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We identified a clinical isolate of Candida glabrata (CG156) exhibiting flocculent growth and cross-resistance to fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB), with MICs of >256, >256, and 32 μg ml−1, respectively. Sterol analysis using gas chromatography-mass spectrometry (GC-MS) revealed that CG156 was a sterol 14α-demethylase (Erg11p) mutant, wherein 14α-methylated intermediates (lanosterol was >80% of the total) were the only detectable sterols. ERG11 sequencing indicated that CG156 harbored a single-amino-acid substitution (G315D) which nullified the function of native Erg11p. In heterologous expression studies using a doxycycline-regulatable Saccharomyces cerevisiae erg11 strain, wild-type C. glabrata Erg11p fully complemented the function of S. cerevisiae sterol 14α-demethylase, restoring growth and ergosterol synthesis in recombinant yeast; mutated CG156 Erg11p did not. CG156 was culturable using sterol-free, glucose-containing yeast minimal medium (glcYM). However, when grown on sterol-supplemented glcYM (with ergosta 7,22-dienol, ergosterol, cholestanol, cholesterol, Δ7-cholestenol, or desmosterol), CG156 cultures exhibited shorter lag phases, reached higher cell densities, and showed alterations in cellular sterol composition. Unlike comparator isolates (harboring wild-type ERG11) that became less sensitive to FLC and VRC when cultured on sterol-supplemented glcYM, facultative sterol uptake by CG156 did not affect its azole-resistant phenotype. Conversely, CG156 grown using glcYM with ergosterol (or with ergosta 7,22-dienol) showed increased sensitivity to AMB; CG156 grown using glcYM with cholesterol (or with cholestanol) became more resistant (MICs of 2 and >64 μg AMB ml−1, respectively). Our results provide insights into the consequences of sterol uptake and metabolism on growth and antifungal resistance in C. glabrata.
The prevalence of infections caused by Candia glabrata among elderly and immunocompromised individuals is increasingly well documented (8, 27, 28, 29). C. glabrata is intrinsically less susceptible to triazole antifungals than other species of Candida (30), and polyene agents (e.g., amphotericin B) constitute one treatment alternative. In contrast to azoles, which disrupt fungal ergosterol biosynthesis through inhibition of sterol 14α-demethylase activity (Fig. 1), polyenes intercalate directly with membrane ergosterol, forming channels that result in leakage of monovalent ions and cellular lysis (6). Today, evidence for the emergence of multidrug, including polyene, resistance in C. glabrata (18) has highlighted the need for a better understanding of the mechanisms that mediate resistance in this species so that more effective antifungals and treatment regimens can be developed.
Studies of azole resistance in clinical isolates of C. glabrata have demonstrated the importance of drug efflux mechanisms (4, 7, 34). Elevated in vitro resistance to azoles, concomitant with increased susceptibility to polyenes, has also been associated with mitochondrial DNA deficiency (petite mutation) in this species (3, 33). However, the universality of this mechanism is unclear given that petite mutants of C. glabrata exhibiting hypersusceptibility to azoles and polyenes have also been identified (39). It has been proposed that aerobic uptake of exogenous sterol (e.g., host cholesterol) contributes to azole (1, 26) and polyene (12) resistance in vivo, and a putative sterol transporter gene for C. glabrata (AUS1) has been identified (26). The genetic controls and biological consequences of obligate and facultative sterol uptake in C. glabrata require further investigation, not least because the potential exists for similarities with other opportunistic pathogens, such as Aspergillus fumigatus (41) and Pneumocystis jirovecii (14).
The present study focuses on a clinical isolate of C. glabrata (CG156) that persisted under treatment with high doses of fluconazole, voriconazole, and amphotericin B (MIC values on yeast-extract-peptone-dextrose [YEPD] of >256, >256, and 32 μg ml−1, respectively). CG156 was found to be an ergosterol-deficient erg11 (sterol 14α-demethylase) mutant, exhibiting flocculent growth, low efflux, and facultative sterol uptake that supported, but was not essential for, growth. Four additional clinical isolates (CG26, CG44, CG388, and CG1012) were selected for comparative studies on the basis of their total cellular sterol composition (>75% ergosterol); this composition indicates sterol 14α-demethylase functionality and is typical of wild-type C. glabrata reference strains (e.g., ATCC 2001) (25).
Ergosterol-deficient, polyene-resistant strains of C. glabrata possessing mutations in ERG1 (squalene epoxidase) (37) and ERG6 (C24-methyl transferase) (38) have been reported; however, unlike CG156, both isolates exhibited increased sensitivity to azoles. Results from this study highlight the potential consequences of sterol uptake on both azole and polyene resistance in C. glabrata and reveal how this opportunistic species might exploit a wide spectrum of exogenous sterols, including several previously uninvestigated cholesterol precursors (Fig. 1D), for survival.
Media.All study isolates (Table 1) were routinely maintained at 30°C on YEPD medium containing 2% (wt/vol) glucose, 2% (wt/vol) Bacto peptone, and 1% (wt/vol) yeast extract (with or without 2% agar). Glucose-based yeast minimal medium (glcYM) containing 1.34% yeast nitrogen base without amino acids (Difco Laboratories) and 2% glucose was prepared as a sterol-free (by gas chromatography-mass spectrometry [GC-MS]) basal medium for use in antifungal susceptibility assays and sterol supplementation experiments. Abbreviations for the sterol supplements used in this study are ergosta 7,22-dienol (7,22), ergosterol (ERG), lanosterol (LAN), cholestanol (CAN), cholesterol (CER), Δ7-cholestenol (CEN), and desmosterol (DES) (medium containing one of these supplements is indicated as, e.g., glcYM + 7,22, glcYM + ERG, etc.). All supplements except cholestenol (Steraloids Inc.) were purchased from Sigma-Aldrich (Poole, United Kingdom). The supplements were dissolved in (1:1) Tween-80-ethanol and added to glcYM to achieve a final concentration of 10 μg sterol ml−1. The morphology of all study isolates was examined using light microscopy following growth on YEPD and glcYM.
The S. cerevisiae (YUG37:erg11) yeast transformants constructed for heterologous expression studies were selected and maintained on glcYM+Trp agar (2% agar) supplemented with 100 μg ml−1 tryptophan. For complementation experiments (Table 2), medium to induce plasmid expression (gal/rafYM+dox) was prepared as previously described (24) using 2% galactose and 1% raffinose instead of glucose and with the addition of 5 μg ml−1 (final concentration) doxycycline.
Culture growth.Growth measurements were made using a Bioscreen C (Oy Growth Curves Ab Ltd., Finland) to record changes in optical density at 600 nm (OD600) over time. Uniform initial culture densities (5 × 105 cells ml−1) were achieved by diluting each sample to 1 × 107 cells ml−1 with filter-sterilized water and adding this cell dilution to the growth medium of interest (50 μl cells plus 950 μl medium). The resulting 1-ml volumes were vortexed, aliquoted into microplate wells (four 200-μl replicates per treatment), and incubated at 30°C in the Bioscreen. Optical density readings (at 600 nm) were taken once every 30 min following the automated (10-s) agitation of the microplate, exported from the Bioscreen in ASCII format, and analyzed using Excel (Microsoft Office 2003). Growth parameters were derived according to the methodology of Weiss et al. (40). Briefly, ΔOD values describe the maximum OD minus the minimum OD; the lag phase is defined as the length of time a culture spends at <10% of the maximum OD; T half max values are equivalent to the time taken to reach half the maximum increase in growth of a culture (ΔOD × 0.5). Maximum doubling times were estimated by dividing the natural logarithm of 2 by the fastest culture growth rates (μ) that were derived from linear trend lines (y = m · x + c, where m μ) fitted to log-transformed OD data.
Antifungal susceptibility assays.The susceptibilities of CG26, CG44, CG156, CG388, and CG1012 to fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB) were determined using standard broth dilution methodology (5). Interpretive breakpoints for susceptibility assays are the following: (i) for FLC, ≤32 μg ml−1 indicates dose-dependent sensitivity and ≥64 μg ml−1 indicates resistant; (ii) for VRC, ≤1 μg ml−1 indicates sensitive and ≥4 μg ml−1 indicates resistant; (iii) for AMB, ≤1 μg ml−1 indicates sensitive. Visual readings for all isolates (except CG156) were made following 48 h of incubation at 30°C; readings for CG156 were made after 72 h. Assays were initially performed using YEPD as the basal growth medium. To investigate the potential contribution of sterol uptake to the resistance phenotypes observed, susceptibility assays for CG156 and CG388 were also conducted using sterol-free and sterol-supplemented glcYM.
