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Natural products biosynthesis often requires the action of multiple CYPs (cytochromes P450), whose ability to introduce oxygen, increasing solubility, is critical for imparting biological activity. In previous investigations of rice diterpenoid biosynthesis, we characterized CYPs that catalyse alternative hydroxylation of ent-sandaracopimaradiene, the precursor to the rice oryzalexin antibiotic phytoalexins. In particular, CYP76M5, CYP76M6 and CYP76M8 were all shown to carry out C-7β hydroxylation, whereas CYP701A8 catalyses C-3α hydroxylation, with oxy groups found at both positions in oryzalexins A–D, suggesting that these may act consecutively in oryzalexin biosynthesis. In the present paper, we report that, although CYP701A8 only poorly reacts with 7β-hydroxy-ent-sandaracopimaradiene, CYP76M6 and CYP76M8 readily react with 3α-hydroxy-ent-sandaracopimaradiene. Notably, their activity yields distinct products, resulting from hydroxylation at C-9β by CYP76M6 or C-7β by CYP76M8, on different sides of the core tricyclic ring structure. Thus CYP76M6 and CYP76M8 have distinct non-redundant roles in orzyalexin biosynthesis. Moreover, the resulting 3α,7β- and 3α,9β-diols correspond to oryzalexins D and E respectively. Accordingly, the results of the present study complete the functional identification of the biosynthetic pathway underlying the production of these bioactive phytoalexins. In addition, the altered regiochemistry catalysed by CYP76M6 following C-3α hydroxylation has some implications for its active-site configuration, offering further molecular insight.
CYPs (cytochromes P450) (EC 1.14.13.x) serve important roles not only in xenobiotic metabolism/detoxification, but also in natural product biosynthesis. In particular, these haem-thiolate mono-oxygenases are often responsible for the insertion of oxygen into unreactive carbon–hydrogen bonds of hydrophobic intermediates. The resulting hydroxy groups then serve to both increase solubility and provide hydrogen-bonding potential that increases the specificity (i.e. binding) of the resulting natural product to its molecular target. Often multiple CYPs are required for the production of bioactive natural compounds and, given their sometimes broad substrate specificity, it can be difficult to determine whether there is a defined or preferred order of reactions, and what effect initial hydroxylation reactions may have on subsequently catalysed reactions. In addition, the homology among CYP paralogues, which can be inferred easily from their grouping into related numbered families and even more closely related lettered subfamilies, and that often exhibit similar biochemical activity, further complicates such analysis, and raises the potential of redundancy, e.g. in plants [1].
Plants produce a vast array of natural products that serve various ecological roles, such as in defence against microbial pathogens. In many cases, the biosynthesis of these compounds is induced by infection with the microbial pathogens against which they exhibit antibiotic activity, leading to their designation as phytoalexins [2]. In the important cereal crop plant rice (Os; Oryza sativa), these phytoalexins are largely members of the LRD (labdane-related diterpenoid) superfamily [3].
LRD biosynthesis is characterized by its initiation via sequential cyclization reactions [4]. First, (bi)cyclization of the general diterpenoid precursor GGPP [(E,E,E)-geranylgeranyl diphosphate], generally to the eponymous labdadienyl/CPP (copalyl diphosphate), catalysed by class II diterpene cyclases (EC 5.5.1.x) then termed CPSs (CPP synthases). This is followed by further cyclization, generally to an olefin, catalysed by class I (di)terpene synthases (EC 4.2.3.x) often termed KSL (ent-kaurene synthase-like) for their derivation from the ancestral enzymatic family member found in all plants for gibberellin phytohormone metabolism. The multicyclic olefins resulting from these initial cyclization reactions are quite hydrophobic, e.g. their logP (partition coefficient) is generally ≥8.5. Accordingly, these are invariably transformed further by the addition of oxygen, generally catalysed by CYPs, to increase their solubility, enabling exertion of biological activity. For example, it has been shown that gibberellin biosynthesis depends on ent-kaurene oxidases from the CYP701A subfamily and subsequently acting ent-kaurenoic acid oxidases from the CYP88A subfamily [5,6]. Similarly, conifer resin acid biosynthesis is dependent on CYP720B subfamily members that act on LRD precursors [7,8].
In the course of functionally characterizing the rice CPS and KSL, we found that consecutively acting OsCPS and OsKSL are close together in the rice genome, along with CYPs [9,10]. Functional characterization of these CYPs revealed their ability to oxygenate diterpene olefins produced by the co-clustered OsCPS and OsKSL [11–13], as well as those resulting from the action of OsKSL found elsewhere in the rice genome [14], with oxy groups found at the targeted positions in the derived natural products. In addition, similar activity has been demonstrated for CYP701A8, a paralogue of the ent-kaurene oxidase required for gibberellin metabolism, that is found elsewhere in the rice genome [15].
Given the alternative positions targeted by the functionally characterized CYPs, it is unclear how even diols, which seem to be the minimal degree of modification required to form bioactive LRDs, are generated. For example, although previous isolation of 3α-hydroxy-ent-sandaracopimaradiene and re-feeding in rice plant cell-free assays indicated its intermediacy in oryzalexin D and E biosynthesis [16], and we have characterized CYP that will catalyse such C-3α (CYP701A8) [15], as well as C-7β (CYP76M5, CYP76M6 and CYP76M8) [14], hydroxylation of the olefin precursor ent-sandaracopimaradiene, their ability to act consecutively remains uncertain (Figure 1). Moreover, the known CYP-catalysed reactions are insufficient to generate the observed range of LRDs, e.g. formation of orzyalexin E requires hydroxylation at C-9, but no CYP catalysing such a reaction with ent-sandaracopimaradiene has been found.
Putative roles of rice CYPs in oryzalexin biosynthesis
In the present study, we provide evidence indicating that oryzalexins D and E are generated by initial C-3α hydroxylation catalysed by CYP701A8, which alters the subsequent reaction catalysed by CYP76M6, such that it hydroxylates C-9β instead of C-7β, generating oryzalexin E (i.e. the 3α,9β-diol), whereas CYP76M8 continues to hydroxylate C-7β, generating oryzalexin D instead (i.e. the 3α,7β-diol). In addition, we examine the implications of the observed change in regiochemistry for configuration of the CYP76M6 active site.
Unless otherwise noted, chemicals were purchased from Fisher Scientific, and molecular biology reagents were from Invitrogen. The logP values presented in the present study were obtained from the SciFinder database (American Chemical Society). GC was performed with a Varian 3900 GC with Saturn 2100 ion trap MS in electron ionization (70 eV) mode for GC–MS analyses, or with an Agilent 6890N GC with FID (flame-ionization detection) for GC–FID analyses. Samples (1 μl) were injected in splitless mode at 50°C and, after holding for 3 min at 50°C, the oven temperature was increased at a rate of 14°C/min to 300°C, where it was held for an additional 3 min. MS data from 90 to 600 m/z were collected starting at 12 min after injection until the end of the run.
Recombinant constructs
The CYP701A8 and CYP76M5–CYP76M8 genes used in the present study are the synthetic fully-codon-optimized and N-terminally modified constructs described recently [14,15]. However, to optimize metabolic flux, a new CYP expression vector was created by insertion of a DEST cassette into the first MCS (multiple cloning site) of pETDuet (Novagen), using the NcoI and NotI restriction sites, as described previously [17]. To enable dual CYP expression, CYP701A8 was inserted into the second MCS of pETDuet/DEST using the NdeI and XhoI restriction sites, creating a pETDuet/DEST/CYP701A8 vector. Alternatively, CYP76M6 or CYP76M8 were inserted into the second MCS of pETDuet/DEST using the NdeI and XhoI restriction sites. In all three of these cases, a rice CPR (CYP reductase) (OsCPR1) was inserted via directional recombination into the DEST cassette, creating pETDuet/DEST::OsCPR1/(CYP701A8, CYP76M6 or CYP76M8) constructs, to enable co-expression for feeding studies. CYP76M5–CYP76M8 were transferred into the pETDuet/DEST/CYP701A8 vector for recombinant expression via directional recombination into the DEST cassette, creating pETDuet/DEST::CYP76M5–CYP76M8)/CYP701A8 constructs for balanced CYP expression. To enable co-production of the CYP olefin substrates investigated in the present study, OsKSL7 and OsKSL10 (producing ent-cassadiene or ent-sandaracopimaradiene respectively) were inserted via directional recombination into the DEST cassette of the pCDFDuet/DEST/OsCPR1 vector described previously [11], creating pCDFDuet/DEST::(OsKSL7 or OsKSL10)/OsCPR1 constructs.
Recombinant expression
All recombinant expression was carried out in the C41 Overexpress strain of Escherichia coli (Lucigen), using the modular diterpene metabolic engineering concept we have described previously [17], and the vectors described above. Specifically, we co-expressed CYP701A8 with each of CYP76M5–CYP76M8, using the pETDuet/DEST::CYP76M(x)/CYP701A8 vectors, which were co-transformed with the compatible pGGeC, to enable production of the upstream GGPP and, subsequently, ent-CPP, as well as the relevant pCDFDuet/DEST::(OsKSL7 or OsKSL10)/OsCPR1 vector, to enable production of the immediate ent-cassadiene or ent-sandaraco pimaradiene respectively, along with the reductase needed for CYP activity. These recombinant strains were grown, typically in 50 ml cultures in TB (12 g yeast extract, 24 g casein hydrolysate and 4 ml glycerol per litre) liquid medium at 37°C to D600 ~0.6, shifted to 16°C for 1 h before induction with 1 mM IPTG, and supplementation with 5 mg/l riboflavin, and 75 mg/l δ-aminolaevulinic acid, then grown for an additional 72 h. The resulting diterpenoids were extracted (from the medium and cells) with an equal volume of hexane, and analysed by GC–MS. In every case, the hydroxylated diterpenoid products were observed as reported previously [14,15], with diols detected in certain cases, as described below.
Diterpenoid production
