Factors associated with the isolation of Nontuberculous mycobacteria (NTM) from a large municipal water system in Brisbane, Australia

Background Nontuberculous mycobacteria (NTM) are normal inhabitants of a variety of environmental reservoirs including natural and municipal water. The aim of this study was to document the variety of species of NTM in potable water in Brisbane, QLD, with a specific interest in the main pathogens responsible for disease in this region and to explore factors associated with the isolation of NTM. One-litre water samples were collected from 189 routine collection sites in summer and 195 sites in winter. Samples were split, with half decontaminated with CPC 0.005%, then concentrated by filtration and cultured on 7H11 plates in MGIT tubes (winter only). Results Mycobacteria were grown from 40.21% sites in Summer (76/189) and 82.05% sites in winter (160/195). The winter samples yielded the greatest number and variety of mycobacteria as there was a high degree of subculture overgrowth and contamination in summer. Of those samples that did yield mycobacteria in summer, the variety of species differed from those isolated in winter. The inclusion of liquid media increased the yield for some species of NTM. Species that have been documented to cause disease in humans residing in Brisbane that were also found in water include M. gordonae, M. kansasii, M. abscessus, M. chelonae, M. fortuitum complex, M. intracellulare, M. avium complex, M. flavescens, M. interjectum, M. lentiflavum, M. mucogenicum, M. simiae, M. szulgai, M. terrae. M. kansasii was frequently isolated, but M. avium and M. intracellulare (the main pathogens responsible for disease is QLD) were isolated infrequently. Distance of sampling site from treatment plant in summer was associated with isolation of NTM. Pathogenic NTM (defined as those known to cause disease in QLD) were more likely to be identified from sites with narrower diameter pipes, predominantly distribution sample points, and from sites with asbestos cement or modified PVC pipes. Conclusions NTM responsible for human disease can be found in large urban water distribution systems in Australia. Based on our findings, additional point chlorination, maintenance of more constant pressure gradients in the system, and the utilisation of particular pipe materials should be considered.

Isolation of nontuberculous mycobacteria (NTM) from household water and shower aerosols in patients with pulmonary disease caused by NTM

It has been postulated that susceptible individuals may acquire infection with nontuberculous mycobacteria (NTM) from water and aerosol exposure. This study examined household water and shower aerosols of patients with NTM pulmonary disease. The mycobacteria isolated from clinical samples from 20 patients included M. avium (5 patients), M. intracellulare (12 patients), M. abscessus (7 patients), M. gordonae (1 patient), M. lentiflavum (1 patient), M. fortuitum (1 patient), M. peregrinum (1 patient), M. chelonae (1 patient), M. triplex (1 patient), and M. kansasii (1 patient). One-liter water samples and swabs were collected from all taps, and swimming pools or rainwater tanks. Shower aerosols were sampled using Andersen six-stage cascade impactors. For a subgroup of patients, real-time PCR was performed and high-resolution melt profiles were compared to those of ATCC control strains. Pathogenic mycobacteria were isolated from 19 homes. Species identified in the home matched that found in the patient in seven (35%) cases: M. abscessus (3 cases), M. avium (1 case), M. gordonae (1 case), M. lentiflavum (1 case), and M. kansasii (1 case). In an additional patient with M. abscessus infection, this species was isolated from potable water supplying her home. NTM grown from aerosols included M. abscessus (3 homes), M. gordonae (2 homes), M. kansasii (1 home), M. fortuitum complex (4 homes), M. mucogenicum (1 home), and M. wolinskyi (1 home). NTM causing human disease can be isolated from household water and aerosols. The evidence appears strongest for M. avium, M. kansasii, M. lentiflavum, and M. abscessus. Despite a predominance of disease due to M. intracellulare, we found no evidence for acquisition of infection from household water for this species.

Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model

Human lymphatic vascular malformations (LMs), also known as cystic hygromas or lymphangioma, consist of multiple lymphatic endothelial cell-lined lymph-containing cysts. No animal model of this disease exists. To develop a mouse xenograft model of human LM, CD34NegCD31Pos LM lymphatic endothelial cells (LM-LEC) were isolated from surgical specimens and compared to foreskin CD34NegCD31Pos lymphatic endothelial cells (LECs). Cells were implanted into a mouse tissue engineering model for 1, 2 and 4 weeks. In vitro LM-LECs showed increased proliferation and survival under starvation conditions (P < 0.0005 at 48 h, two-way ANOVA), increased migration (P < 0.001, two-way ANOVA) and formed fewer (P = 0.029, independent samples t test), shorter tubes (P = 0.029, independent samples t test) than foreskin LECs. In vivo LM-LECs implanted into a Matrigel™-containing mouse chamber model assembled to develop vessels with dilated cystic lumens lined with flat endothelium, morphology similar to that of clinical LMs. Human foreskin LECs failed to survive implantation. In LM-LEC implanted chambers the percent volume of podoplaninPos vessels was 1.18 ± 2.24 % at 1 week, 6.34 ± 2.68 % at 2 weeks and increasing to 7.67 ± 3.60 % at 4 weeks. In conclusion, the significantly increased proliferation, migration, resistance to apoptosis and decreased tubulogenesis of LM-LECs observed in vitro is likely to account for their survival and assembly into stable LM-like structures when implanted into a mouse vascularised chamber model. This in vivo xenograft model will provide the basis of future studies of LM biology and testing of potential pharmacological interventions for patients with lymphatic malformations.

Isolation and characterization of charge-tagged phenylperoxyl radicals in the gas phase : direct evidence for products and pathways in low temperature benzene oxidation

The phenylperoxyl radical has long been accepted as a critical intermediate in the oxidation of benzene and an archetype for arylperoxyl radicals in combustion and atmospheric chemistry. Despite being central to many contemporary mechanisms underpinning these chemistries, reports of the direct detection or isolation of phenylperoxyl radicals are rare and there is little experimental evidence connecting this intermediate with expected product channels. We have prepared and isolated two charge-tagged phenyl radical models in the gas phase [i.e., 4-(N,N,N-trimethylammonium) phenyl radical cation and 4-carboxylatophenyl radical anion] and observed their reactions with dioxygen by ion-trap mass spectrometry. Measured reaction rates show good agreement with prior reports for the neutral system (k(2)[(Me3N+)C6H4 center dot + O-2] = 2.8 x 10(-11) cm(3) molecule(-1) s(-1), Phi = 4.9%; k(2)[(-O2C)C6H4 center dot + O-2] = 5.4 x 10(-1)1 cm(3) molecule(-1) s(-1), Phi = 9.2%) and the resulting mass spectra provide unequivocal evidence for the formation of phenylperoxyl radicals. Collisional activation of isolated phenylperoxyl radicals reveals unimolecular decomposition by three pathways: (i) loss of dioxygen to reform the initial phenyl radical; (ii) loss of atomic oxygen yielding a phenoxyl radical; and (iii) ejection of the formyl radical to give cyclopentadienone. Stable isotope labeling confirms these assignments. Quantum chemical calculations for both charge-tagged and neutral phenylperoxyl radicals confirm that loss of formyl radical is accessible both thermodynamically and entropically and competitive with direct loss of both hydrogen atom and carbon dioxide.

Production and isolation of ligated metal(IV)-oxo ions by tandem mass spectrometry

High valent metal(IV)-oxo species, \[M(=O)(Melm)(n)(OAc)](+) (M = Mn-Ni, MeIm = 1-methylimidazole, n = 1-2), which are relevant to biology and oxidative catalysis, were produced and isolated in gas-phase reactions of the metal(II) precursor ions \[M(MeIm)(n)(OAc)](+) (M = Mn-Zn, n = 1-3) with ozone. The precursor ions \[M(MeIm)(OAc)](+) and \[M(MeIm)(2)(OAc)](+) were generated via collision-induced dissociation of the corresponding \[M(MeIm)(3)(OAc)](+) ion. The dependence of ozone reactivity on metal and coordination number is discussed. Copyright (C) 2010 John Wiley & Sons, Ltd.