Rhodamine 6G efflux assay.The intracellular accumulation of rhodamine 6G (Sigma-Aldrich) in growing isolates was measured according to standard methodology (20). Briefly, cells from an overnight culture (YEPD) were inoculated into fresh medium to an OD600 of ∼0.1 and grown under shaking at 30°C for 4 h. For staining, 10 μM rhodamine 6G (R6G) was added to the culture, and cells were grown for another 2 h, after which they were diluted (1:100) using NaCl. The fluorescence (FL-2) of 1.2 × 104 cells was quantified using a FACScalibur flow cytometer (BD, Heidelberg, Germany). To exclude any bias toward larger cells or agglomerates, efflux values were calculated as the ratio of forward scatter to total fluorescence (FSC/FL-2).
Sterol analysis.Single colonies were used to inoculate 15-ml volumes of media to obtain cultures for sterol analysis. Cells were harvested by centrifugation and washed three times with sterile water prior to sterol extraction and derivatization using a previously optimized methodology (22). Trimethylsilane-derivatized sterols were analyzed using GC-MS and identified with reference to retention times and fragmentation spectra for known standards (23). GC-MS data were analyzed using Agilent software (MSD Enhanced ChemStation; Agilent Technologies Inc.) to determine the sterol composition of all isolates and for derivation of integrated peak areas.
ERG11 gene sequencing.Full-length ERG11 genes for all study isolates were amplified from genomic DNA (single-colony extraction: 0.2% SDS 90°C, 10 min) using forward (F) and reverse (R) primers optimized for use in subsequent heterologous protein expression studies: ERG11F, 5′-ATGTCCACTGAAAACACT-3′; ERG11R, 5′-CTAGTACTTTTGTTCTGG-3′. DNA polymerase with proofreading capacity (High Fidelity Expand; Roche) was used for all PCRs, and sequencing results (Eurofins MWG Operon, United Kingdom) were screened for ambiguous signals prior to translation and alignment against a C. glabrata sterol 14α-demethylase (Erg11p) reference protein (Uniprot accession number P50859).
Heterologous expression of sterol 14α-demethylase (Erg11p) proteins.ERG11 genes for CG26 and CG156 were ligated directly into the yeast expression vector pYES2.1 TOPO (Invitrogen), yielding wild-type and mutated ERG11 plasmid DNA (pCGWT and pCG156, respectively). An S. cerevisiae host (YUG37:erg11), wherein native ERG11 expression is controlled using a doxycycline-regulatable promoter (11, 24), was transformed with pCGWT, pCG156, or empty vector pYES2.1 to create experimental and control strains (YUG37-pCGWT, YUG37-pCG156, and YUG37-pCTRL, respectively), which were selected and maintained on glcYM+Trp agar.
For complementation studies, single colonies from YUG37-pCGWT, YUG37-pCG156, and YUG37-pCTRL transformation plates (all constructs in triplicate) were used to inoculate 15-ml volumes of gal/rafYM+dox; these were incubated for 72 h (30°C, 180 rpm) prior to checks for successful Erg11p complementation, which is evidenced by ergosterol synthesis (GC-MS analysis) concomitant with cell growth.
Sterol uptake experiments.Clinical isolates CG156 and CG388 were cultured using sterol-free and sterol-supplemented glcYM. Optical density data from studies performed with the Bioscreen were used to derive comparative growth parameters for both strains. In addition to antifungal susceptibility assays, GC-MS was used to determine the sterol composition of CG156 and CG388 following growth of 15-ml cultures on supplemented and unsupplemented glcYM (with or without FLC at concentrations equivalent to ×0.5 MIC).
Study isolates.The isolates of C. glabrata included in the present study were retrieved from a collection of pathogenic clinical specimens established as part of an EU FP6 project, the European Resistance Fungal Network (EURESFUN). Specific specimen information is limited; however, it is known that all were isolated from different patients: CG26 and CG388 from blood cultures, CG44 from peritoneal cavity fluid, CG1012 from a central venous catheter, and CG156 from a urine sample.
Growth characteristics and cell morphology.During routine culture maintenance using YEPD, CG156 exhibited slow growth, forming small (<1-mm diameter), flat colonies that were only visible 36 to 48 h after each subculture. All other study isolates were readily culturable, forming larger (typically >2-mm diameter) colonies in <18 h. Bioscreen growth experiments indicate that the lag phases and maximum doubling times for CG156 cultured on YEPD broth were at least double the length of those recorded for CG26, CG44, CG388, and CG1012 (Table 1). Microscope examination of cells from these experiments revealed that CG26, CG44, CG388, and CG1012 all displayed yeast-like growth characterized by solitary blastoconidia and normal cell budding (Fig. 2A). CG156 cultures were highly flocculent and exhibited abnormal cellular budding (Fig. 2B).
The typical growth rates and culture densities supported by glcYM were lower for CG156 than those recorded using complex YEPD (Table 1 and 3). It was noted that CG388 and CG156 were both culturable using sterol-free glcYM (Table 3; also see Fig. 4). CG26, CG44, CG388, and CG1012 exhibited normal and CG156 abnormal cellular budding regardless of the growth medium used (i.e., including when cultured on sterol-free and sterol-supplemented glcYM).
Antifungal resistance phenotypes: YEPD media.When YEPD was used as the basal growth medium (Table 1), CG44, CG388, and CG1012 displayed identical sensitivity to FLC, VRC, and AMB (MICs of 128, 2, and 2 μg ml−1, respectively). CG26 was relatively more resistant to FLC and VRC but not to AMB (MICs of 256, 16, and 2 μg ml−1, respectively). These results are consistent with R6G efflux values for these isolates (Table 1), which indicate that CG26 is a high-efflux strain. Despite being the most azole-resistant isolate (MICs of >256, >256, and 32 μg ml−1 for FLC, VRC, and AMB, respectively), CG156 exhibited lower R6G efflux than all other study isolates (except CG44). This suggested that additional or alternative (nonefflux) mechanisms contribute to its antifungal resistance phenotype.
Sterol composition: YEPD media.Initial GC-MS analysis of YEPD-grown cultures indicated that CG26, CG44, CG388, and CG1012 all had a sterol composition similar to that of wild-type strains (possessing functional sterol 14α-demethylase [Erg11p]), wherein 14α-demethylated sterols accounted for >95% of the total cellular sterol fraction and ergosterol was always the most abundant sterol (Table 1, boldface values). Conversely, CG156 was found to be an erg11 mutant (lacking functional Erg11p), wherein 14α-methylated sterol intermediates (lanosterol was >80% of the total) were the only cellular sterols detectable.
ERG11 sequence and complementation studies.A single missense mutation (G944A) which resulted in the replacement of glycine 315 with aspartate was detected in the Erg11p sequence for CG156 (Fig. 3A). No amino acid substitutions were identified in the translated Erg11p sequences for CG26, CG44, CG388, or CG1012.
The S. cerevisiae YUG37-pCGWT yeast transformant (expressing the ERG11 gene [encoding wild-type Erg11p] from CG26) was readily culturable using gal/rafYM+dox; mutant YUG37-pCG156 and negative control YUG37-pCTRL cultures were not. Real-time PCR (data not shown) verified that both wild-type and mutant C. glabrata ERG11 genes were expressed at comparable levels in YUG37-pCGWT and YUG37-pCG156 cultured using gal/rafYM+dox. GC-MS sterol chromatograms revealed that ergosterol comprised >50% of the total sterol fraction in gal/rafYM+dox-cultured YUG37-pCGWT. In contrast, neither ergosterol nor any other 14α-demethylated sterols was detectable in YUG37-pCG156 and YUG37-pCTRL transformants (Fig. 3B). That the fungistatic sterol 14α-methyl ergosta 8,24(28)-dien-3β-6α-diol comprised >50% of the sterol fraction in the YUG37-pCG156 construct is consistent with the failure of mutated (G315D) CG156 Erg11p to complement the function of S. cerevisiae sterol 14α-demethylase.
Influence of sterol supplementation on growth kinetics.Little variation was seen between the final culture densities reached by CG388 grown using sterol-free and sterol-supplemented glcYM (typical ΔOD values of 1.28). However, some differences between the maximum doubling times and shortest lag phases for CG388 were observed (Table 3 and Fig. 4A). It is possible that the presence of extracellular sterols does affect the regulation of growth and endogenous ergosterol production; however, any specific or mechanistic inferences regarding growth rate variability in CG388 are beyond the scope of the current study.
CG156 grew relatively slowly when cultured using sterol-free glcYM and glcYM + LAN (Fig. 4B), with lag phases (typically ≥60 h) and doubling times (≥2.8 h) being similar on both media. Optimal growth parameters (i.e., shortest lag phases, fastest doubling times, and highest cell densities) were achieved for CG156 cultured using glcYM + CER or glcYM + 7,22 (Table 3). Despite reaching optimal final densities (ΔOD of ∼ 1.20), CG156 samples cultured using glcYM supplemented with ERG, CAN, CEN, or DES had slower maximum doubling times (typically >3.5 h) than those grown using glcYM + CER or glcYM + 7,22 (both ≤2.5 h).