To obtain the observed dual CYP product in sufficient amounts for NMR analysis, flux was increased into isoprenoid metabolism and the recombinant culture volumes also simply scaled up. To increase flux, the recombinant strain was transformed further with the pMBI vector encoding the bottom half of the mevalonate dependent isoprenoid precursor pathway [18]. This is compatible with those mentioned above, and feeding 10 mM mevalonolactone to the resulting recombinant cultures greatly increases (di)terpenoid yield, as previously described [19]. The dihydroxylated diterpenoid was extracted and purified much as previously described [11], using GC–MS to detect the presence of the desired compound (i.e. in various fractions). Briefly, 20×1 litre cultures were extracted twice with equal volumes of hexanes, and the combined organic extract then dried by rotary evaporation. The resulting residue was dissolved in 10 ml of hexanes, which was then extracted thrice with equal volumes of acetonitrile, and the combined acetonitrile extract dried under a gentle stream of N2. This was resuspended in 5 ml of hexanes, which was then fractionated via flash chromatography over a 4-g silica column using a Reveleris system (Grace) with UV detection and a hexanes to acetone gradient as the mobile phase. The dihydroxylated diterpenoid eluted in the 20% acetone fraction, which was dried under N2 and resuspended in 1 ml of acetonitrile. Further purification was carried out using an Agilent 1200 series HPLC instrument equipped with autosampler, fraction collector and diode array UV detection, over a ZORBAX Eclipse XDB-C8 column (4.6 mm×150 mm; 5 μm) at a 0.5 ml/min flow rate. The column was pre-equilibrated with 50% acetonitrile/ddH2O (double-distilled water), sample loaded, then the column washed with 50% acetonitrile/ddH2O (0–2 min), and eluted with 50–100% acetonitrile (2–7 min), followed by a 100% acetonitrile wash (7–10 min), leading to purification of a final estimated ~1 mg of the dihyroxylated diterpenoid.
Chemical structure identification
NMR spectra were recorded at 25°C on a Bruker Avance 700 spectrometer equipped with a 5-mm HCN cryogenic probe for detection of 1H and 13C. The purified compound was dried under a gentle stream of N2, and then dissolved in 0.5 ml deuterated chloroform (CDCl3; Sigma–Aldrich), with this evaporation-resuspension process repeated once more to completely remove the protonated acetonitrile solvent. This sample was placed in a NMR microtube (Shigemi) for analysis, and chemical shifts were referenced using known chloroform-d (13C 77.23, 1H 7.24 p.p.m.) signals offset from TMS (tetramethylsilane). Structural analysis was performed using 1D 1H, and 2D DQF-COSY (double-quantum-filtered correlation spectroscopy), HSQC (heteronuclear single-quantum coherence), HMQC (heteronuclear multiple-quantum coherence), HMBC (heteronuclear multiple bond correlation) and NOESY experiment spectra acquired at 700 MHz, and 1D 13C and DEPT-135 (distortionless enhancement by polarization transfer 135) spectra (174 MHz) using standard experiments from the Bruker TopSpin v1.4 software. Correlations from the HMBC spectra were used to propose a partial structure, whereas connections between protonated carbons were obtained from DQF-COSY data to complete the partial structure and assign proton chemical shifts. The structure was further verified using HSQC and DEPT-135 to confirm the chemical shift assignments.
Feeding studies
Substrates (ent-sandaracopimaradiene, 3α-hydroxy-ent-sandaracopimaradiene and 7β-hydroxy-ent-sandaracopimaradiene) were obtained via extraction and purification from metabolically engineered strains of E. coli as described recently [14,15]. Feeding studies with CYP701A8, CYP76M6 and CYP76M8 were then carried out as follows. The CYPs were co-expressed with OsCPR1, using the pETDuet/DEST::OsCPR1/(CYP701A8, CYP76M6 or CYP76M8) constructs described above, in the C41 Overexpress strain of E. coli. These recombinant cultures (50 ml) were supplemented with the addition of 1 mM thiamine, 5 mg/l riboflavin and 75 mg/l δ-aminolaevulinic acid at the time of induction, then grown for 24 at 16°C. The relevant substrates were then fed to a final concentration of 50 μM (added from 100× stocks in methanol). The cultures were allowed to ferment for an additional 48 h, and then extracted with an equal volume of hexanes, following which the resulting products were analysed by GC–MS. All studies were run in duplicate.
Substrate competition assays
For in vitro assays, cells co-expressing CYP76M6 or CYP76M8 with OsCPR1 were harvested 72 h after induction via centrifugation (15 min at 5000 g), with the cells then resuspended in one-tenth of the culture volume of buffer A (0.1 M Tris/HCl, pH 7.2, 20% glycerol and 0.5 mM EDTA) and passed through a French press homogenizer (Emulsiflex-C5; Avestin) three times at 15000 psi (1 psi=6.9 kPa). The resulting lysates were clarified via centrifugation (15 min at 15000 g), and the supernatant used for substrate competition assays. These contained 50 μM 3α-hydroxy-ent-sandaracopimaradiene, along with various concentration of ent-sandaracopimaradiene, ranging 5–80 μM, and were carried out in 0.5 ml volumes, containing 100 μl CYP preparation, with reactions initiated by the addition of NADPH (to 0.4 mM). After 10 min of incubations at 30°C, the reactions were terminated by the addition of 50 μl of 1 M HCl, then extracted thrice with an equal volume of hexanes. In all cases, the enzymatic products were analysed by GC–MS. The relative specificity constant (kcat/Km) for ent-sandaracopimaradiene compared with 3α-hydroxy-ent-sandaracopimaradiene was calculated from the resulting data, as described previously [20], with the observed R2>0.9 in each case.
Bioinformatic analysis of subcellular localization
The potential differential subcellular localization of CYP701A8 compared with CYP76M6 and CYP76M8 was investigated by bioinformatics analysis using four distinct algorithms: CholoroP (http://www.cbs.dtu.dk/services/ChloroP/), Predotar (http://urgi.versailles.inra.fr/predotar/predotar.html), PCLA (http://www.andrewschein.com/cgi-bin/pclr/pclr.cgi) and iPSORT (http://ipsort.hgc.jp/). As controls, the immediately upstream OsKSL7 and OsKSL10 were included, as was the CYP701A3 family member shown previously to be in the plastid [21]. The corresponding sequences are CYP76M6 (NP_001047192), CYP76M8 (NP_001047184), CYP701A8 (NP_001057905), CYP701A3 (NP_197962), OsKSL7 (NP_001047186) and OsKSL10 (NP_001066799).
Probing consecutive hydroxylation reactions via metabolic engineering
The potential sequential activity of CYP701A8 with CYP76M5- CYP76M8 in oryzalexins A–F and/or phytocassanes A–E biosynthesis (e.g. Figure 1) was investigated via a synthetic biology approach. In particular, we used codon-optimized genes for these CYP to enable functional expression in E. coli [14,15]. Using a modular metabolic engineering system developed previously [17], CYP701A8 was then co-expressed with each of CYP76M5–CYP76M8, along with the requisite CPR, and biosynthetic enzymes for production of the corresponding LRD olefin precursor (i.e. either ent-cassadiene or ent-sandaracopimaradiene). Although only trace amounts of any dihydroxylated product were observed, in the course of these initial studies we noted that the amount of hydroxylated product was dependent on which plasmid the relevant CYP was cloned into, with higher yields observed when this was the pET-derived pDEST14 vector as opposed to pCDFDuet. Accordingly, we incorporated use of the pETDuet vector to enable expression of dual CYPs, subcloning the invariable CYP701A8 into the second MCS and a DEST cassette into the first MCS to enable modular assembly of constructs for balanced co-expression of CYP701A8 with each of CYP76M5–CYP76M8.
Using these improved constructs, it was possible to observe reasonable production of both of the expected hydroxylated products from CYP701A8 and any of CYP76M5–CYP76M8 with either ent-cassadiene or ent-sandaracopimaradiene in the appropriately engineered E. coli. Moreover, although no dihydroxylated products were observed with ent-cassadiene, such products were observed with ent-sandaracopimaradiene. In particular, products were observed resulting from the activity of CYP701A8 with either CYP76M6 or CYP76M8 (Figure 2). Notably, despite the fact that CYP76M6 and CYP76M8 both catalyse C-7β hydroxylation with ent-sandaracopimaradiene [14], the dihydroxylated products were not the same, indicating a change in regiochemistry for one of the hydroxylation reactions. Comparison of these products with an authentic standard of oryzalexin E (a gift from Professor Robert M. Coates emeritus, Department of Chemistry, University of Illinois, Urbana-Champaign, IL, U.S.A.), demonstrated that CYP701A8 and CYP76M6 together produce this 3α,9β-diol, indicating a change in regiochemistry, from C-7 to C-9, of the CYP76M6 catalysed hydroxylation reaction following C-3α hydroxylation of ent-sandaracopimaradiene by CYP701A8.
Production of diterpenoid diols by co-expression of CYP701A8 and CYP76M6 or CYP76M8
Identification of CYP701A8 and CYP76M8 product as oryzalexin D
To enable production of sufficient amounts of the unknown dihydroxylated diterpenoid product of CYP701A8 and CYP76M8 acting on ent-sandaracopimaradiene for structural analysis by NMR, we increased flux into terpenoid metabolism in the metabolically engineered E. coli. Specifically, the endogenous methylerythritol phosphate isoprenoid precursor supply pathway was supplemented by incorporation of the bottom 'half' of the mevalonate-dependent pathway, along with feeding of mevalonate, which significantly increases yield, as described previously [19]. This enabled production of approximately 1 mg of protein from not unreasonable quantities of recombinant culture (20 litres), with the resulting diterpenoid extracted and purified by flash chromatography and HPLC. From the subsequent NMR analysis (Figure 3 and Table 1), it was possible to assign the position of the two hydroxy groups to C-3α and C-7β, with the resulting diol then corresponding to the oryzalexin D expected from the originally observed olefin hydroxylation reactions (see Figure 1).
Structural analysis of oryzalexin D by NMR.
|δC (p.p.m.) .
|δH (p.p.m.), multiplicity .
|J (Hz) .
|1.212(dt), 1.723(dt)
|3.2, 13.2
|1.546(m), 1.658(m)
|4.8, 11.6
|1.589(m), 1.788(m)
|1.476(m), 1.610(m)
|1.369(dt), 1.467(m)
|3.1, 12.5
|10.3, 17.5
|4.912(d), 4.923(d)
|10.3, 17.5
|δC (p.p.m.) .
|δH (p.p.m.), multiplicity .
|J (Hz) .
|1.212(dt), 1.723(dt)
|3.2, 13.2
|1.546(m), 1.658(m)
|4.8, 11.6
|1.589(m), 1.788(m)
|1.476(m), 1.610(m)
|1.369(dt), 1.467(m)
|3.1, 12.5
|10.3, 17.5
|4.912(d), 4.923(d)
|10.3, 17.5
Defining CYP reaction order
Although the observed change in CYP76M6 catalytic regiochemistry presumably results from action on 3α-hydroxy-ent-sandaracopimaradiene rather than ent-sandaracopimaradiene, the order of reactions in oryzalexin D biosynthesis was uncertain. Feeding experiments demonstrated the expected conversion of 3α-hydroxy-ent-sandaracopimaradiene to oryzalexin E by CYP76M6. Similarly, CYP76M8 readily converts this to oryzalexin D. By contrast, CYP701A8 only poorly reacts with 7β-hydroxy-ent-sandaracopimaradiene, producing only trace amounts of oryzalexin D (Figure 4).
Substrate feeding indicates reaction order
The increased solubility of 3α-hydroxy-ent-sandaracopimaradiene relative to ent-sandaracopimaradiene complicated comparison of the kinetic constants obtained in reactions with these as substrates. Thus we turned to direct comparison in substrate competition assays [20]. In particular, running reactions with the CYP76M6 and CYP76M8 that are active with both, in the presence of a constant amount of 3α-hydroxy-entsandaracopimaradiene (50 μM) and varying levels of ent-sandaracopimaradiene. These studies revealed that CYP76M6 exhibits a clear preference for 3α-hydroxy-ent-sandaracopimaradiene compared with ent-sandaracopimaradiene, with a relative catalytic efficiency for ent-sandaracopimaradiene relative to 3α-hydroxy-ent-sandaracopimaradiene of 0.03 (i.e. ~33-fold increase in kcat/Km). By contrast, CYP76M8 seems to exhibit a slight preference for ent-sandaracopimaradiene, with a relative catalytic efficiency of 3.3 for this compared with 3α-hydroxy-ent-sandaracopimaradiene.