Stability of endoglucanases from mesophilic fungus and thermophilic bacterium in acidified polyols

Recent developments in chemical pretreatments of lignocellulosic biomass using polyols as co-solvents (e.g., glycerol and ethylene glycol) at temperatures less than 100 °C may allow the effective use of thermostable and non-thermostable cellulases in situ during the saccharification process. The potential of biomass saccharifying enzymes, endoglucanases (EG) from a thermophilic bacterium (Thermotoga maritima) and a mesophilic fungus (Trichoderma longibrachiatum), to retain their activity in aqueous buffer, acidified glycerol, and acidified ethylene glycol used as co-solvents at pretreatment temperatures at or below 100 °C were examined. The results show that despite its origin, T. longibrachiatum EG (Tl-EG) retained 75% of its activity after exposure to 100 °C for 5 min in aqueous buffer while T. maritima EG (Tm-EG) retained only 5% activity. However, at 90 °C both enzymes retained over 87% of their activity. In acidified (0.1% (w/w) H2SO4) glycerol, Tl-EG retained similar activity (80%) to that obtained in glycerol alone, while Tm-EG retained only 35%. With acidified ethylene glycol under these conditions, both Tl-EG and Tm-EG retained 36% of their activity. The results therefore show that Tl-EG is more stable in both acidified glycerol and ethylene glycol than Tm-EG. A preliminary kinetic study showed that pure glycerol improved the thermal stability of Tl-EG but destabilized Tm-EG, relative to the buffer solution. The half-lives of both Tl-EG and Tm-EG are 4.5 min in acidified glycerol, indicating that the effectiveness of these enzymes under typical pretreatment times of greater than 15 min will be considerably diminished. Attempts have been made to explain the differences in the results obtained between the two enzymes.

Methods for isolation and cultivation of filamentous fungi

Filamentous fungi are important organisms for basic discovery, industry, and human health. Their natural growth environments are extremely variable, a fact reflected by the numerous methods developed for their isolation and cultivation. Fungal culture in the laboratory is usually carried out on agar plates, shake flasks, and bench top fermenters starting with an inoculum that typically features fungal spores. Here we discuss the most popular methods for the isolation and cultivation of filamentous fungi for various purposes with the emphasis on enzyme production and molecular microbiology.

Isolation and characterization of tumor cells from the ascites of ovarian cancer patients : molecular phenotype of chemoresistant ovarian tumors

Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12-14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions.

A sensor fault detection and isolation method in interior permanent-magnet synchronous motor drives based on an extended Kalman filter

Interior permanent-magnet synchronous motors (IPMSMs) become attractive candidates in modern hybrid electric vehicles and industrial applications. Usually, to obtain good control performance, the electric drives of this kind of motor require one position, one dc link, and at least two current sensors. Failure of any of these sensors might lead to degraded system performance or even instability. As such, sensor fault resilient control becomes a very important issue in modern drive systems. This paper proposes a novel sensor fault detection and isolation algorithm based on an extended Kalman filter. It is robust to system random noise and efficient in real-time implementation. Moreover, the proposed algorithm is compact and can detect and isolate all the sensor faults for IPMSM drives. Thorough theoretical analysis is provided, and the effectiveness of the proposed approach is proven by extensive experimental results.