Influence of sterol supplementation on antifungal resistance phenotypes.When sterol-free glcYM or sterol-supplemented glcYM + LAN was used as the basal growth medium, MICs (for FLC and VRC) identical to those recorded using YEPD were observed for both CG156 and CG388. CG388 was equally sensitive to AMB when grown using YEPD, glcYM, and glcYM + LAN. Conversely, YEPD-cultured CG156 was found to be less sensitive to AMB-cultured than glcYM- and glcYM + LAN-cultured cells (MICs of 32 and 16 μg ml−1, respectively).
The azole MICs recorded for CG388 cultured using all sterol-supplemented media (except glcYM + LAN and glcYM + CAN) increased from 128 and 2 μg ml−1 to >256 and >256 μg ml−1 for FLC and VRC, respectively (Table 3). In contrast, there were no differences between the AMB MICs (2 μg ml−1) recorded for CG388 on any media. CG156 retained strong cross-resistance to FLC and VRC (MICs of >256 μg ml−1 for both azoles) regardless of the growth medium used; however, variation was observed in its sensitivity to AMB. Compared to those cultured using YEPD (AMB MIC of 32 μg ml−1), glcYM, or glcYM + LAN (AMB MICs of 16 μg ml−1), CG156 grown on glcYM + CAN and glcYM + CER exhibited elevated resistance to AMB (MIC, 64 μg ml−1). Conversely, CG156 samples grown on glcYM + DES and glcYM + CEN were more sensitive (MICs of 4 μg AMB ml−1); CG156 samples grown using glcYM + ERG and glcYM + 7,22 were again more sensitive to AMB, exhibiting MICs (2 μg ml−1) identical to those of CG388.
Validation of sterol uptake by GC-MS.The sterol composition of CG156 and CG388 cultured using sterol-free glcYM (and glcYM + LAN) is consistent with that of YEPD-grown cells, except for a lower ergosterol content (60 to 65% of the total sterol fraction) and higher proportions of 14α-demethylated sterol intermediates in CG388 (Tables 1 and 4). This result is indicative of the effects of nutrient limitation on cell growth.
CG156.GC-MS chromatograms for CG156 cultured in the absence of FLC (Fig. 5B to H) provide evidence for facultative sterol uptake and the metabolism of exogenously supplied sterols. Negligible (<1% of the total sterol fraction) quantities of ergosta 7,22-dienol were detected in untreated CG156 cultured using glcYM + 7,22; the second most abundant sterol (after lanosterol) identified in these cultures was ergosterol (12.8%). Exogenously supplied ergosterol comprised approximately 25% of the sterol fraction of CG156 cultured using glcYM + ERG (Table 4); ergosterol was not detected in CG156 grown on any other sterol-supplemented medium (except glcYM + 7,22). CG156 cultured using glcYM + CER incorporated relatively large quantities of exogenously supplied cholesterol, such that it comprised approximately 31% of the total sterol fraction in untreated cells (Table 4). Cholesterol was also detected as a sterol metabolite (comprising 2.8% of the total) in CG156 grown on glcYM + CAN; in these cultures the original cholestanol substrate constituted approximately 20% of the total sterol fraction. Lathosterol (Δ7-cholestenol) was only detected in trace quantities (<2% of the total sterol fraction) in CG156 cultured using glcYM + CEN; no potential sterol metabolites of CEN (e.g., cholesterol) were detected in these cells. Conversely, while similarly low levels of desmosterol (2.8% of total) were detected in CG156 cultured using glcYM + DES, metabolites of desmosterol ordinarily not synthesized by C. glabrata (ergosta 5,22-dienol and ergosta 5-enol) were detected in relative abundance, comprising 16.3 and 10.7% of the total sterol fraction, respectively.
In FLC-treated CG156 cultured using sterol-free glcYM and all sterol-supplemented media (except glcYM + CEN), the proportions of exogenously supplied sterols and their respective sterol metabolites (detailed above) are very similar to those recorded in untreated cells (Table 4, bracketed and underlined values). GC-MS data indicate that FLC-treated CG156 incorporated approximately 5-fold more Δ7-cholestenol (9.5% of total sterol fraction) than untreated CG156 grown on glcYM + CEN; however, as seen in untreated CG156 cultures, potential sterol metabolites of CEN were again absent.
CG388.In the absence of FLC, CG388 exhibited wild-type sterol composition (Fig. 5A) on both sterol-free and sterol-supplemented glcYM; this reflects the continuation of endogenous ergosterol biosynthesis. Conversely, sterol profiles for FLC-treated CG388 indicate azole inhibition of Erg11p (evidenced by decreased ergosterol content and the accumulation of 14α-methylated sterol intermediates) in addition to the uptake and metabolism of exogenously supplied sterols. The ergosterol content of FLC-treated CG388 cultured using sterol-free glcYM decreased to 62% of that of untreated cells, comprising 37 and 59.7% of the total cellular sterol fraction, respectively. The levels of ergosterol detected in FLC-treated CG388 cultured using glcYM + 7,22 and glcYM + ERG were relatively similar (45.5 and 43.3%, respectively). The depletion of ergosterol in all other FLC-treated CG388 cultures was more significant; ERG comprised at most 8% (glcYM + DES) and typically <3% of the total cellular sterol fraction (Table 4).
Results from this study of five clinical isolates of C. glabrata (CG26, CG44, CG156, CG388, and CG1012) complement findings from work with engineered laboratory strains (10, 25, 26) and provide novel evidence regarding the versatility and consequences of facultative sterol uptake for growth and antifungal resistance in C. glabrata.
CG156, the most azole- and polyene-resistant isolate (Table 1), was found to be an erg11 (sterol 14α-demethylase) mutant completely lacking 14α-demethylated sterols (including ergosterol) and harboring a single-amino-acid substitution (G315D) which nullified the function of Erg11p. This substitution occurs in the highly conserved αI helix of Erg11p and other cytochromes P450 (Fig. 3A), affecting the structure of substrate recognition site IV (36). An equivalent substitution also results in loss of function in S. cerevisiae sterol 14α-demethylase (13). It was initially suspected that CG156 persists by scavenging trace amounts of ergosterol from the YEPD growth medium on which it was maintained. However, using sterol-free (glcYM) minimal medium, we found that CG156 could grow in the absence of exogenously supplied sterol (Table 3) with 14α-methylated sterol intermediates (lanosterol was >80% of the total), again constituting the entire cellular sterol fraction. This finding is consistent with reports of other erg11 strains of C. glabrata (10) and S. cerevisiae (9), which remain aerobically viable and are able to grow on lanosterol-type sterols. It is possible that changes in overall membrane composition (e.g., proportions of fatty acid, phospholipids, and sphingolipids) compensate for perturbations in sterol content in ergosterol-deficient yeast; this is now an avenue for further investigation. Such plasticity in membrane composition might well enhance the capacity of C. glabrata to withstand extended periods of starvation stress (31) and underscores data showing that Erg11p is not an ideal drug target in C. glabrata (25).
Facultative sterol uptake supported higher cell densities in CG156 samples cultured using all (except glcYM + LAN) sterol-supplemented glcYM than in samples grown on sterol-free glcYM (Table 3), indicating that exogenously supplied sterols could serve bulk functions (35) in C. glabrata. The elevated growth rates of CG156 cultured using cholesterol-containing (glcYM + CER) media demonstrate how this species is well adapted for survival in the human host (31). The ability of both CG156 and CG388 to sequester Δ7-cholestenol (lathosterol) and to further metabolize both cholestanol and desmosterol (Table 4) is also significant. All three cholesterol precursors accumulate within the body in certain inborn human malformation syndromes, including lathosterolosis, desmosterolosis, and cerebrotendinous xanthomatosis (17, 32).
In contrast to CG388, wherein the uptake of exogenously supplied sterols (observed only in azole-treated CG388) (Table 4) correlated with an increase in resistance to fluconazole and voriconazole (Table 3), sterol uptake had no effect on azole MICs recorded for CG156. Furthermore, while clear differences were observed between the sterol composition of untreated and FLC-treated CG388 (Table 4), the sterol content of CG156 grown on all sterol-supplemented media was similar in the absence and presence of FLC (Table 4). It has been proposed that facultative sterol uptake contributes to elevated azole resistance in C. glabrata (1, 26); however, it does not appear to be essential for the high-level azole resistance observed in CG156. Interestingly, the proportions of exogenously supplied cholestanol, cholesterol, Δ7-cholestenol, and desmosterol (and their respective sterol metabolites) detected in FLC-treated CG388 and CG156 are highly comparable (Table 4, bracketed and underlined values). It is possible that these sterols serve a compensatory role (e.g., stabilizing membrane integrity) in azole-treated cells but that they are only required in threshold amounts.
