae β-tubulin: interaction between residue 167 and benzimidazole compounds

AbstractBenzimidazoles are widely used as anthelmintic agents and systemic fungicides. In susceptible organisms, benzimidazoles bind to β-tubulin and block microtubule polymerization. To further characterize this interaction, site-directed mutagenesis followed by gene replacement was used to change Saccharomyces cerevisiae β-tubulin residue Phe-167 to Tyr. Consistent with previous studies, this mutation resulted in at least 3–4-fold decreased sensitivity to the benzimidazole derivatives carbendazim and nocodazole. The Tyr-167 mutant was cold sensitive, implying a direct effect on benzimidazole binding rather than a nonspecific increase in microtubule stability. Surprisingly, the mutant had 8-fold increased sensitivity to the derivative benomyl, which is structurally identical to carbendazim except at position 1. This suggests that residue 167 interacts with benzimidazoles in the vicinity of the 1-position

Evaluation of Polymorphic Locus Sequence Typing for Candida glabrata Epidemiology.

The opportunistic yeastCandida glabratais increasingly refractory to antifungal treatment or prophylaxis and relatedly is increasingly implicated in health care-associated infections. To elucidate the epidemiology of these infections, strain typing is required. Sequence-based typing provides multiple advantages over length-based methods, such as pulsed-field gel electrophoresis (PFGE); however, conventional multilocus sequence typing (targeting 6 conserved loci) and whole-genome sequencing are impractical for routine use. A commercial sequence-based typing service forC. glabratathat targets polymorphic tandem repeat-containing loci has recently been developed. These CgMT-J and CgMT-M services were evaluated with 56 epidemiologically unrelated isolates, 4 to 7 fluconazole-susceptible or fluconazole-resistant isolates from each of 5 center A patients, 5 matched pairs of fluconazole-susceptible/resistant isolates from center B patients, and 7 isolates from a center C patient who responded to then failed caspofungin therapy. CgMT-J and CgMT-M generated congruent results, resolving isolates into 24 and 20 alleles, respectively. Isolates from all but one of the center A patients shared the same otherwise rare alleles, suggesting nosocomial transmission. Unexpectedly, Pdr1 sequencing showed that resistance arose independently in each patient. Similarly, most isolates from center B also clustered together; however, this may reflect a dominant clone since their alleles were shared by multiple unrelated isolates. Although distinguishable by their echinocandin susceptibilities, all isolates from the center C patient shared alleles, in agreement with the previously reported relatedness of these isolates based on PFGE. Finally, we show how phylogenetic clusters can be used to provide surrogate parents to analyze the mutational basis for antifungal resistance

Determination of MLN0128, An Investigational Antineoplastic Agent, in Human Plasma by LC–MS/MS

MLN0128, an mTOR kinase inhibitor, is currently undergoing clinical investigation for treatment of a variety of cancers. To support this work, an LC–MS/MS method has been developed for the determination of MLN0128 in human plasma. A structural analog STK040263 was used as the internal standard. Both MLN0128 and the IS were first extracted from plasma using methyl tert-butyl ether; then separated on a Waters XTerra® MS C18 column using a mobile phase consisting of methanol–acetonitrile–10.0 mm ammonium formate (34:6:60, v/v/v) at a flow rate of 0.300 mL min−1. Quantitation of MLN0128 was done by positive electrospray ionization tandem mass spectrometry in multiple-reaction-monitoring mode. This method has a total run time of \u3c4 min with the retention times of 1.95 and 2.94 min for the IS and MLN0128, respectively. The method has been validated per the US Food and Drug Administration guidance for bioanalytical method validation. It has a calibration range of 0.100–50.0 ng mL−1 in human plasma with a correlation coefficient \u3e 0.999. The overall assay accuracy and precision were ≤ ± 4 and ≤8%, respectively. The IS normalized recovery of MLN0128 was 98–100%. The stability studies showed that MLN0128 was stable under all tested conditions. The method developed may be useful for clinical studies of MLN0128