Although this kinetic analysis leaves some uncertainty in preferred reaction order, at least for biosynthesis of the 3α,7β-diol, 3α-hydroxy-ent-sandaracopimaradiene is readily detectable in planta [15], whereas 7β-hydroxy-ent-sandaracopimaradiene was not detected previously [14]. In the present study, through pre-fractionation, we were able to detect 7β-hydroxy-ent-sandaracopimaradiene, but it is only present at ~1% of the amount of 3α-hydroxy-ent-sandaracopimaradiene. These results then indicate that oryzalexin biosynthesis proceeds via initial hydroxylation of ent-sandaracopimaradiene by CYP701A8 at the C-3α position, followed by alternative hydroxylation reactions catalysed by CYP76M6 or CYP76M8 at the C-9β or C-7β positions, producing oryzalexins E and D respectively (Figure 5).
Proposed biosynthetic pathway for oryzalexins D and E
Plant diterpenoid, as well as monoterpenoid, biosynthesis is initiated in plastids, whereas CYPs are almost invariably localized to the endoplasmic reticulum [22]. For example, the limonene (monoterpene) hydroxylases (CYP71B subfamily members) from Mentha species [23] and the CYP720B subfamily members involved in conifer diterpenoid resin acid biosynthesis [24]. However, it has been reported previously that CYP701A3, the ent-kaurene oxidase required for gibberellin phytohormone biosynthesis in Arabidopsis thaliana [25,26], is localized on the plastid membrane, providing it ready access to its ent-kaurene substrate [21]. Hypothesizing that similar plastid localization and increased substrate access for the rice CYP701A8 might help explain the observed higher levels of 3α-hydroxy-ent-sandaracopimaradiene relative to 7β-hydroxy-ent-sandaracopimaradiene in planta, we carried out bioinformatics analysis to predict subcellular localization. Strikingly, CYP701A8 is predicted to be in the plastid by two of the four utilized algorithms, just as is CYP701A3, whereas CYP76M6 and CYP76M8 are not (Table 2). Given the clear plastid localization of the immediately upstream OsKSL10 (as well as OsKSL7), such co-localization of CYP701A8 may provide preferential access to the diterpene olefins, leading to the observed accumulation of its product, relative to that of CYP76M6 and CYP76M8, in planta.
The use of CYPs is widespread in natural product biosynthesis, and is especially prevalent with terpenoids, where the production of highly hydrophobic olefin intermediates essentially necessitates the action of these membrane-associated mono-oxygenases. However, we have relatively limited understanding of how these operate, and it can be particularly opaque how multiple CYPs act in common biosynthetic processes. For example, although gibberellin biosynthesis follows a defined CYP reaction order [5,6], as does biosynthesis of the sesquiterpenoid lactone costunolide [27], the reaction order for the multiple relevant CYPs is not entirely clear in biosynthesis of the diterpenoid paclitaxel/taxol [28], as well as the triterpenoid glycyrrhizin [29].
Although the work reported in the present study directly applies to biosynthesis of the rice LRDs, which act as phytoalexins and allelochemicals [30,31], the use of LRDs, at least as phytoalexins, seems to extend beyond rice and be widespread throughout the Poaceae plant family [3]. For example, wheat (Triticum aestivum) contains expanded CPS and KSL gene families, and the transcription of certain members is induced by at least UV irradiation, with the encoded enzymes further exhibiting biochemically diverse function [32–34], much like that of the rice homologues involved in phytoalexin biosynthesis [3]. Moreover, maize (Zea mays) not only has been found to contain at least a CPS whose transcription is induced by fungal infection [35], but also more directly demonstrated to produce LRD phytoalexins, i.e. kauralexins A1–3 and B1–3, which exhibit anti-fungal activity [36]. Thus understanding LRD biosynthesis has broader importance.
Although oxygen can be incorporated during the reactions catalysed by class II diterpene cyclases [37–40] and/or class I diterpene synthases [41–47], LRD biosynthesis generally proceeds through a diterpene olefin intermediate [4]. These are strongly hydrophobic (logP≥8.5), requiring the incorporation of oxygen to increase polarity and solubility. Moreover, although addition of a single oxygen does increase solubility, the resulting compounds resemble cholesterol (e.g. 3α-hydroxy-ent-sandaracopimaradiene), and are then expected to still partition into membranes. Hence it perhaps is not surprising that the bioactive rice LRDs contain a minimum of two oxygen molecules, which reduces their logP to ~5 (e.g. 5.0 and 4.9 for oryzalexins D and E respectively), indicating that these will exhibit significant solubility in natural settings.
Given the invariable use of CYPs to introduce these oxygen molecules, investigation of their consecutive action is then critical. In the present study, we not only provide evidence elucidating the biosynthesis of bioactive LRDs, but also demonstrate flux through dual plant CYP-mediated steps in bacteria engineered to complete such metabolism, further supporting the utility of this synthetic approach to investigating biosynthetic pathways.
We have hypothesized that phytocassane biosynthesis would proceed via early C-3α and C-11α hydroxylation [3], which can be catalysed with the ent-cassadiene olefin precursor by CYP701A8, and CYP76M7 and CYP76M8 respectively [11,14,15]. However, we do not observe production of any dihydroxylated ent-cassadiene in cultures co-expressing CYP701A8 and CYP76M7 (or CYP76M8), despite the presence of the expected C-3α- and C-11α-(mono)hydroxylated-ent-cassadiene, demonstrating that both CYPs are active. This suggests that phytocassane biosynthesis may not proceed via this pair of reactions and/or utilizes other enzymes for this purpose.
On the other hand, the production of dihydroxylated ent-sandaracopimaradiene products, with co-expression of CYP701A8 and CYP76M6 or CYP76M8, does provide some insight into orzyalexin biosynthesis. Interestingly, this extends beyond the potential roles indicated by their activity with the ent-sandaracopimaradiene olefin precursor (Figure 1). In particular, the presence of the C-3α-hydroxy group changes substrate orientation in CYP76M6 such that it targets C-9β instead of C-7β, which is targeted by both CYP76M6 and CYP76M8 with ent-sandaracopimaradiene and, in the case of CYP76M8, also with the derived C-3α-hydroxylated compound. Accordingly, our results clarify the biosynthesis of not only oryzalexin D (i.e. the originally expected 3α,7β-diol), but that of oryzalexin E (the 3α,9β-diol) as well (Figure 3).
The change in regiochemistry of the CYP76M6 catalysed reactions prompted us to examine more closely the underlying alteration in substrate orientation. Although C-7 and C-9 are on opposite 'sides', the common β position/hydrogen target does fall on same 'face' of the middle 'B' ring. Examination of 3D models for the alternative substrates highlights the proximity of these positions, indicating that the catalysed insertion of oxygen occurs via very similar binding modes, with only a slight perturbation in orientation in the CYP76M6 active site imposed by the presence of a C-3α-hydroxyl group. We hypothesize that this is mediated via the presence of a polar group in the CYP76M6 active site that either hydrogen bonds to the hydroxy group, leading to reaction at C-9, or binds a water molecule that then drives the observed alternative regiospecificity for C-7 with ent-sandaracopimaradiene (Figure 6).
Proposed configuration of CYP76M6 active site
We suggest in the present study that oryzalexin biosynthesis proceeds via initial C-3α hydroxylation. This has been suggested by previous isolation of 3α-hydroxy-ent-sandaracopimaradiene and rice plant-derived cell-free assays demonstrating its conversion to oryzalexins D and E [16], along with the relative abundance of this intermediate compared with the alternative 7β-hydroxy-ent-sandaracopimaradiene in planta reported in the present paper. However, although feeding experiments are consistent with this suggestion, kinetic analysis indicates that such a reaction order is not driven by intrinsic substrate preference, at least in the case of orzyalexin D (3α,7β-diol) biosynthesis. Intriguingly, it has been suggested previously that members of the CYP701A subfamily, which generally act as ent-kaurene oxidases in gibberellin phytohormone biosynthesis, are localized to plastids [21], where diterpene olefin intermediates are produced [22]. Bioinformatic analysis of CYP701A8 indicates that this is similarly targeted to the plastid, whereas CYP76M6 and CYP76M8 are not (Table 2). Thus we speculate that initial C-3α hydroxylation by CYP701A8 occurs, at least in part, due to preferential access through its co-localization to the plastid, where ent-sandaracopimaradiene is initially produced. CYP76M6 and CYP76M8 then have only secondary access due to their more typical endoplasmic reticulum localization. Accordingly, these catalyse subsequent alternative hydroxylations, CYP76M6 at C-9β and CYP76M8 at C-7β, yielding oryzalexins E (3α,9β-diol) or D (3α,7β-diol) respectively (Figure 5).
In any case, rather than the previously suggested redundancy [14], it now appears that CYP76M6 and CYP76M8 play separate roles in rice LRD metabolism, particularly orzyalexin biosynthesis, by reacting with distinct sides of the middle 'B' ring in 3α-hydroxy-ent-sandaracopimaradiene. Moreover, our results then elucidate the complete biosynthetic pathways leading to these bioactive phytoalexins, adding to the rather few plant natural products for which such knowledge has been accumulated.
copalyl diphosphate
CYP reductase
CPP synthase
cytochrome P450
distortionless enhancement by polarization transfer 135
double-quantum-filtered correlation spectroscopy
flame-ionization detection
(E,E,E)-geranylgeranyl diphosphate
heteronuclear multiple bond correlation
heteronuclear single-quantum coherence
ent-kaurene synthase-like
labdane-related diterpenoid
multiple cloning site
- Os
Oryza sativa
The work described was largely carried out by Yisheng Wu, building on preliminary results obtained by Qiang Wang, while Matthew Hillwig provided advice and carried out the described NMR structural analysis, all of which was under the guidance of Reuben Peters.
We thank Meimei Xu for construction of the pETDuet/DEST vector, and Professor Robert M. Coates (University of Illinois, Urbana-Champaign, IL, U.S.A.) for kindly providing oryzalexin E.
This work was supported by grants from the USDA (U.S. Department of Agriculture)-NIFA (National Institute of Food and Agriculture)-NRI (National Research Initiative)/AFRI (Agriculture and Food Research Initiative) [grant number 2008-35318-05027] and NIH (National Institutes of Health) [grant number GM086281] to R.J.P.
Author notes
Present address: Conagen Inc., 1005 North Warson Road, St Louis, MO 63132, U.S.A.
Present address: Department of Plant Physiology, Sichuan Agricultural University, Chengdu 625014, China
Present address: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA 15260, U.S.A.