Owing to the continuation of endogenous ergosterol biosynthesis, CG388 exhibited uniform sensitivity to amphotericin B (MIC, 2 μg ml−1) on all media; conversely, the sensitivity of CG156 to AMB was affected by the uptake and metabolism of specific exogenously supplied sterols. CG156 cultured using media containing ergosterol (and ergosta 7,22-dienol) were more sensitive to AMB than CG156 grown on cholesterol (or cholestanol) (MICs of 2 and >64 μg ml−1, respectively). The presence of ergosterol (the binding target of polyene antifungals [6]) and cholesterol (weaker binding to polyenes) in CG156 cultured using glcYM + 7,22 and glcYM + CAN likely reflects the activity of endogenous ERG3 protein (C5-desaturase) on ergosta 7,22-dienol and cholestanol precursors (Fig. 1D) and would account for the decreased sensitivity to AMB we observed. This is the first time that sterol uptake has been shown to alter AMB sensitivity in C. glabrata; it highlights the potential to exploit sterol transport proteins for the uptake and intracellular targeting of antifungal compounds.
Aside from demonstrating the ability of C. glabrata to sequester several previously uninvestigated sterols, results from the present study provide novel insights into endogenous sterol metabolism. That significant (>5%) amounts of Δ7-cholestenol were only sequestered by azole-treated CG388 and CG156 suggests that it is not an ideal bulk sterol. That no potential products of Δ7-cholestenol metabolism (e.g., ergosterol) were detected in glcYM + CEN-grown cells indicates that C. glabrata lacks the enzymatic machinery required to further metabolize Δ7-cholestenol (e.g., to ergosterol or cholesterol). Azole-treated CG156 and CG388 cultured using glcYM + DES contained only negligible amounts of desmosterol (≤3% of the total sterol fraction). Nonetheless, significant proportions of ergosta 5-enol (indicative of endogenous ERG6 protein [C24-methyl transferase] activity on desmosterol) and ergosta 5,22-dienol (indicative of endogenous ERG5 protein [C22-desaturase] activity on ergosta 5-enol) were detected in both isolates.
Sterol metabolism data suggest that (i) despite binding with high affinity to heterologously expressed C. glabrata C22-desaturase (Erg5p) (19), FLC is not a potent inhibitor of Erg5p activity in vivo, and (ii) that CG156 also possesses functional Erg6p. That C22-desaturase activity is apparent in both CG388 and CG156 is important, because Erg5p (CYP61) is a heme-thiolate cytochrome P450 enzyme (19). It remains possible that the clinical isolates, including CG156, harbor additional mutations; however, heme biosynthetic mutations detected in other studies (1, 9, 10) can be excluded for CG156. The functionality of Erg6p is relevant because a clinical isolate of C. glabrata with reduced susceptibility to polyenes, harboring a missense mutation in ERG6 and exhibiting pseudohyphal growth, has been reported (38). The polyene resistance and the flocculent growth of CG156 thus appear to have a different molecular basis. An ace2 mutant of C. glabrata exhibiting clumpy growth and hypervirulence in a murine model of invasive candidiasis has been described (15). This isolate was able to cause systemic infections characterized by fungal escape from the vasculature, tissue penetration, and proliferation in vivo. Although poorly documented, the molecular basis and consequences of phenotypic switching in C. glabrata now require fuller investigation, not least because it is understood to be a key virulence factor in Candida albicans (2).
Taken as a whole, results from this study indicate that some isolates (here, CG156) might persist as slow-growing agents of chronic infections, because they (i) possess mutated Erg11p that has low or no affinity for azoles; (ii) lack cellular ergosterol and thus exhibit high-level resistance to polyenes; (iii) can survive without sterol auxotrophy through growth on lanosterol-type sterols; and/or (iv) can opportunistically exploit a wide spectrum of host/environmental sterols for growth. That CG156 was found to be a low-efflux isolate (Table 1) is particularly interesting. It is possible that the altered cellular sterol composition of CG156 affects intracellular signaling and trafficking pathways, including the distribution and function of membrane-anchored proteins. The latter could include efflux machinery (3, 7, 34) and any transport proteins that are proposed to mediate azole import via facilitated diffusion (21). The investigation of these possibilities represents an avenue for further work.
This research was supported by the EU FP6 project EURESFUN (European Resistance Fungal Network).
Strains CG26, CG44, CG388, and CG1012 were obtained through the German National Reference Center for Systemic Mycoses. CG156 was kindly contributed to the EURESFUN strain collection by E. Mellado (ISCII, Madrid, Spain).
Technical support was from N. J. Rolley, and analytical facilities were provided by the EPSRC National Mass Spectrometry Service Centre (Swansea University, United Kingdom).
- Received 28 November 2011.
- Returned for modification 26 January 2012.
- Accepted 12 May 2012.
- Accepted manuscript posted online 21 May 2012.
- Copyright © 2012, American Society for Microbiology. All Rights Reserved.


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Searchlight Chromia Led 10 Disc Ceiling Bar Light - Chrome - Frosted Acrylic
This LED 10 Disc Bar Light with chrome circles and frosted acrylic shades is an ultra-modern and stylish focal point to any modern living space. The design is based on interlocking geometric circles of various sizes and different heights, which join together to produce a clear warm light which would great above a modern dining area or breakfast bar. The ten frosted acrylic discs look great with the chrome finish trim and the fitting is illuminated with the latest energy saving LED technology.
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- Maximum Wattage: 38
- Bulb Type: LED
- Bulb Shape: LED
- Bulb Included: YES
- Dimmable: NO
- Switch Location: N/A
- IP Rating: IP20
- Height: 1170
- Width: 265
- Length / Depth: 1000
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subset: commoncrawl


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Vahé Tachjian
Vahé Tachjian is a historian. Born in Lebanon, he earned his Ph.D. in History and Civilization at the Ecole des Hautes Etudes en Sciences Sociales (EHESS) in Paris. His research covers the period of the French occupation of Cilicia, Syria and Lebanon between the two World Wars, the last years of the Ottoman Empire, refugee problems in the Middle East, and Kurdish-Armenian relations. He has carrried out extensive research in several archives in Paris, Nantes, London, Cairo, Beirut, Aleppo, and Yerevan. Among his publications are "The Prince": The Life and Political Activity of Ishkhan of Van, Yerevan, 1994, 210 pages (in Armenian); La France en Cilicie et en Haute-Mésopotamie. Aux confins de la Turquie, de la Syrie et de l'Irak, Karthala Editions, Paris, 2004, 465 pages; with Raymond Kévorkian and Michel Paboudjian, Introduction and Notes to Ohannès Pacha Kouyoumdjian, Le Liban à la veille et au début de la guerre. Mémoires d'un gouverneur, 1913-1915, Beirut, 2003; with Raymond Kévorkian and Lévon Nordiguian, eds., Les Arméniens, 1917-1939. La quête d'un refuge, Presses de l'Université Saint-Joseph, Beirut, 2006, 320 pages. He was one of the organizers of the photographic exhibition held in Beirut in 2006: The Armenians, 1917-1939: The Quest for a Shelter in the Near East. He is currently employed as a research associate in the Nubarian Library, Paris.


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Saratoga Race Course: Will Take Charge wins Travers
It was supposed to be 1982 all over again.
It turned into 1995, instead.
Anticipation was high for a Travers Stakes with three distinct Triple Crown race winners running against each other, but that delicious scenario dissolved when Preakness winner Oxbow was pulled with an ankle injury 21⁄2 weeks ago.
Will Take Charge proved to be an able stand-in for his stablemate, though, by winning the 144th Travers by a nose in a very close photo finish over 31-1 Moreno before 47,597 under pure blue skies at Saratoga Race Course on Saturday.
That rang a bell for Hall of Fame trainer D. Wayne Lukas, who had to scratch Preakness winner Timber Country because of a high temperature the night before the 1995 Travers, only to watch stablemate Thunder Gulch win the race on his way to the 3-year-old championship.
"We went down and looked at that little Thunder Gulch and said, 'Ball is in your court, here you go,' " Lukas said. "That's kind of what happened this time. We sent Oxbow home for some freshening and cranked this one out."
Cranked, indeed.
Jim Dandy third-place finisher Moreno proved to be more than a worthy contender while trying to win the $1 million Travers on the front end, chased by Kentucky Derby winner Orb, Belmont Stakes winner Palace Malice and 3-2 post-time favorite Verrazano, the Haskell winner.