Genetic Reconstruction of Protozoan rRNA Decoding Sites Provides a Rationale for Paromomycin Activity against Leishmania and Trypanosoma

Aminoglycoside antibiotics target the ribosomal decoding A-site and are active against a broad spectrum of bacteria. These compounds bind to a highly conserved stem-loop-stem structure in helix 44 of bacterial 16S rRNA. One particular aminoglycoside, paromomycin, also shows potent antiprotozoal activity and is used for the treatment of parasitic infections, e.g. by Leishmania spp. The precise drug target is, however, unclear; in particular whether aminoglycoside antibiotics target the cytosolic and/or the mitochondrial protozoan ribosome. To establish an experimental model for the study of protozoan decoding-site function, we constructed bacterial chimeric ribosomes where the central part of bacterial 16S rRNA helix 44 has been replaced by the corresponding Leishmania and Trypanosoma rRNA sequences. Relating the results from in-vitro ribosomal assays to that of in-vivo aminoglycoside activity against Trypanosoma brucei, as assessed in cell cultures and in a mouse model of infection, we conclude that aminoglycosides affect cytosolic translation while the mitochondrial ribosome of trypanosomes is not a target for aminoglycoside antibiotics

A broad distribution of the alternative oxidase in microsporidian parasites

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of ironsulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosome

Reciprocal regulation of GAS5 lncRNA levels and mTOR inhibitor action in prostate cancer cells.

This is the peer reviewed version of the following article: Yacqub-Usman, K., Pickard, M. R., & Williams, G. T. (2015). Reciprocal regulation of GAS5 lncRNA levels and mTOR inhibitor action in prostate cancer cells. Prostate, 75(7), 693-705. DOI: 10.1002/pros.22952, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1002/pros.22952/abstract;jsessionid=B9892067CCD9082275B1EFFFC59926F6.f02t02. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-ArchivingBACKGROUND: New therapies are required for castrate-resistant prostate cancer (CRPC), and growth-arrest specific 5 (GAS5) lncRNA, which riborepresses androgen receptor action, may offer novel opportunities in this regard. This lncRNA promotes the apoptosis of prostate cancer cells and its levels decline as prostate cancer cells acquire castrate-resistance, so that enhancing GAS5 expression may improve the effectiveness of chemotherapies. Since GAS5 is a member of the 5' terminal oligopyrimidine gene family, we have examined mTOR inhibition as a strategy to increase GAS5 expression. Furthermore, we have determined if GAS5 itself mediates the action of mTOR inhibitors, as demonstrated for other chemotherapeutic agents in prostate cancer cells. METHODS: The effects of mTOR inhibitors on GAS5 lncRNA levels and cell growth were determined in a range of prostate cancer cell lines. Transfection of cells with GAS5 siRNAs and plasmid constructs was performed to determine the involvement of GAS5 lncRNA in mTOR inhibitor action. RESULTS: First generation mTORC1, combined mTORC1/mTORC2 and dual PI3K/mTOR inhibitors all increased cellular GAS5 levels and inhibited culture growth in androgen-dependent (LNCaP) and androgen-sensitive (22Rv1) cell lines, but not in androgen-independent (PC-3 and DU 145) cell lines. The latter exhibited low endogenous GAS5 expression, and GAS5 silencing in LNCaP and 22Rv1 cells decreased the sensitivity to mTOR inhibitors, whereas transfection of GAS5 lncRNA sensitized PC-3 and DU 145 cells to these agents. CONCLUSION: mTOR inhibition enhances GAS5 transcript levels in certain prostate cancer cell lines. This selectivity is likely to be related to endogenous GAS5 expression levels, since GAS5 lncRNA is itself required for mTOR inhibitor action in prostate cancer cells.Prostate Cancer Collaborative, United Kingdom (PCUK

Sur7 Promotes Plasma Membrane Organization and Is Needed for Resistance to Stressful Conditions and to the Invasive Growth and Virulence of Candida albicans