text:
Allergies are becoming increasingly more common, with a majority of them being related to food. It can be nerve-wracking for parents sending their child with an allergy to camp. Especially when you're unsure of how they accommodate children with allergenic and medical needs. Here are some tips to give you peace of mind your child will be safe this summer:
1. Speak with prospective camp counselors at the earliest convenience to get as much information as possible.
2. Ask about the allergy policies and emergency protocols. Check to see if the camp have a written policy to ensure no allergens are brought into the camp environment (either through campers, staff, or suppliers). Ask if they have previous experience with children who may have had an allergic reactions? Also ask how an emergency situation would be handled?
3. Check with the camp as to the counselor to camper ratio. It is also good to know if the staff is trained on how to avoid allergens and to recognize symptoms of an allergic reaction. An important question to ask is your child allowed to carry their own medication or is this left in the care of the counselors?
4. Some camps will provide you with details for the camp medical staff or medical office. Have these numbers handy should you need to follow up with the camp.
5. Two more good points to check with the camp staff about is for day camps, do children bring their own food and how is this controlled? And for overnight campers, how do they keep from cross-contamination with potential allergens in their facilities?
The above list is all important questions which you need the answers for and these will help when determining the best camp for your child. Once you've made your decision and booked your child's spot, remember to submit the medical forms as soon as possible. Don't be afraid to go over this information with the camp director in order to ensure there is a plan in place your child. Most importantly, let your child know in an emergency to tell the camp staff. Let them know the staff is there to help and care for them. By keeping your children at ease about their allergies, you will help keep their camp experience a positive one.