Will Take Charge closed well to get within a length of Palace Malice for second in the Jim Dandy, and the extra furlong of the mile-and-a-quarter Travers was just enough for Will Take Charge to get his nose in front of Moreno in the final stride.
"Yeah, I didn't know if I had won," said Luis Saez, Will Take Charge's fourth different rider in his last four races. "When I was coming to the wire, I thought I could win it, maybe, if I rode a little harder. So I did, and then when I crossed the wire, I didn't know. I had to ask the outrider, and he told me I won."
"Brutal, huh? Last jump," said Eric Guillot, Moreno's trainer.
Willis Horton's Will Take Charge, who ran in all three Triple Crown races with nothing better than a seventh, 16 lengths behind Oxbow in the Preakness, to show for it, went off at 9-1 and led off a $5,098.00 superfecta on a day when the ninth-largest crowd in Travers history wagered almost a quarter of the $41,363,760 all-sources betting handle, the first time Saratoga has surpassed $40 million.
Most of the Travers money went in the direction of Verrazano and Palace Malice, the formidable pair of colts from the barn of Todd Pletcher.
Runaway Groom beat Gato Del Sol, Aloma's Ruler and Conquistador Cielo in the 1982 Travers, the only time in history that three Triple Crown race winners have been beaten in the same race.
Despite the loss of Oxbow, the 2013 Travers still had plenty of star power and intrigue, especially since Verrazano was beginning to show signs of being perhaps the best of this year's 3-year-old crop.
Will Take Charge appeared to
have a few things going against him, not the least of which was a jockey switch to Saez, who had never won a Grade I race, from Jim Dandy rider Junior Alvarado.
"You look terrible if it doesn't work, but it's sweet if it does," Lukas said.
The other significant jockey change in the Travers was Jose Lezcano on Orb.
Joel Rosario was supposed to ride, but a broken left foot suffered on Friday wiped out not only a big weekend in which he was supposed to ride Game On Dude in the Pacific Classic today, but the next six weeks of work for him.
Guillot promised that Moreno would be on the lead, just as he was in a gritty Jim Dandy effort, and Jose Ortiz made good on that, getting Moreno through ideal splits of 24.40 for the first quarter-mile, 48.88 for the half and 1:13.43 for six furlongs.
While tracking in fourth and fifth, Saez stayed in close enough contact with Moreno to have a shot to catch him. Barely.
As Moreno came off the second turn, Orb cut the corner inside and took a slim lead and appeared to be in perfect position to go up the rail and win.
Moreno didn't falter, though, and regained the lead about six strides from the wire.
Meanwhile, Will Take Charge angled slightly toward the middle of the track from behind them and just caught Moreno as Orb finished three-quarters of a length back in third.
"Who wouldn't have wanted to win on this day, on this stage?" Guillot said. "Like I say, you've got to pay the piper for all the stuff I talk, right? But I backed it up pretty damn good running second at 30-1 in the Travers, right? You can look at it both ways."
"At this time of the year, when horses have had hard campaigns, these races are special," Lukas said. "There has never been a bad Travers. But they don't put up a million bucks on a Grade I and just hand it to you. You better get on your belly and have a horse ready, or you're not going to win one of these."
Pletcher's pair had trouble starting and finishing.
Jockey Mike Smith said Palace Malice, who was fourth, essentially lost the race when he broke flat-footed.
"All I could do is sit back there, creep up, creep up, creep up and see if I could get him there," he said. "It was just too much to make up."
Jockey John Velazquez said Verrazano had a good trip, but by the half-mile pole, "he just went through the motions. He really didn't put in much effort."
"I thought Verrazano got a beautiful trip," Pletcher said. "The only difference was we thought he'd be stalking Palace Malice, who, Mike said, missed the break. Mike thought he was much the best, but his break killed him.
"It's horse racing. It happens every day, every race — 14 times a day, sometimes. We've had a great meet, and we're not going to cry about it."


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Established in 2012, the International Centre for Technological Innovations (ICTI) is a unique initiative that boosts research, innovation and entrepreneurship for sustainable development. Our activities are implemented in partnership with community based organizations, academia, research labs, government agencies, companies and small medium enterprises. Together, we develop innovative products and services, start new companies, and nurture future leaders.
International Centre for Technological Innovations (ICTI) is a research organisation. Our work focus on changing peoples lives by developing new products and services based on technological innovations and commercialising it through spinoffs, technology transfer and other outreach initiatives.


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This Friday through Monday, I'll be attending Lifelight Music Festival in Worthing, South Dakota. It's a free Christian music festival that happens every year over Labor Day weekend.
Since I first began praying for Jahar in April, I've been striving to build up an army of prayer warriors for him. I figured what better way to do that then to try to reach out to people at Lifelight, where the crowds at times exceed 100,000 people.
I designed a T-shirt to wear and made some business cards to hand out if people express interest in Christians United for Jahar after seeing my shirt or talking to me. I'm including some photos of the shirt and the business cards at the end of the post.
Please pray that the shirt and business cards will be effective in getting more Christians to pray for Jahar. Also pray that I would be able to withstand any hate that might come my way. If I can get even one person to pray for Jahar, it's worth suffering through an abundance of hate. He's worth that much to me.


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Utah: Poll of Likely Voters Shows 73% Support for a Medical Cannabis Ballot Initiative
By Michael Bachara
Hemp News
A February poll of 402 Utahns found that 73% of voters support a medical cannabis ballot initiative, with only 20% opposed and 7% undecided.
"The poll results show overwhelming and broad support for medical cannabis in Utah," said DJ Schanz, director of Utah Patients Coalition, a political campaign committee formed to support a 2018 ballot initiative to establish a medical cannabis program in Utah. "Voters believe that patients should be able to safely and legally access the medicine they need."
Voters from virtually all demographic groups expressed support, including 64% of Republican voters, 63% of active LDS voters, and 75% of voters age 50 and above. Nearly 80% of all voters support medical cannabis in principle. The poll found 72% support for allowing doctors to recommend medical cannabis as a treatment for chronic pain.
Between 1999 and 2010, states with medical cannabis laws had a 24.8% lower rate of opioid overdose deaths than states without medical cannabis laws, a 2014 study published in the Journal of the American Medical Association found.
"The opioid epidemic has already taken too many lives in our state," said campaign spokesperson Christine Stenquist. "We should allow medical cannabis as a treatment for chronic pain for two urgent reasons. First, medical cannabis is a more effective treatment for many patients. And second, it can potentially play a significant role in reducing the rate of opioid overdose deaths in Utah."
The poll was conducted with live callers on both landline and wireless phones and was commissioned by the Marijuana Policy Project, a national marijuana reform organization that is supporting the 2018 Utah campaign. Fairbank, Maslin, Maullin, Metz & Associates, an opinion research firm, conducted the poll.
Recent blog posts
- Study debunks claims against cannabis retail
- Michigan: Cannabis Industry Association Capitol Protests
- The First Obituary of Steven Hager
- Washington: Adult Cannabis Cultivation Bill, HB 2559, Seeking Public Comment
- U.S.: Former US Deputy Attorney General Cole's reaction to Sessions' Rescission
- Vermont: Senate Passes Recreational Cannabis Bill, Governor Expected to Sign
- Global: Oregonian Being Held In El Salvador for 36 Grams of Cannabis
- U.S.: Attorney General Sessions to Rescind Memo on State Cannabis Laws
- Wisconsin: Medical Cannabis Advocate, Jacki Rickert, Passes Away
- South Carolina: Twenty Farmers Selected for Industrial Hemp Pilot Program


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The company with experience in making signs will help you have good signs.You will have an assurance of quality since, if a company has experience.
The Best Advice About Signs I've Ever Written


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Samuel's Testimonial:
I came into Green City Force not knowing what to expect. I wasn't working or going to school, but I knew that I needed to do something with myself. Going out into service and helping the underprivileged, I found a purpose in helping those that need it the most. In a sense, I saw myself the same way I see those we serve: someone in need of help. It's more than just light bulbs and recycling bags, you're creating opportunities in areas that are in need, bringing change in neighborhoods that haven't seen it. You, as a Green City Force Corps Member, are bringing hope back into these communities, and that's the greatest service of all.


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It might be confusing for some, but AI refers to all things that exhibit some sort of intelligence. From your phone's calculators to supercomputers to self-driving cars, artificial intelligence is everywhere even if you don't realize it.
And, the applications of AI and its uses are only going to increase in the time to come. This makes up for exciting times for all those interested in building intelligent machines.
We have recently launched an online guide to getting started with artificial intelligence for beginners. However, to cover some of the advanced topics, we invited Anand Chandrasekaran, CTO, MadStreetDen, for a podcast to discuss and answer popular questions on AI and its applications.