The human fungal pathogen Candida albicans causes lethal systemic infections because of its ability to grow and disseminate in a host. The C. albicans plasma membrane is essential for virulence by acting as a protective barrier and through its key roles in interfacing with the environment, secretion of virulence factors, morphogenesis, and cell wall synthesis. Difficulties in studying hydrophobic membranes have limited the understanding of how plasma membrane organization contributes to its function and to the actions of antifungal drugs. Therefore, the role of the recently discovered plasma membrane subdomains termed the membrane compartment containing Can1 (MCC) was analyzed by assessing the virulence of a sur7Δ mutant. Sur7 is an integral membrane protein component of the MCC that is needed for proper localization of actin, morphogenesis, cell wall synthesis, and responding to cell wall stress. MCC domains are stable 300-nm-sized punctate patches that associate with a complex of cytoplasmic proteins known as an eisosome. Analysis of virulence-related properties of a sur7Δ mutant revealed defects in intraphagosomal growth in macrophages that correlate with increased sensitivity to oxidation and copper. The sur7Δ mutant was also strongly defective in pathogenesis in a mouse model of systemic candidiasis. The mutant cells showed a decreased ability to initiate an infection and greatly diminished invasive growth into kidney tissues. These studies on Sur7 demonstrate that the plasma membrane MCC domains are critical for virulence and represent an important new target for the development of novel therapeutic strategies

Repurposing drugs to fast-track therapeutic agents for the treatment of cryptococcosis

Many infectious diseases disproportionately affect people in the developing world. Cryptococcal meningitis is one of the most common mycoses in HIV-AIDS patients, with the highest burden of disease in sub-Saharan Africa. Current best treatment regimens still result in unacceptably high mortality rates, and more effective antifungal agents are needed urgently. Drug development is hampered by the difficulty of developing effective antifungal agents that are not also toxic to human cells, and by a reluctance among pharmaceutical companies to invest in drugs that cannot guarantee a high financial return. Drug repurposing, where existing drugs are screened for alternative activities, is becoming an attractive approach in antimicrobial discovery programs, and various compound libraries are now commercially available. As these drugs have already undergone extensive optimisation and passed regulatory hurdles this can fast-track their progress to market for new uses. This study screened the Screen-Well Enzo library of 640 compounds for candidates that phenotypically inhibited the growth of Cryptococcus deuterogattii. The anthelminthic agent flubendazole, and L-type calcium channel blockers nifedipine, nisoldipine and felodipine, appeared particularly promising and were tested in additional strains and species. Flubendazole was very active against all pathogenic Cryptococcus species, with minimum inhibitory concentrations of 0.039-0.156 μg/mL, and was equally effective against isolates that were resistant to fluconazole. While nifedipine, nisoldipine and felodipine all inhibited Cryptococcus, nisoldipine was also effective against Candida, Saccharomyces and Aspergillus. This study validates repurposing as a rapid approach for finding new agents to treat neglected infectious diseases

Engineering the rRNA decoding site of eukaryotic cytosolic ribosomes in bacteria

Structural and genetic studies on prokaryotic ribosomes have provided important insights into fundamental aspects of protein synthesis and translational control and its interaction with ribosomal drugs. Comparable mechanistic studies in eukaryotes are mainly hampered by the absence of both high-resolution crystal structures and efficient genetic models. To study the interaction of aminoglycoside antibiotics with selected eukaryotic ribosomes, we replaced the bacterial drug binding site in 16S rRNA with its eukaryotic counterpart, resulting in bacterial hybrid ribosomes with a fully functional eukaryotic rRNA decoding site. Cell-free translation assays demonstrated that hybrid ribosomes carrying the rRNA decoding site of higher eukaryotes show pronounced resistance to aminoglycoside antibiotics, equivalent to that of rabbit reticulocyte ribosomes, while the decoding sites of parasitic protozoa show distinctive drug susceptibility. Our findings suggest that phylogenetically variable components of the ribosome, other than the rRNA-binding site, do not affect aminoglycoside susceptibility of the protein-synthesis machinery. The activities of the hybrid ribosomes indicate that helix 44 of the rRNA decoding site behaves as an autonomous domain, which can be exchanged between ribosomes of different phylogenetic domains for study of function