subset: commoncrawl


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text:
Posts Tagged 'London magazine'
Missing Stevenson articles in London magazine, part 2
The 'Inland Voyage' Notebook
The 'Inland Voyage' notebook in the Beinecke Library contains a page of income for 1878, the first part drawn up in ink in early June 1878 (since the last item written in ink—the opening episode of the 'Later-Day Arabian Nights'—was published on 8 June), and continued afterwards in pencil:
Generously leaving the pencil additions on the right of the page and the totals top right and bottom left to be explained by another researcher, the main list can be transcribed as follows:.
The first thing to note is that the list does not cover all Stevenson's publications in 1878, as we can see if we look at the table below of 1878 publications in chronological order, with items on the Notebook list in bold:
The Notebook list is in chronological order, except for Inland Voyage in third place, probably because payment for this book was made before publication. However, the list does not name any titles after 'English Admirals'; the three unnamed final payments in the Notebook list must be a continuation of the series of payments for 'New Arabian Nights'.
Can we find evidence for 1878 payments in other documents?
Charles Baxter acted as Stevenson's banker and investment manager, and kept a record of receipts and payments in his 'Accounts Current between Robert Louis Stevenson […] and Mitchell and Baxter W.S.' (these records in the The Harry Ransom Center at the Univesity of Texas). However, it seems clear that a number of payments did not pass through Baxter's Accounts but were kept by RLS for his current expenses, so this record will not be complete. Additional evidence of payments comes from letters (in this case, Henley's letters to RLS and to Baxter).
Baxter's 'Accounts Current' for 23 August record a payment of £3 7s 'Cheque from Mr Sutton [Alfred Sutton, the publisher of London], London'. This does not correspond to any item on the list, suggesting that the the latter ends before 23 August, or the number of London that the payment refers to: assuming this was a payment for the 'Latter-Day Arabian Nights', and since there was no episode on 24 August, it must have been a late payment for the episode of 17 August or an early payment, on receipt of the MS, for the episode of 31 August. We'll look at this later when we've worked out the probable rate of payment made by London. [Added later: it's probably for 17 August episode; £3 7s works out as 28 pence per hundred words, a very similar rate to that of the other 'Arabian' episodes.]
A letter written by Henley to Baxter dated 12 September 1878 (Yale, B 4555) mentions that it includes two payments of £3 5s and £2, but this does not correspond to any payment on the list and would confirm that the Notebook list covers only part of the year.
Baxter's Accounts also have a payment dated 13 September of £8 8s. 'in payment of contribution to "Cornhill"', which although identical to the Cornhill payment for 'The English Admirals' on the list must be for 'Child's Play', published in the same magazine that month.
In an earlier letter of Henley to RLS of 20 June 1878 (Atkinson 51), WEH send £4 in sovereigns and owes 7/6 because he 'can't post three half-crowns', even though he has received them (from the publisher to forward to contributors): this must correspond to the £4 payment of item 10 on the Notebook list, corresponding to the 'Arabian' episode of 15 June (see below, 'Payments for the "Latter-Day Arabian Nights'). The list, if this is true, records only actual payments received and could be seen as Stevenson's way of keeping account of his limited income and expenditure while living with Fanny Osbourne in Paris.
Payments for articles in London
Let's take 'A Plea for Gas Lamps' and its payment of £1 11s 6d or 378 pence, and see how this relates to the number of words (payment by column or column-inch would be proportionate to this). The essay has 1325 words, so this works out as 29 pence per hundred words. 'Pan's Pipes' receives an identical fee, but is slightly longer: 1430 words (it is also longer in columns or column inches). This immediately tells us that any measurement of length was made roughly. The rate here is 26 pence per hundred words. 'El Dorado' is paid less, £1 6s or 312 pence, and is in fact shorter at 1166 words, which works out at 27 pence per hundred words.
It looks at this point as if these three contributions are paid by length at about 27 pence per hundred words or 19 pence per column inch, with length roughly measured or rounded up and down by a system we cannot know. We can get a better idea of the rate by looking at the payments for the 'Latter-Day Arabian Nights'.
Payments for the 'Latter-Day Arabian Nights'
If the payments without any title in the Notebook list all refer to the 'Latter-Day Arabian Nights' then there are 9 payments for 'Arabs'. One of them, however, is for £9 so it must be for two episodes. If the list contains all the payments for the first ten episodes, then it goes from 8 June to 10 August, the date when the list was abandoned (soon after Stevenson returned from France), thus leaving 8 episodes unrecorded, including the £3 7s recorded by Baxter on 23 August:
If we calculate the rate per hundred words of these episodes, then the £9 covers episodes 3 and 4 very neatly and there is a close approximation of weeks with a higher rate per hundred words (56, 34, 33, 35) and the weeks (if this association of payments and episodes is correct) when the Notebook marks an 'article also':
The payment for the first five episodes comes out as 29, 27, 29 and 29 pence per hundred words, which looks very close to the payment for the three essays in London, 29, 26 and 27 pence per hundred words. The second payment has a slightly lower rate—but if this is the £4 which Henley paid on 20 June (Atkinson, 51), then the full payment owed would be £4 7s 6d, which works out as 29 pence per hundred words for this episode also. Hence, for the calculations of the payments we'll take 29 pence per hundred words as the standard payment for 'Arabs'. This will probably not be the actual rate (paid by page or column) but will give us results that are roughly correct.
It should be said that the words have been calculated from the sections published in London (the list of opening and closing words of each episode kindly supplied by Roger Swearingen), as they were reprinted in the 1882 volume New Arabian Nights—it may well be that these sections were slightly longer or shorter in the book version, so for the moment (and I am generously leaving the counting of the words in the London version to another researcher) these must be seen as rough calculations only, though probably not too far from the actual numbers.
Taking then these numbers of words for the episodes in London, we see that payments for episodes 6—9 (13 Jul—3 Aug) are clearly accompanied by payments for additional items. The notebook list, however, suggests the additional items were associated with episodes 5—8 (6 Jul—27 Jul). The first of these, 6 July, is one of the payments of £4. Could it be that this is the payment of £4 made by Henley on 20 June? £4, as we see, covers payment for 'Arab' episode six at 29 pence per hundred words (henceforth, I'm afraid: 'pphw'); in this case—and the reader will here be aware that I am adding a series of possibilities one on the other, a structure which, like a house of cards, has its limits; but who knows, we may arrive at a single convincing hypothesis at the end covering everything satisfactorily—in this case, the 7s 6d that Henley owed Stevenson would be for that 'article also', published on the 6 July, and (using the rough rates discovered) just over 300 words long. This would be about 25 lines or a third of a column in length, so a very short piece, probably in the weekly odd items section called 'Whispering Gallery'.
Unfortunately in looking through the 6 July number, RLA and myself had not noted anything that seemed to be by Stevenson. The only item that caught our attention in that number was 'The Ethics of Lying', which might have started him thinking about 'Truth of Intercourse'—but, apart from the un-Stevensonsonian style, this is much longer, about 1800 words in length. Perhaps the short item in question was published on 13 July?
Additional Stevenson contribution to the 13 July number?
There was in fact an item in the 13 July number of London that both RLA and myself thought was almost certainly by Stevenson. It is unfortunately just under 300 words, 272 words to be exact. Anyway, here it is, from the 'Whispering Gallery' section of the magazine:
And by the way, what amazing devil (to quote the late Charles Dickens) is it that impels the distinguished lady who wrote "Adam Bede" to write such bitter dulness as "A College Breakfast Party"? Why should this woman of genius in prose imperil her soul by endeavouring to be also a woman of genius in verse? Surely if she be not all-wise herself, she has friends who are wise enough to save her from such a shame? Or is it true that the wicked world must approach her on its bended knees, and that her words are one and all oracular? I am not an enthusiast of hers, but I even am sorry. Her blank verse always reminds me of deals in a timber yard, or bricks stacked up for use at a builder's; and never a whit of poetry. Before "A College Breakfast Party" even Professor Dowden and Mr. G. H. Lewes, I should think, would "tremble and turn and be changed." But there's no accounting for tastes, and no disputing them neither. What to you and me, dear reader of my heart, is only dulness and awkwardness and a mistake, may be to others one of the greatest works of the human mind. It is amazing how desperately a personal interest will modify one's views! And let us not forget that while we (you and I, that is) are thanking heaven we are not as they are, they—the elect—are thanking heaven (or its equivalent) they are not as we are. And so the world wags on, and bad literature is let look for half a minute like good. (London, 13 July 1878, p. 36)
'A College Breakfast Party' had been published in Macmillan's Magazine for July 1878. The view of George Eliot in this brief London note is similar to that expressed by Stevenson in a letter of December 1877, in which he also uses the phrase 'woman of genius':
George Eliot: a high but, may we not add? − a rather dry Lady.[…] Hats off, all the same, you understand: a woman of genius. (Letters 2, 228)
The piece also contains some unexpected phrases that sound Stevensonian: 'bitter dulness', 'I even am sorry', and 'let look' ('bad literature is let look for half a minute like good')—the second and third are examples of how Stevenson invents new syntactical combinations that are perfectly understandable but strange at the same time. The third-to-last sentence ('It is amazing how desperately a personal interest will modify one's views!') and the sentence that follows are also reminiscent of the passage in 'Virginibus Puerisque' (from 1876) about how everyone believes their own opinions to be true:
The word "facts" is, in some ways, crucial. I have spoken with Jesuits and Plymouth Brethren, mathematicians and poets, dogmatic republicans and dear old gentlemen in bird's-eye neckcloths; and each understood the word "facts" in an occult sense of his own. Try as I might, I could get no nearer the principle of their division. What was essential to them seemed to me trivial or untrue. We could come to no compromise as to what was, or what was not, important in the life of man. Turn as we pleased, we all stood back to back in a big ring, and saw another quarter of the heavens, with different mountain-tops along the sky-line and different constellations overhead. We had each of us some whimsy in the brain, which we believed more than anything else, and which discoloured all experience to its own shade. How would you have people agree, when one is deaf and the other blind?
The cheekiness of 'thanking heaven (or its equivalent)' is also reminiscent of Stevenson's 1870s essays, as is the slightly crazy nonchalance of 'Her blank verse always reminds me of deals in a timber yard, or bricks stacked up for use at a builder's; and never a whit of poetry'.
Adding up possible Stevensonian contributions to the 13 July number
The payment of £5 (1200 pence) for an 'Arabian' episode and 'an article also' would correspond to 4138 words (at 29 pphw). If it refers to the 13 July number of London, does this payment correspond to Stevenson's certain and probable contributions to that number? i.e. episode six of the 'Arabs', 'A Story-teller' (see Part 1 of this report) and this brief notice on George Eliots's 'A College Breakfast Party'? Let's see:
We are still 749 words short of the quantity of 'copy' that should correspond to the £5 payment. Zut, alors! In our examination of the bound volumes in October, neither RLA nor myself identified anything else clearly by Stevenson in this number, and—alas!—we did not make scans (so inexpensive and so easy to make in the new British Library Newsroom!) which we could now be examining at our leisure. However, there is another clue from a letter by Henley to RLS dated 12 July. Talking about the contents for 13 July number:
Don't tax me with 'Ce Que Se Dit'. I only brushed it up. In doing so, I've made it presentable, but I've broken the author's heart. (Atkinson, 52)
Atkinson's footnote to 'Ce Que Se Dit' says 'A three verse unsigned poem in the issue of 13 July'. Judging by Henley's comment it could have been a poem by RLS that Henley revised without consultation and is now trying to pre-empt criticism by making a joke about it. Unfortunately, we don't have a scan of this page, nor do we have any idea of the rate of payment for verse: it's unlikely to be 749 words but perhaps it was paid by the column inch.
And the 20 July number?
RLA and myself identified several connected short items probably by Stevenson in the 'Whispering Gallery' section about Paris and the 1878 Paris Exhibition in the 20 July number of London. The series of short items begins like this:
On 7 June 1878, RLS had left Edinburgh for Paris, where he was secretary to Fleeming Jenkins, a member of the Jury for the Paris Exposition. He stayed in Hotel Mirabeau, moving to Hotel du Val de Grace, rue St Jacques in late June/early July. On 11 July he left Paris for Grez.
He must have written these notes just before leaving, as Henley writes to him on 12 July 'I have received notes and Hansom Cabs. […] Shall print and pay for Whispers. Next week' (Atkinson, 52). By 'next week' he meant the issue of 20 July, when the first part of 'The Adventure of the Hansom Cabs' appeared and also these Notes from Paris in the 'Whispering Gallery' section. The notes include a section about the good-natured festive crowd on what must be the Avenue de l'Opéra lit by electric lights; as he mentions the singing of the Marseillaise, this must be the celebration of the Republic on 30 June, which was also a popular expression of national pride after the defeat of 1870, with music and singing in the streets until late and much waving of flags (celebrated in a painting by Monet). He describes the hotel chambermaid preparing to go out into the festive streets (the opening comment is by Henley):
We can see Stevenson's typical unexpected epithet in 'the conspicuous morning'; the French way of proclaiming feelings is also commented on in 'Forest Notes' (1876)—people recommended Grez for its beauty, adding ' "Il y a de l'eau," […] with an emphasis, as if that settled the question'.
The Notes also contain a description of the first post-Revolutionary 14 July celebrations—officially comemorating the centenary of the death of Rousseau, after the govenment had forbidden any street celebrations for the fall of the Bastille—including a description of the main celebration in the Cirque Myers led by Louis Blanc. This is perhaps not by Stevenson, who would have had to return to Paris from Grez and then send a supplementary note. Excluding this from his contribution also makes sense from the point of view of the payment.
In fact, 3414 words (for the 'Arab') and 575 words (Paris Notes without the 14 July event) make 3989 words. Paid at 29 pphw, this number of words would correspond to 1157 pence—practically identical with the 1152 pence paid (£4 16s).
27 July?
This gets a bit embarassing. For 27 July, if our assignment of dates to the Notebook entries is correct, 3852 words (for the 'Arab') needs to be complemented by 534 words ('an article also') to make the 4386 words that correspond to the 1272 pence (£5 6s) paid. This is assuming that everything is paid at 29 pphw (the extra article would be 574 words if paid at 27 pphw).
Unfortunately, we only noted one item in the 27 July number that we thought could be by Stevenson, a review entitled 'History of the Indian Mutiny' (p. 90) and we gave it a vote of two out of five, so practically excluded it, though we know that Stevenson was interested in this episode of history. I have no record of its length, nor (alas!) a scan, which would have been so easy to make…
And 3 August?
Similarly for 3 August the payment of 1080 pence (£4 10s) covers the 3108 words of the 'Arab' episode and 744 words (or a bit more if the rate of payment was lower) of the extra 'article'.
Here, we noted the following as possible candidates: 'The Humours of "Bradshaw"' (pp. 111-12), 'Bohemia: Emile Augier III. The Dramatist' (pp. 112-13), 'Modern Frenchmen' (pp. 113-14). We gave a vote of two to the first; the same vote to the second (which, however, is the third of a series of articles), and one to the third. We did not make a scan for these pages either.
The assignment of dates to the 'Latter-Day Arabian Nights' episodes in the Notebook list finds a good correspondence between payments and the length of the 'Arab' episodes and has also helped us to identify three contributions by Stevenson.
The unique copies of London in the British Library contain much of interest: the many poems written by Henley and perhaps by Stevenson; the contributions by Katherine De Mattos and Walter Ferrier (much admired by Stevenson), possible contributions from Bob, as well as Henley's important input. It deserves a study of its own, to which the present study has made a small and incomplete contribution. (If only we'd made those scans…)


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Naperville Taxi : Driver Information
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Users last evening reported issues during session. Loss of vacuum to chamber and isolation
of FE-Gun section.
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asking for shipment on today's FED-X for overnight. Will they make the truck?!!