Anand is a neuroscientist who went on to study artificial intelligence. The two fields aren't completely unrelated but it is quite unlikely for people into either field to make a transition. Anand, however, was intrigued by the similarities between brain and the capabilities of the computer that enabled it to emulate the functions of the brain.
The excerpts of the podcast can be accessed below:
Anand's views on AI as a field of study and how he embarked on the journey of AI
Relationship between neuroscience and AI
Advantages and disadvantages of changing disciplines.
Work done at MadStreetDen in context of AI
How computer vision evolved over the years and scope of its improvement?
Self-learning capabilities of computers and progress in the field
Real time scenario handling in terms of AI
Problems with Siri and google that deteriorate their popularity
Anand's advice to AI aspirants on getting started and succeeding


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Senior Member
Last Activity: 19th April 2017 09:27 AM
About Me
- About mindlesSheep
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Home > About us
Ningbo Bigmouth Robotics Technology Co., Ltd (BM in short) has been in the line of international trade for more than 30 years. Each year, BM creates more than 125M sales around the world.
BM has registered its own brands Auxiwa, Simenmax, Kawany and Buteny in the United States. To better establish its brand image and reputation, BM has shared its ownership of brands with retailers around the world.
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Are your friends always telling you that you should get this or get that for their birthday? And when you look at what they want, it's usually something way out of your budget? In between all your student loans, credit card bills, and car payments, you'd actually save money by NOT seeing them on their birthday. This birthday card is for those people expecting too much.
DJ Khaled, the self-proclaimed "BEST" at everything, has put together some of the biggest hip hop collaborations of all time. Songs such as "We Takin' Over" and "All I Do Is Win" have grown to become the beloved anthems of champions. And while the songs bear DJ Khaled's name, he doesn't really rap on them, but instead blesses us with his famous catchphrases such as "WE THE BEST" and of course "ANOTHER ONE" to let you know he has another hit single on his hands.
In 2015, DJ Khaled stepped his game up, releasing everyday tips and advice via SnapChat titled "Keys to Success." On November 26, 2015, Music Choice released a motivational video featuring DJ Khaled and his compilation of motivation to help you win at life.
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East Berlin
Early one cold and overcast November day in 1983 I crossed through Check Point Charlie for a day in East Berlin.
After the obligatory look at the Brandenburg Gate, my next priority was Karl Marx's old alma mata, the Humboldt University of Berlin, just down the road on the Unter den Linden. I was most keen to wander around the corridors of the spiritual home of Hegel and the intellectual birthplace of Marxism. Given the communist state had restored the building after the predations of the war I was quite sure that the University would have made a feature of such history.
It must have been time for lectures as I arrived - students were milling around outside and climbing the steps to go inside. An opportunity to sit in on a lecture I thought - fond memories of my undergraduate days – so I joined the throng of remarkably clean cut and quiet students, climbed up the steps and entered the foyer.
As I did so from the shadows appeared at my sides two sets of muscle in identical suits: no you cannot enter – said one in English – did I look English with my beard, longish hair, duffel jacket, camera and note book? I want to see where Karl Marx studied – It is verboten - You are not a student - Go now please. Please? I had no option as they physically escorted me down the steps. So much for intellectual solidarity with the leader. I settled for a cup of coffee and cake in a grubby café over the road.
As I thought about the incident over my cup of German workers coffee I was struck by the irony of exclusion and paranoia in the Workers State. I could almost hear old Karl groaning all the way from Highgate.
It was not until very recently that I learnt that Humboldt University, during the heady days of the East German Workers Paradise, catered for those ideologically pure students destined for higher things in the State. The authorities took their charges most seriously and instituted steps to ensure that the State's future intellectuals were not infected with any liberal western ideas, such as curiosity for curiosities sake. There would have been no room for young Hegelians at Humboldt then.


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Victory/Divine Helpers
Your Divine Helpers are present, bearing the good news that whatever you've been working on will succeed.
The bad news .. well, there really isn't any bad news, but there is reality, which in your case suggests that you can fully enjoy your success only if you temper your ego and recognize that you didn't get where you are by yourself.
This isn't to suggest that you must diminish your light or be falsely modest in your achievements - not by a long shot.
You deserve to shine, so please do. Your Divine Helpers simply remind you to be grateful, and acknowledge the contributions of those who have helped you get where you are.
This includes your spirit helpers and guides as well. One final word: With success comes the challenge of standing alone in your light. Of course you've heard the saying 'It's lonely at the top.'
But it doesn't have too be if you remember how you got there. Your Divine Helpers' message: 'Share the spotlight!'
Love and Blessings, Dallas <3
Leave a Reply.
Dallas Hodder Franklin is an Intuitive/Medium and Channel Writer counseling souls along the way.
March 2020


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Trade view / 29 September 2017 at 11:48 GMTStrategic trade/BuyThe US housing boom continues as KB Home reported forecast-beating third-quarter results yesterday and kept a positive outlook. US building permits are still rising and have around 20% more upside from current levels as family formation increases and permits swing back to the long-term average from 1960. Among North America home builders, we believe DR Horton is the most undervalued, with around 28% upside over the next 12 months.Read the Trade View
Article / 07 December 2016 at 2:23 GMT
Morning Report APAC: Asian shares rise, AUD sputtersAsian shares were mostly up this morning, following the leads from overseas. The ASX was also holding its own, with the only crack coming in the form of the AUD, which lost 0.6% based on third quarter gross domestic product which declined 0.5% from the prior quarter - the first contraction since 2011.Read the article
Article / 02 September 2016 at 11:55 GMT
Recent changes to the equity portfolioOur equities portfolio remains predominantly net-long with several new positions, but note some changes that will be made in the event of an underwhelming nonfarm payrolls print.Read the article3yHi Peter! Does this means you recommending to close the short position of GDX ETF on the market opening now shortly? Please clarify3yHi Peter. Is the open positions board available on tradingfloor? Or how can it be accessed?3yDear Peter. I bought some EOAN shares at 8.20 EUR. How do you see spin off 1:10 made by E.ON and Uniper? EOAN -14.5% today. Will it...
Video / 25 August 2016 at 9:17 GMT
US housing boom to continue: GarnryPeter GarnryRecent data on new US home sales showed a big surprise to the upside, according to Saxo Bank head of equity strategy Peter Garnry.
New US home sales have reached their highest level in 9 years but this is still below the natural rate.
Garnry believes that the robust employment trend could spell a continuation of the US housing boom. And this trend could prove positive for companies like Home Depot and Toll Brothers.
Garnry is positive on Toll Brothers and his trade idea is to go long with a target of $40 and a stop at $30.
Trade view / 24 August 2016 at 13:39 GMTStrategic trade
#SaxoStrats: US housing boom bodes well for buildersAmerican constructors are building homes at a faster pace and more and more consumers are buying them. This bodes well for construction companies like Toll Brothers and other housing-related stocks.Read the Trade View
Trade view / 18 May 2015 at 13:46 GMTMedium term
Toll Brothers looks to build a rallyLike many interest-rate sensitive parts of the stock market, home builder stocks suffered in April as US interest rates rose due to the violent global bond sell-off. The bond smack-down, however, looks to have been overdone in the near-to-medium term, which should help home builder stocks to push higher.Read the Trade View
Video / 24 February 2015 at 15:04 GMT
Who's next after Home Depot's billion dollar buy-back?Serge BergerA happy day for Home Depot shareholders, who can look forward to a USD 18 billion share buyback after the company posted fourth quarter profits that topped analysts' estimates. Serge Berger takes a look at other companies set to gain from the rising US housing prices and how to trade them.5yThis comment has been redacted
Squawk / 03 September 2014 at 9:02 GMTToll Brothers Q3 revenue $1.06B vs. $978M estimated with gross margin improving 175-200 bps


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United States District Court, D. Hawaii.
August 22, 2016
UNITED STATES OF AMERICA, Plaintiff,v.DONALDSON ENTERPRISES, INC. (01); CHARLES DONALDSON (02); and CARLTON FINLEY (03), Defendants.