Controlling electronic and adiabatic isolation of quantum dots from the substrate : an ionization-energy theoretic study

Recent controversy on the quantum dots dephasing mechanisms (between pure and inelastic) is re-examined by isolating the quantum dots from their substrate by using the appropriate limits of the ionization energy theory and the quantum adiabatic theorem. When the phonons in the quantum dots are isolated adiabatically from the phonons in the substrate, the elastic or pure dephasing becomes the dominant mechanism. On the other hand, for the case where the phonons from the substrate are non-adiabatically coupled to the quantum dots, the inelastic dephasing process takes over. This switch-over is due to different elemental composition in quantum dots as compared to its substrate. We also provide unambiguous analysis as to understand why GaAs/AlGaAs quantum dots may only have pure dephasing while InAs/GaAs quantum dots give rise to the inelastic dephasing as the dominant mechanism. It is shown that the elemental composition plays an important role (of both quantum dots and substrate) in evaluating the dephasing mechanisms of quantum dots.

Isolation, structure determination and cytotoxicity studies of tryptophan alkaloids from an Australian marine sponge Hyrtios sp

Mass-guided fractionation of the MeOH extract from a specimen of the Australian marine sponge Hyrtios sp. resulted in the isolation of two new tryptophan alkaloids, 6-oxofascaplysin (2), and secofascaplysic acid (3), in addition to the known metabolites fascaplysin (1) and reticulatate (4). The structures of all molecules were determined following NMR and MS data analysis. Structural ambiguities in 2 were addressed through comparison of experimental and DFT-generated theoretical NMR spectral values. Compounds 1–4 were evaluated for their cytotoxicity against a prostate cancer cell line (LNCaP) and were shown to display IC50 values ranging from 0.54 to 44.9 μM.

Isolation of microvascular endothelial cells from cadaveric corneal limbus

Limbal microvascular endothelial cells (L-MVEC) contribute to formation of the corneal-limbal stem cell niche and to neovascularization of diseased and injuries corneas. Nevertheless, despite these important roles in corneal health and disease, few attempts have been made to isolate L-MVEC with the view to studying their biology in vitro. We therefore explored the feasibility of generating primary cultures of L-MVEC from cadaveric human tissue. We commenced our study by evaluating growth conditions (MesenCult-XF system) that have been previously found to be associated with expression of the endothelial cell surface marker thrombomodulin/CD141, in crude cultures established from collagenase-digests of limbal stroma. The potential presence of L-MVEC in these cultures was examined by flow cytometry using a more specific marker for vascular endothelial cells, CD31/PECAM-1. These studies demonstrated that the presence of CD141 in crude cultures established using the MesenCult-XF system is unrelated to L-MVEC. Thus we subsequently explored the use of magnetic assisted cell sorting (MACS) for CD31 as a tool for generating cultures of L-MVEC, in conjunction with more traditional endothelial cell growth conditions. These conditions consisted of gelatin-coated tissue culture plastic and MCDB-131 medium supplemented with fetal bovine serum (10% v/v), D-glucose (10 mg/mL), epidermal growth factor (10 ng/mL), heparin (50 μg/mL), hydrocortisone (1 μg/mL) and basic fibroblast growth factor (10 ng/mL). Our studies revealed that use of endothelial growth conditions are insufficient to generate significant numbers of L-MVEC in primary cultures established from cadaveric corneal stroma. Nevertheless, through use of positive-MACS selection for CD31 we were able to routinely observe L-MVEC in cultures derived from collagenase-digests of limbal stroma. The presence of L-MVEC in these cultures was confirmed by immunostaining for von Willebrand factor (vWF) and by ingestion of acetylated low-density lipoprotein. Moreover, the vWF+ cells formed aligned cell-to-cell ‘trains’ when grown on Geltrex™. The purity of L-MVEC cultures was found to be unrelated to tissue donor age (32 to 80 years) or duration in eye bank corneal preservation medium prior to use (3 to 10 days in Optisol) (using multiple regression test). Optimal purity of L-MVEC cultures was achieved through use of two rounds of positive-MACS selection for CD31 (mean ± s.e.m, 65.0 ± 20.8%; p<0.05). We propose that human L-MVEC cultures generated through these techniques, in conjunction with other cell types, will provide a useful tool for exploring the mechanisms of blood vessel cell growth in vitro.

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.