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Hi, guys, it's Neringa, the founder of Kensington Nutrition. I would love to share my knowledge with you and help you finally get the clean and lean body you have always dreamed off. So here is why I created the programme called "Lose weight and keep it off". This is not another fad diet, but an amazing lifestyle change.
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- Key eating strategies. Why eating less and exercising more is not always the answer.
- Key foods to avoid and key foods to include in your daily eating habits to maximise weight loss.
- How to lose fat fast without going hungry
- Snacks, fruits - which one to eat
- Best alkalising foods for weight loss
- Learn how to make easy and delicious food
- Recipes, shopping list
- Discover the best weight loss secrets and methods
This programme will guide you safely through your weight loss programme journey. You will learn why you should eat certain foods and avoid others in order to clean your body from the inside which will then inevitably show on the outside. No matter if you are vegan, or vegetarian or paleo - this programme will benefit you greatly in order to lose weight and get in your best shape ever - all of that without starvation or by counting calories!
If you are looking how to lose weight and keep it off, there is a link below to my website for you to check out. I think you will find it really helpful.


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Healthy Holiday Desserts The holidays aren't really the holidays without scrumptious desserts, luscious cookies and mouthwatering cakes and pies. You can have your cake and eat it too with these waist watcher desserts. Fresh Fruit Wreaths Low in calories and full of fiber and vitamins, fresh fruit is perfect for dessert. Up the presentation by arranging the fruit in a … [Read more...]
Do You Love Peanut Butter as Much as I do? Open up!
Anyone who knows me, knows that I LOVE peanut butter so I thought I would do a fun peanut butter recipe roundup before the holiday's. For you chocolate and peanut butter lovers, this one's for you. You can thank me later. Here's to some simple baking inspiration for the holiday season! If you haven't finished coming up with your Thanksgiving dessert menu, these ideas would … [Read more...]
Tempt Your Tastebuds with Delicious Zucchini Recipes
Do you love zucchini as much as I do? There are so many ways to enjoy zucchini. Our yard was used by family this year to grow a garden and one of the veggies that were plentiful was ZUCCHINI. So so delicious. Here are some delicious zucchini recipes I wanted to share with you. Garlic Parmesan Zucchini Chips Chicken Enchilada Zucchini Boats Ricotta Zucchini … [Read more...]
Learn How to Make Healthy Rice! Delicious Recipe Inside.
How to Make Healthy Rice with Curry Powder or Turmeric Dinner side dishes like rice are a great way to get extra nutrients into your family's diet. Instead of making plain old rice, you can add an array of nutrient-dense foods and spices to all of your rice dishes to really perk up the flavor and give tons of healthy benefits. Not only that, but your family will appreciate … [Read more...]
Simple Homemade Salad Dressing Recipe
Thanks to Kristina from Fully Raw I did it. I made my own homemade salad dressing. And it turned out good. You can watch this video for the recipe and more of her yummy creations. She made it so easy for someone like me who really doesn't cook. Here it is. Then look at this beauty. All those delicious veggies. I did add a little spice to the top … [Read more...]


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"He….encouraged them all that with purpose of heart they should continue with the Lord." Acts 11:23
Failure Is An Event – Not A Person!
Part 3
During Times of Failure We Must Remember The Greatness of God
Note: The greatness of God is the primary factor in ministry – it is not ministry if God is not in it!
[Read more…] about The Barnabas File – Volume 4 Number 11 9/24/04


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Also found in: Thesaurus, Medical, Acronyms, Encyclopedia, Wikipedia.
rime 1(rīm)
1. A white incrustation of ice formed when supercooled water droplets freeze almost instantly on contact with a solid surface.
2. A coating, as of mud or slime, likened to a frosty film: "A meal couldn't leave us feeling really full unless it laid down a rime of fat globules in our mouths and stomachs" (James Fallows).
tr.v. rimed, rim·ing, rimes
To cover with or as if with frost or ice: "heavy [shoes] rimed with mud and cement ... from the building site" (Seamus Deane).
[Middle English rim, from Old English hrīm.]
rim′y adj.
rime 2(rīm)
n. & v.
Variant of rhyme.
(Physical Geography) frost formed by the freezing of supercooled water droplets in fog onto solid objects
(Physical Geography) (tr) to cover with rime or something resembling rime
[Old English hrīm; related to Dutch rijm, Middle High German rīmeln to coat with frost]
n, vb
(Poetry) an archaic spelling of rhyme
n., v. rimed, rim•ing. n.
2. to cover with rime or hoarfrost.
[before 900; Middle English rim, Old English hrīm; c. Dutch rijm, Old Norse hrīm]
rim′y, adj. rim•i•er, rim•i•est.
n., v.t., v.i. rimed, rim•ing.
Past participle: rimed
Gerund: riming
ThesaurusAntonymsRelated WordsSynonymsLegend:
Switch to new thesaurus
|rime - ice crystals forming a white deposit (especially on objects outside)
|rime - correspondence in the sounds of two or more lines (especially final sounds)
poem, verse form - a composition written in metrical feet forming rhythmical lines
versification - the form or metrical composition of a poem
internal rhyme - a rhyme between words in the same line
alliteration, beginning rhyme, head rhyme, initial rhyme - use of the same consonant at the beginning of each stressed syllable in a line of verse; "around the rock the ragged rascal ran"
assonance, vowel rhyme - the repetition of similar vowels in the stressed syllables of successive words
consonance, consonant rhyme - the repetition of consonants (or consonant patterns) especially at the ends of words
double rhyme - a two-syllable rhyme; "`ended' and `blended' form a double rhyme"
eye rhyme - an imperfect rhyme (e.g., `love' and `move')
|rime - be similar in sound, especially with respect to the last syllable; "hat and cat rhyme"
correspond, gibe, jibe, match, tally, agree, fit, check - be compatible, similar or consistent; coincide in their characteristics; "The two stories don't agree in many details"; "The handwriting checks with the signature on the check"; "The suspect's fingerprints don't match those on the gun"
assonate - correspond in vowel sounds; rhyme in assonance; "The accented vowels assonated in this poem"
|rime - compose rhymes
create verbally - create with or from words
tag - supply (blank verse or prose) with rhymes
alliterate - use alliteration as a form of poetry
n (liter) → (Rauh)reif m


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509 7th Street NW, 3rd Floor
Washington D.C. 20004
Follow this class to get an email the next time it's scheduled.
Sign-up not required
509 7th Street NW, 3rd Floor
Washington D.C. 20004
Follow this class to get an email the next time it's scheduled.
Sign-up not required
The Excel at Excel workshop is designed for students who are relatively familiar with other office productivity software, for example Microsoft Word. (The students who succeed the most are comfortable using a computer and navigating around desktop programs on their own). It introduces a range of Excel topics giving students a taste of the potential that can be achieved with Excel. It touches briefly on everything from navigation and layout to using functions and charts. This workshop was not designed to go over any topic in extreme detail, but more as a "sampler" of topics. The expectation is for students to be able to leave and be familiar with several of the Excel skills necessary for most positions that require some "basic to intermediate" Excel knowledge.
Agenda (not necessarily taught in this order) - Terminology and Navigation - Filtering, Sorting, Formatting - Removing Duplicates - Hiding and Grouping Data - Conditional Formatting - Introduction to Formulas - Basic Math Functions - PivotTables - Charts and Graphs
Students should have a general familiarity with Excel, including knowing how to navigate a spreadsheet, and how to copy & paste. Please bring a laptop with Microsoft Excel installed (PC recommended, Mac acceptable). Most functions/features also work on a Mac although there may be minor differences. The instructor will be using a PC. It is also recommended that you bring a mouse. This is an interactive class, and your participation and questions are encouraged.
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Land of Lincoln Goodwill Industries- Springfield, IL
Land of Lincoln Goodwill Industries is teaming up with the ALICE Training to offer a special two-day training course designed to teach participants how to survive violent intruder or active shooter incidents on October 7 and 8. Officials say the training course will take place at Goodwill's Administrative Offices in Springfield. The training aims to provide individuals with survival-enhancing options during violent situations. ALICE officials say participants will receive ALICE Training certification upon completion of the course. Read more