Oki Mollway United States District Judge
case relates to permitting requirements that the Government
says applied to fireworks seized by the Government. On April
8, 2011, there was an explosion at the Waileke bunker where
the fireworks were located. Five people were killed. The
Superseding Indictment asserts criminal charges against
Donaldson Enterprises, Inc., the company allegedly
responsible for storing and disposing of the fireworks, and
two individuals, Charles Donaldson and Carlton Finley
(collectively, "Defendants").
three Defendants are charged in Count 1 of the Superseding
Indictment of August 6, 2014, with conspiracy to treat and
store hazardous waste without a permit in violation of the
Resource Conservation and Recovery Act ("RCRA"),
42 U.S.C. § 6928(d)(2)(A) and 18 U.S.C. § 371.
See ECF No. 43. Counts 2 and 5 of the Superseding
Indictment charge Defendants with treating hazardous
waste without a permit in violation of 42 U.S.C. §
6928(d)(2)(A) and 18 U.S.C. § 2. Count 3 charges
Defendants with storing hazardous waste without a
permit in violation of 42 U.S.C. § 6928(d)(2)(A) and 18
move for dismissal of the charges, arguing that their due
process rights are being violated. The court denies the
case involves Defendants' alleged failure to comply
with RCRA and Hawaii's Hazardous Waste Management Plan.
is a comprehensive environmental statute that governs the
treatment, storage, and disposal of solid and hazardous
waste." Meghrig v. KFC W., Inc., 516 U.S. 479,
483 (1996). "RCRA's primary purpose . . . is to
reduce the generation of hazardous waste and to ensure the
proper treatment, storage, and disposal of that waste which
is nonetheless generated, 'so as to minimize the
present and future threat to human health and the
environment.'" Id. (quoting 42 U.S.C.
§ 6902(b)). To that end, 42 U.S.C. § 6925(a)
requires that a permit be obtained for the treatment,
storage, or disposal of hazardous waste. Criminal penalties
are set forth in 42 U.S.C. § 6928(d)(2)(A) for any
person who "knowingly treats, stores, or disposes of
any hazardous waste identified or listed under this
subchapter [42 U.S.C. §§ 6921 to 6939g]--(A)
without a permit under this subchapter or pursuant to title I
of the Marine Protection, Research, and Sanctuaries Act (86
Stat. 1052) [33 U.S.C. §§ 1411 to 1421]."
Defendants are charged with violating 42 U.S.C. §
As for
the elements of a 42 U.S.C. § 6928(d)(2)(A) crime, the
Ninth Circuit has held with respect to the mens rea
requirement that the Government must prove a
defendant's knowledge only of the treatment, storage,
or disposal of hazardous waste, not of the permit status.
See United States v. Hoflin, 880 F.2d 1033, 1039
(9th Cir. 1989); see also United States v.
Heuer, 4 F.3d 723, 732 (9th Cir. 1993)
("knowledge that the material is waste is also a
requirement for conviction under §
6928(d)(2)(A)"). Additionally, the Ninth Circuit has
approved of a jury instruction listing as an element that the
defendant knew the waste had the potential to be harmful to
others or to the environment and was not just an innocuous
substance like water, reasoning that this sufficiently
described "hazardous" for purposes of the
statute. Hoflin, 880 F.2d at 1039. Both the
Government and Defendants therefore agree that another
element of a § 6928(d)(2)(A) charge is that the
defendant knew the waste had the potential to be harmful to
others or to the environment. ECF No. 84-1, PageID # 275; ECF
No. 89, PageID # 433.
to prove a violation of § 6928(d)(2)(A), the Government
must prove beyond a reasonable doubt that: 1) a defendant
knowingly treated, stored, or disposed of hazardous waste; 2)
the defendant knew the material had the potential to be
harmful to others or to the environment; 3) the material was
identified or listed by the United States Environmental
Protection Agency as hazardous waste pursuant to RCRA; and 4)
the defendant acted without a permit.
42 U.S.C. § 6912, the Administrator of the Environmental
Protection Agency is authorized to prescribe regulations to
implement RCRA, and also has the job of identifying and
listing hazardous wastes. The Environmental Protection Agency
has accordingly issued regulations set forth in Title 40,
Part 261 of the Code of Federal Regulations.
federal Government may authorize a state to administer and
enforce RCRA through the state's hazardous waste
program. See 42 U.S.C. § 6926. Hawaii received
federal approval to administer and enforce the state's
hazardous waste management plan on November 1, 2001.
See 66 Fed. Reg. 55115-01, 2001 WL 1337427. The
regulations for Hawaii's hazardous waste management
plan are set forth in Title 11 of the Hawaii Administrative
Rules, Chapters 260 to 280.
One of
the main issues in this case is whether the seized fireworks
qualify as "hazardous waste" for purposes of
RCRA, which defines "hazardous waste" as:
a solid waste, or combination of solid wastes, which because
of its quantity, concentration, or physical, chemical, or
infectious characteristics may--
(A) cause, or significantly contribute to an increase in
mortality or an increase in serious irreversible, or
incapacitating reversible, illness; or
(B) pose a substantial present or potential hazard to human
health or the environment when improperly treated, stored,
transported, or disposed of, or otherwise managed.
term "solid waste" is further defined by RCRA as
"[S]olid waste" means any garbage, refuse, sludge
from a waste treatment plant, water supply treatment plant,
or air pollution control facility and other discarded
material, including solid, liquid, semisolid, or contained
gaseous material resulting from industrial, commercial,
mining, and agricultural operations, and from community
activities, but does not include solid or dissolved material
in domestic sewage, or solid or dissolved materials in
irrigation return flows or industrial discharges which are
point sources subject to permits under section 1342 of Title
33, or source, special nuclear, or byproduct material as
defined by the Atomic Energy Act of 1954, as amended (68
Stat. 923) [42 U.S.C.A. § 2011 et seq.].
addition to the above statutory language, the Environmental
Protection Agency's regulations define "solid
waste" as any "discarded material" that is
not excluded by other provisions, including any material:
(A) Abandoned, as explained in paragraph (b) of this section;
(B) Recycled, as explained in paragraph (c) of this section;
(C) Considered inherently waste-like, as explained in
paragraph (d) of this section; or
(D) A military munition identified as a solid waste in §
40 C.F.R. § 261.2. Hawaii's Administrative Rules
define "solid waste" similarly. See Haw.
Admin. R. 11-261-2.
In 40
C.F.R. § 261.20 of the RCRA regulations, a "solid
waste" (as defined in 40 C.F.R. § 261.2) is a
"hazardous waste" when it is not excluded by the
regulations and exhibits any of the following
characteristics: 1) ignitability (40 C.F.R. § 261.21);
2) corrosivity (40 C.F.R. § 261.22); 3) reactivity (40
C.F.R. § 261.23); or toxicity (40 C.F.R. § 261.24).
Hawaii's Administrative Rules define when a
"solid waste" is a "hazardous waste"
similarly. See Haw. Admin. R. 11-261-3.
parties have focused on the characteristic of reactivity. A
"solid waste" demonstrates reactivity when
"[i]t is capable of detonation or explosive reaction if
it is subjected to a strong initiating source or if heated
under confinement, " or "[i]t is readily capable
of detonation or explosive decomposition or reaction at
standard temperature and pressure." 40 C.F.R. §
261.23(a)(6) and (7). Hawaii's administrative rules
define "reactivity" similarly, stating that a
solid waste exhibits the characteristic of reactivity when
"[i]t is capable of detonation or explosive reaction if
it is subjected to a strong initiating source or if heated
under confinement, " or "[i]t is readily capable
of detonation or explosive decomposition or reaction at
standard temperature and pressure." Hawaii
Administrative Rule § 11-261-23.
Hazardous Waste Permit Program is managed by the Department
of Health, Solid & Hazardous Waste Branch, and is set
forth in Chapter 270 of the Hawaii Administrative Rules.
See Declaration of Gracelda M. Simmons ¶ 2, ECF
No. 128-1, PageID # 1447. Section 11-270-1(b) of the Hawaii
Administrative Rules states, "Treatment, storage, or
disposal of hazardous waste by any person who has not applied
for or received a hazardous waste management permit is
is an exclusion from Hawaii's permitting requirements
when there is
[a]n immediate threat to human health, public safety,
property, or the environment from the known or suspected
presence of military munitions, other explosive material, or
an explosive device, as determined by an explosive[s] or
munitions emergency response specialist as defined in section
Haw. Admin. R. § 11-270-1(c)(3)(i)(D). Section 11-260-10
defines "explosives or munitions emergency response
specialist" as
an individual trained in chemical or conventional munitions
or explosives handling, transportation, render-safe
procedures, or destruction techniques. Explosives or
munitions emergency response specialist include U.S.
Department of Defense (DOD) emergency explosive ordnance
disposal (EOD), technical escort unit (TEU), and
DOD-certified civilian or contractor personnel, and other
federal, state, or local government, or civilian personnel
similarly trained in explosives or munitions emergency
When no
"explosives or munitions emergency response
specialist" determines that explosive material poses an
immediate threat to human health, public safety, property, or
the environment, the Hawaii Administrative Rules do not
automatically require a regular Hazardous Waste Permit.
Instead, an Emergency Hazardous Waste Permit (sometimes
called a "Temporary Emergency Permit") is
available under section 11-270-61 of the Hawaii
Administrative Rules when there is "an imminent and
substantial endangerment to human health or the
December 10, 2007, U.S. Customs and Border Protection, an
agency within the Department of Homeland Security, seized 11
pallets of fireworks (the "Lindsey Fireworks").