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Up next we have the Tony Vlachos Award for Most Likely to Find an Idol.
This category is pretty self-explanatory. For better or worse, we all know how valuable Hidden Immunity Idols can be in the game of Survivor, and there are certain people that just have a knack for finding them. Survivor: Cagayan winner, Tony Vlachos, powered his way through the game with his lying and scheming, but he also had an expert ability at finding idols – and because of those idols he was able to maneuver his way to the Final Tribal Council. But idols can also be a curse – remember in Survivor: China, James Clement was blindsided and sent home with two idols in his possession. Finding an idol is one thing, but knowing how to best utilize an idol is the key thing.
In Survivor: Cambodia – Second Chance, only 5 of the 20 contestants found Hidden Immunity Idols in their original season – and only one of them is a woman, which makes these nominations even tougher. Also, there are four players returning in Cambodia that first played the game before hidden idols even came into existence. How will they adapt to this unknown element? As always, I have nominated the two men and two women I think are most likely to find an idol.
ATTENTION SURVIVOR FANS – You can SAVE 10% on the purchase of any past Survivor Buff courtesy of PlanetBuff.com when you use the exclusive coupon code: INSIDESURVPAST
And the nominees are…
Spencer Bledsoe – in Survivor: Cagayan, perpetual underdog Spencer kick-started an island-wide idol hunt after he discovered a clue at a reward. It was probably the most panicked idol searching ever, but even with the entire tribe looking for the idol, Spencer was able to find it and keep it secret from his fellow tribe-mates. He may not have played the idol correctly the first time around, but I believe Spencer will have a great chance to redeem himself in that regard this season. He's a superfan, he knows the type of places the producers hide idols, and he has experience in searching for them.
Terry Deitz – the circumstances were a little different back when Terry found his Hidden Immunity Idol in Survivor: Panama. It was only the second time an idol was introduced, and unlike the idol we are most familiar with today, this one you could play after the votes were read at Tribal Council. Terry also found this idol, not at camp, but on Exile Island. However, Terry's determination to find the idol is what makes me believe he will be committed to finding any idol that is hidden on the beaches of Koh Rong Island. Terry is someone that needs to be in control, and having the idol will give him that control.
Abi-Maria Gomes – the only woman in the Second Chance cast that actually found a Hidden Immunity Idol in her original season. Abi not only found the idol in Survivor: Philippines but she correctly used it to save herself at Tribal Council. Even when she didn't have an idol, she made up a lie pretending she did (after she won an advantage at the Survivor Auction). Abi is someone who very much understands the power of an idol and how deep they can take you in this game. I expect her to be looking for it on Day 1.
Shirin Oskooi – as I said, none of the other women in the cast have previous experience of finding an idol, but if anyone on this cast who should know where idols are hidden it's Shirin Oskooi. One of the biggest superfans to ever play the game, in Survivor: Worlds Apart, Shirin effectively broke the Survivor Auction with her superfan knowledge, and she could just as easily apply that knowledge to locating idols. Much like Spencer, she should have a good idea of the kind of places the producers tend to hide these things.
Vote below for which of these four nominees you think is Most Likely to Find an Idol in Survivor: Cambodia. Results will be announced in September.
Check out our Survivor Second Chance interviews here and Second Chance Cast Retrospectives.
Photo Credit: Rosmarie Voegtli via Compfight cc
Martin is a 31-year-old writer from England. He's represented by Berlin Associates for comedy writing and writes about TV and entertainment, currently for TV Insider and Vulture, previously ET Canada and Yahoo. A finalist for the Shortlist Sitcom Search in 2012 for "Siblings," Martin received his BA in English with Creative Writing from The University of Hull.


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Technology is often at the heart of every modern business. With the persuasions of going environmentally friendly and leaving a positive carbon footprint to the effective forms of communication and delivery with customers, it is almost impossible to fault the integration of technology in business.
According to a report titled 2016 Snapshot of the Australian Workplace by Dr Lindsay McMillan OAM, "over 40% of workers are relying on technology to perform over 75% of their work." Reference to technology is made in relation to both hardware and software and include desktop computers, laptops, printers, servers, telecommunications systems, and software programs.
The type of technology required to operate a business will naturally vary from one to another. For example, an accounting business may require the loan of laptops, a server, networking equipment, a printer, as well as an accounting software and other customer relationship management software.
In contrast, a small moving company may only require a desktop computer, networking equipment, a printer, and a customer relationship management software. It is recommended that consultations should be made with a technology expert to help determine the most effective and appropriate technology equipment for your business and situation.
Regardless of the size of your business, it is a known fact the business environment is an extremely volatile one. With costs of operations ranging from manageable to relatively demanding, having an option for technology financing can help alleviate any unnecessary stress on your business.
Essentially, a technology loan is a form of financing that is specifically tailored for technological equipment. In some cases, companies may specify that the loan is for business purposes while others may not.
In order to gain the most out of a technology financing solution, it is recommended that the technology loan is tailored to suit your needs. A technology loan company that is willing to assess the current situation of you and your business to help tailor your specific needs is not impossible to find. In fact, some technology loan companies are cooperative enough to look past a bad credit history in attempts to work together with genuine business owners.
You will also want to ensure that the technology loan provider you sign up to stocks the latest technological equipment to ensure that your equipment is current with modern technology. Other points to look out for in a good technology loan provider include the option of up to 100% financing without any upfront costs, whether any add-ons are included on top of your technology such as GST, and if there are any tax deductions on interest and depreciation.
Before you decide to commit to a technology loan, be sure to read the fine print to determine the ownership of the technological equipment once you purchase it. You will want to ensure that ownership of the equipment belongs to you once a purchase is made.
Article provided by Positive Lending Solutions


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Have you ever dreamed of tying the knot at IKEA, or are you after a seriously unique wedding? If you answered yes to either of these questions, then keep on reading!
The wedding bells are ringing and IKEA are on the hunt for the perfect couple to tie the knot at one of their stores in Sydney. So what would an IKEA wedding involve? The venue, reception, decorations and food and drink for 80 guests is included, PLUS $1000 towards your dream wedding dress and a $5000 IKEA gift card! Don't think it ends there…to make it a truly special event, they are throwing in a honeymoon to a surprise destination.
So if you would love to have your wedding at IKEA don't muck around!! Entries close on November 15th and you need to be an IKEA Family Member to enter. All you need to do to enter is <a href="email@example.com;">email a photo of you and your partner, plus 100 words or less, why you would love to have your wedding at IKEA. The happy couple should also be comfortable sharing their experience each step of the way with all of their fellow IKEA Family Members.
Image Courtesy of IKEA Family


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Author Archives: attraversiamodarmaz
For the Love
I haven't been here in awhile. Good Friday seemed like a fine time to show up. I started the year all ready to be more intentional about goals and plans and resolutions and I was going to be the girl … Continue reading
Posted in Uncategorized 1 Comment
It's all about the story…
Holidays can be so tricky…schedules, budgets, expectations, travel, blah blah blah. I am ever seeking the best way to make it matter. I also try to be reasonable and have a general "less is more" approach. I find myself easily … Continue reading
I sort of love breaking rules
I create rules…because I love a good plan…and then I break the rules when they no longer serve me well. Today was a lovely day. There were many great moments. Yet, it wasn't exploding with grateful photos. I am a … Continue reading
Thank you for your service
I think all too often it is easy to forget why we have our choices. I can say I am not comfortable with violence and choose to solve problems in other ways. I can care for my children and my … Continue reading
The great thing about today.
It's warm!! I had lunch with a beautiful soul I have known since kindergarten. I started the day in a classroom…I love that. I am on my way to workout…I love that too (but if today is not your workout … Continue reading
I gave you grace and I can snatch it right back…wait…
It was one of those days… You know the kind? Mostly it is good. Many lovely things happened. Why can't I focus on that? Nope. Instead I play in my head over and over again these two parenting moments that … Continue reading
I miss you!
I missed a few days. I didn't want to write this one. The most beautiful soul died so very suddenly. She was only 42. She taught right next door to me my first year in KC. She shook her head … Continue reading


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Rusty Grand blends inspirational lyrics with groovy sounds
on new mixtape 'Traffic Music'
ST. HELENA ISLAND, SC – A long time ago when Rusty Grand was just a child, his mother would punish him for bad grades by making him stay in his room with no TV or video games. All he could do to pass the time was listen to the radio. Little did she know that this plant seeds in a young boy's life that would one day grow into a full-fledged music career.
Today, Rusty Grand is a fast-rising hip-hop artist whose unique blend of styles and sounds makes him one of the musicians to pay attention to. After a four-year stint with the U.S. Army, Rusty Grand is about to finish his bachelor's degree at Full Sail University for recording arts and entertainment business, and he's announcing himself to the world with new music releases. His most recent project is a mixtape called "Traffic Music" that he's releasing free to the world in early January 2019.
"It's a motivational tape for everyone," he said. "It's the kind of project that makes you want to get up in the morning and go make something of your life. If you get aggravated while you're driving in traffic, you can put this in and get your focus on. It's the kind of project that makes you feel better about yourself instead of tearing you down. People today feel like they don't have any hope left, and I want to help people in the world to make changes in their lives."
Rusty said he named the project "Traffic Music" as a nod to that motivational idea of getting people to become better and do what they have to do to get ahead. It features his unique sound and style, which is something he's been working on for the better part of the past decade. At age 15 he saved up enough money to purchase his first recording equipment, and he hasn't looked back since. And along the way he's learned that while he's musically gifted, his true art comes in the whole process of being an entertainer. That's why he launched his company Too Much Entertainment Studios – with aspirations of signing other artists, creating films, producing events and making a big splash through all mediums of the entertainment industry.
"Ever since the age of 13, I have been working toward getting into the entertainment industry – not just being involved with music but the many aspects of entertainment," he said. "Music has always been my main niche to where I thought I could actually get in the door somewhere, but my main goal is to gain enough knowledge to create a pocket in the industry to actually help people – like Gucci Mane or even Jimmy Iovine. I just do what I love, have fun, and help the world where it's needed."
To listen to Rusty Grand's music, or to follow him on social media, please visit:


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The Montana State University professor is so happy to uncover a duckbilled species, which was found in the Judith river formation, Montana. In a recent research, it confirms that duckbilled dinosaurs were lived 79 million years ago. The paper published in the PLOS ONE stats, this giant creature brought an evolutionary change from the duckbilled ancestors.
A few years ago, Freedman Fowler (Paleontologist) removed mud from the bed earth. The site was located in north central Montana, where he found the duckbilled dinosaur fossil.
The recreated image of the fossil clearly shows the view of duckbilled dinosaurs. A team of researchers stats, the evolutionary species seem to be herbivores.
According to the Jack Honer (Montana State University Panleontologist),
"It is confirmation that Dr. Freedman Fowler is an excellent paleontologist, helping further cement MSU's reputation for offering graduate students a chance to be part of something extraordinary".
They like to name the newly discovered species as "Probrachylophosaurus bergei" or "Superduck", said Jack Honor and Fowler. It also confirms that, Superduck will have a missing link with the Acristavus, which walked on earth 81 million years ago. In a a quick look, Probrachylophosaurus bergei crest is smaller and got triangular shape. If you put together Aristavus and Probrachylophosaurus bergei, both the discovered skull bones are much similar to one another.
Liz Freedman Fowler says,
"It's always exciting to have a new species of dinosaur. What's most exciting about this one is that it's showing the process of evolution in action and that's what makes this is a very special new animal,"


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It's time to rethink how Australians are creating content. The funding from the Australian Government is not sufficient; not enough money is put into the marketing of films. Creating less Australian content and more genre films and finding new ways to distribute films are all necessary to reach Australian audiences that "have turned their back on them [Australian Media Industry]" (Roach, 2014). These are aspects that were discussed as Australia's Box Office returns were poor. By improving on these elements it suggests, the Australian Film Industry will be able to reconnect with audiences and produce an 'industrial transition into a niche market' (Verhoeven D et al. 2015).
The industry has struggled to keep up with the constant changes in technology and the shifts in media consumption. De Roeper and Luckman (2009), suggest that the industry has responded to changes in four ways – denial, panic, embrace and co-create. De Roeper and Luckman (2009) propose that rather than denying the past and panicking about the future, the film industry should embrace opportunities and co-create in the 'shareable world' (p. 12). Co-creation has seen emerging patterns of media consumption and digital storytelling become an ever-expanding feature in the film industry. With the expansion of Netflix, Stan and other streaming websites as well as illegal downloads, the Australian Film Industry has not been able to embrace the changes to the way that people are viewing content online. This has become a problem for the film industry because the Australian audience has kept up with the technological changes and have chosen to continuously watch content online. Australian content has lagged in securing spots online and on streaming services.
In 2017, the Australian government released a parliamentary inquiry discussing how to grow the media industry and balance international and national productions in a way to benefit the Australian Media Industry. More specifically the parliamentary inquiry recognised that the technological advances within the industry have significantly changed the way that audiences are viewing and accessing content through digital platforms instead of cinema experiences. Therefore, these digital changes are shifting release patterns and audience behaviours. For example, 'streaming online via Netflix, Amazon, Hulu (and Stan in Australia) have impacted these behaviours as most films are not making the cinema screens and are going straight online for a wider range audience' (Commonwealth of Australia, 2017, Chapter 2). Chapter 2 concluded that the 40% offset is not enough because producers 'simply do not have the budget to release their productions in a cinema and cannot make money if they do' (Commonwealth of Australia, 2017, Chapter 2).
Chapter 4 in the parliamentary inquiry discusses the international aspect and co-production of the media industry. Significantly, they note that the competition to gain funding from Screen Australia is extremely hard. This suggests, going offshore is the way to go for emerging talent for more opportunities. Treaties are welcomed by the government as Verhoeven et al. (2015) suggests that it "promotes improved international relations between nations". Australia already has treaties with 12 countries which is an "important source of finance and training and opportunities… making cultural connections both domestically and internationally" (Commonwealth of Australia, 2017, Chapter 4). While barriers, such as language and cultural differences, can prove a challenge for productions, co-productions are highly regarded and considered beneficial to the future of the Australian film industry. Productions may also be considered as "'footloose productions', productions that are not made in their country of origin, bring considerable economic benefit" (Commonwealth of Australia, 2017, Chapter 4).
The Australian Film Industry is just as successful as any other film industry although the quality of the films lags, compared to Hollywood films. With the provided funding given to producers specifically for "Significant Australian Content", producers don't have much of a chance to bring out their creative skills, market effectively and distribute the films efficiently reaching a wider audience.
Commonwealth of Australia, 2017, Report on the inquiry into the Australian film and television industry, Chapters 1, 2, and 4, Last viewed 31 January 2018, <https://www.aph.gov.au/Parliamentary_Business/Committees/House/Communications/AustralianfilmandTV/Report>.
De Roeper, J Luckman, S 2009, Future audiences for Australian stories: industry responses in a post-web 2.0 world, Media International Australia, no. 130, pp. 5-16.
Quinn, K 2017, More for TV, less for film, foreign actors OK: Inquiry recommends sweeping changes, The Sydney Morning Herald, December 2017, last viewed 31 January 2018, <http://www.smh.com.au/entertainment/movies/more-for-tv-less-for-film-foreign-actors-ok-inquiry-recommends-sweeping-changes-20171208-h01bl0.html>.
Roach, V 2014, Local audiences snub Australian filmmakers yet Hollywood loves them, News.com, September 2014, last viewed 31 January 2018, <http://www.news.com.au/entertainment/movies/box-office/local-audiences-snub-australian-filmmakers-yet-hollywood-loves-them/news-story/95338f7d82412c0ea02688fc8952d0d0>.
Verhoeven, D Davidson, A Coate, B 2015, Australian films at large: expanding the evidence about Australian cinema performance, Studies in Australasian Cinema, vol. 9, no. 1, pp. 7-20.


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The U.S. Ex-Im Bank and its president Fred Hochberg have never met a coal project they didn't like.
At times it's so bad we don't know what century the institution thinks it is operating in. Now, despite a worldwide uproar over the Bank's interest in one of the world's largest coal 'mega-mines' in Australia, it has been linked to another Australian mega mine that would bring coal across the Great Barrier Reef. Underwriting this potential destruction of the Great Barrier Reef is an unacceptable use of U.S. tax payer dollars — and it's time Ex-Im Bank came clean on its involvement.
It's not surprising to hear over-eager developers link the Ex-Im Bank to these projects because the institution has a long history of supporting fossil fuel projects. And that track record is getting worse. It got so bad that the Sierra Club wrote an open letter to President Fred Hochberg after we witnessed first hand the destruction these projects are wreaking on communities and livelihoods (check out our blog on the Sasan coal project in India).
But our pleas were callously ignored as President Hochberg okay-ed a massive expansion of coal finance in every corner of the globe. From Kusile in South Africa, to Sasan in India, to Xcoal in the U.S., to the recently proposed mines in Australia, it appears that Ex-Im Bank cares little for the environmental damage the institution is causing around the world — not to mention the reputation of this administration.
The problem, however, is that the public does. And that public stretches from Australia where the mining would take place, to India where the coal would be burned, to the U.S. where the financing would come from. This global outcry was captured in part by Avaaz's petition to #savethereef (consider taking a minute to contact Fred Hochberg personally via twitter: @fredhochberg), but also by media scrutiny in India, the U.S., the UK and Australia. It appears that while President Hochberg may consider designations like "World Heritage Site" pesky obstacles, the global public considers them treasures.
Which is why Greenpeace Australia's recent report on massive coal export expansion plans that would trample the Great Barrier Reef — and the global climate — was so damning. They found that if underwritten by institutions like Ex-Im Bank, the world would add emissions equivalent to a country the size of Canada while increasing traffic through one of the world's greatest natural wonders. For an excellent visual representation of this lunacy check out Greenpeace's short video:
So how did President Hochberg react? By quietly talking with another Indian company to finance another mega mine — this time with Adani group whose record of violations would give most institutions pause. Not Ex-Im though. After all, it makes sense that being linked to a scandal plagued Indian coal sector beset by "coal-gate" investigations would be of little consequence if you are willing to finance the destruction of a global treasure like the reef.
But at the end of the day, these may be over-eager developers looking to increase perception in the financial community that their projects have legs. The truth is, with prices at near two-year lows, the Australian mining community is understandably worried that these projects are no longer viable. That is why the vociferous claims from Adani and GVK may be making their way into the press at this opportune moment.
But if that's true, it's easy enough for President Hochberg to condemn these public statements, and publicly dissociate the bank from this scandal. Instead he has been silent — leaving a huge question mark over U.S. involvement. But let's not put words in anyone's mouth. Let's let President Hochberg speak for himself.
So Mr. Hochberg, where do you stand on these projects? The world is waiting for your answer.
Justin Guay is head of the Sierra Club's international program. This post was originally published at Sierra Club's India Environment Post and was reprinted with permission.


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Sunday, November 2, 2014
Spook-tacular Week in Art
Much to the chagrin of my students, we did not do any Halloween art lessons this week (the curriculum IS our driving force!). However, I didn't hear much complaining because they all continued to work on their clay or printmaking lessons (definite favorites of the year). I had a wonderful week enjoying all of the outfits my donned by my students. This past week was Red Ribbon week at our school and each day had a special theme. Here are some of the jewels of my class this week, in their awesome get-ups!
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What can a Dental Hygiene Therapist do?
Our Dental Hygiene Therapist is able to carry out a wide range of treatments to improve your oral health.
Since in Dental Therapists were introduced in 2002, Dental Hygiene Therapists play a vital role within the dental team and they work alongside a dentist. They undergo the same training as dentists in the treatments that they are able to carry out and are an important member of the team improving patient's oral care by increasing access, availability of appointments and choice of clinician.
Dental Hygiene Therapists are able to do the treatments that a Dental Hygienist can, such as; provide oral health advice, hygiene treatments such as scale and polish's, deeper cleaning to help treat gum disease, take x-rays, carry out tooth whitening, take impressions, apply materials to teeth such as fissure sealants and fluoride. Our Hygiene Therapist has also had training in implant maintenance.
The Dental Therapy aspect includes; fillings on deciduous and permanent teeth, extractions on children, placing crowns on deciduous teeth and carrying out pulp therapy on deciduous teeth. They are also able to deliver local anaesthetic to carry out such procedures.