See Superseding Indictment ¶ 11, ECF No. 43,
PageID # 142. On January 13, 2010, Homeland Security
Investigations seized 17 pallets of other fireworks (the
"Chang Fireworks"). See id.; ECF No.
131-2, PageID # 1517 (Custody Receipt for Seized Property).
The Chang Fireworks had been originally detained on December
18, 2009, by U.S. Customs and Border Protection. See
ECF No. 131-1, PageID # 1515; ¶ 1, ECF No. 131-11,
PageID # 1594. The Chang Fireworks were assigned seizure
number 2010-3205-000012-01. Decl. of Lisa Leung ¶ 4, ECF
No. 131-11, PageID # 1594. Defendant Donaldson Enterprises,
Inc. ("DEI"), sometimes incorrectly identified
the Chang Fireworks as
2010-3205-000013-01. See
E-mail from Carlton Finley to Darin Yamamoto (Dec. 13, 2010)
(stating that the number was wrong and should be corrected).
Inc., had a nationwide contract with the U.S. Government to
transport, store, and dispose of materials seized by the
Department of Homeland Security. See Superseding
Indictment ¶ 11, ECF No. 43, PageID # 141-42. On January
13, 2010, through Lisa Leung, a Fines, Penalties, and
Forfeitures Officer with U.S. Customs and Border Protection,
issued a Disposition Order to "consign the seized
fireworks to the VSE contractor." ECF No. 131-3, PageID
# 1519; Leung Decl. ¶ 5, ECF No. 131-11, PageID # 1594.
According to Leung, her job includes tracking seized property
through forfeiture proceedings, issuing notices of seizures
to interested parties, and managing and directing the process
of handling seized property. See Leung Decl. ¶
2, PageID # 1594.
DEI is
a Hawaii corporation that provides unexploded ordnance
disposal and environmental services. See Superseding
Indictment ¶ 12, ECF No. 43, PageID # 142. Its director
of operations is Defendant Charles Donaldson. Id.
¶ 13. Defendant Carlton Finley is DEI's project
manager responsible for DEI's obligations under
VSE's contract with DEI. See Superseding
Indictment ¶ 14.
March 18, 2010, DEI contracted with VSE to transport, store,
and dispose of the Lindsey Fireworks and the Chang Fireworks.
See Superseding Indictment ¶ 15, ECF No. 43,
PageID # 142; ECF No. 131-4 (copy of contract). As accepted
by Michelle Eugenio, DEI's treasurer, the ...
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Hellow guys, I'm back, Happy New Years for ya! And let's start the year with a useless news for who doesn't have a 3DS or a DS Read bellow 🙂 Continue reading "Chocolate Box for your Nintendo DS/3DS's Games"
Tag: 3ds
This year's 46th issue of Shueisha's Weekly Young Jump magazine is announcing that Terrformars manga is getting a new game for Nintendo 3DS on the next spring. Continue reading "New Game of the Manga Terra Formars is Coming to 3DS"
A new video advertisement for the New 3DS, released by Nintendo Japan, show us a little better the new features of the New 3DS. Continue reading "New Commercial of the New 3DS Showing off its New Features"
The Legend of Legacy is a new j-RPG being developed by FuRyu, the creators of the SaGa series, Exstetra, Unchained Blades and others nice games. The game is coming for 3DS in collaboration with V-Jump magazine and the release date is January 22 in Japan for 5,908 yens, approximately US$ 60. The game will have turn-based combats, looks similar to Continue reading "New JRPG "The Legend of Legacy" for 3DS from the SaGa Team"
The Great Ace Attorney (Dai Gyakuten Saiban: Ryuunosuke Naruhodo no Bouken), the next game from the "Phoenix Wright" Series will take the player back to Meiji Era Japan and will have "Sir Arthur Conan Doyle"'s legendary fictional detective Sherlock Holmes with Iris Watson at his side, an Continue reading "Next Ace Attorney Game "The Great Ace Attorney" will takes us back to Meiji Era Japan with Sherlock Holmes"
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Any list about the "greatest" at anything is sure to spark a debate. Outside of LeBron James vs. Michael Jordan, there's no fiercer "greatest ever" debate in sports than that of Tiger Woods or Jack Nicklaus. The 2 PGA Tour legends with a combined 32 majors were the clear choices for No. 1 and No. 2 on this list of the greatest golfers, but in which order? Brace yourself for a trip deep into the PGA's history.
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The future of tax is coming. In many businesses tax remains the "forgotten child" of the finance function; often lagging behind other parts of the business in terms of technology investment. With the introduction of MTD and a wealth of Making Tax Digital software solutions available, we explore how HMRC is not only moving to a digital future themselves, but is kick-starting the whole industry on a long and prosperous tech journey with them.
Originally published for the Tax Transformation Summit 2018.
HMRC has ambitions to become "one of the most digitally advanced tax administrations in the world". They have some ground to cover; some firms still submit paper returns, whereas in countries such as Spain SAF-T is underway, where submissions happen electronically every four days. HMRC's ambition is admirable, and it's our view at Arkk Solutions that Making Tax Digital should be embraced and treated as an important strategic initiative.Traditionally, many firms have a short-termist stance when it comes to investing in Tax technology. It's easy to see why – not much has existed in the market, and the usability of these products is questionable at best. Accountants are good at accounting but not necessarily good at building software. Often, the outcome is a point solution with an expiry date.
That's why, when Making Tax Digital for VAT creeps up on the agenda, API-only tools can seem an acceptable solution for many. They functionally work, are incredibly cheap, and sit on the end of the existing, manual process.
This might work initially, but as this market develops at pace, we are starting to see many organisations appreciate the benefit of taking a long-term view. Bridging software leaves gaps which will not tackle the changes we are set to see in the tax function of the future.
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What Are You—Media Person or Tech Person?
Are you a tech person or a media person? Rob Beeler is happy to be both, and says ops should embrace the responsibility they have to support quality media.Read More
Are you a tech person or a media person? Rob Beeler is happy to be both, and says ops should embrace the responsibility they have to support quality media.Read More
Is LinkedIn the New Facebook (for Content Distribution)? LinkedIn is reportedly firming up relationships with publishers in the U.S. and the U.K., according to Digiday, and a growing number of publishers are getting more serious about using LinkedIn as a content distribution channel. (They're not just B2B publishers, either). LinkedIn…Read More
Real talk for a second: Whenever you hear me griping about the myriad things Facebook doesn't do well, either for users or for its publisher partners, I'm probably subconsciously trying to convince myself of something. I'm a social media addict, and I suppose I have been ever since the Geocities…Read More
Back on Jan. 11, Facebook announced it was switching up its news feed algorithm to prioritize original posts from users, and to de-prioritize links to news stories. Following uproar and confusion from publishers and marketers about how much good or ill that will do to users, Facebook came back this…Read More
There's a report from DCN (Digital Content Next) that's been making the rounds in recent weeks, breaking down what the whole process of moving content into distribution platforms has meant for publisher revenue so far. Pulling data and commentary from 17 of DCN's publisher members during the first half of…Read More
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Interviewing Ernesto Schmitt of Zeebox for the previous Screen Grab, I was struck by what he said about media buying becoming more complicated, because of the need to identify the multiple user journeys on the second screen. What made me pause was not the idea of multiple user journeys themselves,…Read More
Olympic fever seems to have hit every person in the world except for me. As an avid skier, I love the Winter Games, but I just can't get into all this running and shot-putting and pole-vaulting that the summer games offer. Where's the luge, dude? Why would I want to…Read More
Ernesto Schmitt is co-founder and CEO of social TV app company Zeebox. It launched in October last year and in January reported it had 250,000 users. In the same month it sold a 10% stake to BSkyB.The first thing I asked Schmitt was what he thinks are the key opportunities…Read More
The world of Social Media advertising continues to evolve at an unprecedented pace, which, at times, can leave brands confused and unsure of the best strategy for them. Given the recent announcement about the launch of Facebook's RTB Exchange, a wealth of opportunities have been opened up to brands. However,…Read More


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The "Tag" Toque
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An uber soft hand knit toque. This toque includes an ever so fluffy colourful 6″ faux fur pom pom you will wear everywhere!
Toque Colour: Black
Pom Pom: Pink or Blue
Fit: one size fits most
Size (laying flat): 7.5″ wide x 9.5″ tall – excluding pom pom
Material: 85% acrylic, 15% nylon, faux fur
Washing: hand wash cold, lay flat to dry, try not to fully submerged pom pom, spot wash and refluff with blow dryer on low setting
Do you want to make your own? Head over to the blog for the free knit pattern! 🙂
Additional information
|.1 kg
|30 × 25 × 5 cm
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