Three-dimensional geological modelling and multivariate statistical analysis of water chemistry data to analyse and visualise aquifer structure and groundwater composition in the Wairau Plain, Marlborough District, New Zealand

Concerns regarding groundwater contamination with nitrate and the long-term sustainability of groundwater resources have prompted the development of a multi-layered three dimensional (3D) geological model to characterise the aquifer geometry of the Wairau Plain, Marlborough District, New Zealand. The 3D geological model which consists of eight litho-stratigraphic units has been subsequently used to synthesise hydrogeological and hydrogeochemical data for different aquifers in an approach that aims to demonstrate how integration of water chemistry data within the physical framework of a 3D geological model can help to better understand and conceptualise groundwater systems in complex geological settings. Multivariate statistical techniques(e.g. Principal Component Analysis and Hierarchical Cluster Analysis) were applied to groundwater chemistry data to identify hydrochemical facies which are characteristic of distinct evolutionary pathways and a common hydrologic history of groundwaters. Principal Component Analysis on hydrochemical data demonstrated that natural water-rock interactions, redox potential and human agricultural impact are the key controls of groundwater quality in the Wairau Plain. Hierarchical Cluster Analysis revealed distinct hydrochemical water quality groups in the Wairau Plain groundwater system. Visualisation of the results of the multivariate statistical analyses and distribution of groundwater nitrate concentrations in the context of aquifer lithology highlighted the link between groundwater chemistry and the lithology of host aquifers. The methodology followed in this study can be applied in a variety of hydrogeological settings to synthesise geological, hydrogeological and hydrochemical data and present them in a format readily understood by a wide range of stakeholders. This enables a more efficient communication of the results of scientific studies to the wider community.

Expression Pattern of the Alpha-Kafirin Promoter Coupled with a Signal Peptide from Sorghum bicolor L. Moench

Regulatory sequences with endosperm specificity are essential for foreign gene expression in the desired tissue for both grain quality improvement and molecular pharming. In this study, promoters of seed storage α-kafirin genes coupled with signal sequence (ss) were isolated from Sorghum bicolor L. Moench genomic DNA by PCR. The α-kafirin promoter (α-kaf) contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC, and TATA boxes required for α-kafirin gene expression in sorghum seeds. The constructs pMB-Ubi-gfp and pMB-kaf-gfp were microprojectile bombarded into various sorghum and sweet corn explants. GFP expression was detected on all explants using the Ubi promoter but only in seeds for the α-kaf promoter. This shows that the α-kaf promoter isolated was functional and demonstrated seed-specific GFP expression. The constructs pMB-Ubi-ss-gfp and pMB-kaf-ss-gfp were also bombarded into the same explants. Detection of GFP expression showed that the signal peptide (SP)::GFP fusion can assemble and fold properly, preserving the fluorescent properties of GFP.

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor α (TNFα) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNFα is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasmamembrane, and we investigated a possible role for lipid rafts in TNFα trafficking and secretion. TNFα surface delivery and secretion were found to be cholesterol- dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasmamembrane, particularly on filopodia. Imaging the early stages of TNFα surface distribution revealed these puncta to be the initial points of TNFα delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.

Syntaxin 6 and Vti1b form a novel SNARE complex, which is upregulated in activated macrophages to facilitate exocytosis of TNF.

A key function of activated macrophages is to secrete proinflammatory cytokines such as TNF; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNF� vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNF� trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion.

Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities*

20 and 26 S proteasomes were isolated from rat liver. The procedure developed for the 26 S proteasome resulted in greatly improved yields compared with previously published methods. A comparison of the kinetic properties of 20 and 26 S proteasomes showed significant differences in the kinetic characteristics with certain substrates and differences in the effects of a protein substrate on peptidase activity. Observed differences in the kinetics of peptidylglutamyl peptide hydrolase activity suggest that the 26 S complex cannot undergo the conformational changes of 20 S proteasomes at high concentrations of the substrate benzyloxycarbonyl (Z) -Leu-Leu-Glu-b-naphthylamide. Various inhibitors that differentially affect the trypsin-like and chymotrypsin-like activities have been identified. Ala-Ala-Phe-chloromethyl (CH2Cl) inhibits chymotrypsin-like activity assayed with succinyl (Suc) -Leu-Leu-Val-Tyr-AMC, but surprisingly not hydrolysis of Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC). Tyr-Gly-Arg-CH2Cl inhibits Suc-Leu-Leu-Val-Tyr-AMC hydrolysis as well as trypsinlike activity measured with t-butoxycarbonyl (Boc) -Leu-Ser-Thr-Arg-AMC, while Z-Phe-Gly-Tyr-diazomethyl (CHN2) was found to inhibit only the two chymotrypsin- like activities. Radiolabeled forms of peptidyl chloromethane and peptidyl diazomethane inhibitors, [3H]acetyl-Ala-Ala-Phe-CH2Cl, [3H]acetyland radioiodinated Tyr-Gly-Arg-CH2Cl, and Z-Phe-Gly- Tyr-(125I-CHN2), have been used to identify catalytic components associated with each of the three peptidase activities. In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3,4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by twodimensional polyacrylamide gel electrophoresis followed by autoradiography/fluorography and immunoblotting with subunit-specific antibodies. In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in different relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities. The results strongly suggest a relationship between trypsin-like activity and chymotrypsin-like activity. They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase activities

A study of ionic liquids for dissolution of sugarcane bagasse

Over the last decade, Ionic Liquids (ILs) have been used for the dissolution and derivatization of isolated cellulose. This ability of ILs is now sought for their application in the selective dissolution of cellulose from lignocellulosic biomass, for the manufacture of cellulosic ethanol. However, there are significant knowledge gaps in the understanding of the chemistry of the interaction of biomass and ILs. While imidazolium ILs have been used successfully to dissolve both isolated crystalline cellulose and components of lignocellulosic biomass, phosphonium ILs have not been sufficiently explored for the use in dissolution of lignocellulosic biomass. This thesis reports on the study of the chemistry of sugarcane bagasse with phosphonium ILs. Qualitative and quantitative measurements of biomass components dissolved in the phosphonium ionic liquids (ILs), trihexyltetradecylphosphonium chloride ([P66614]Cl) and tributylmethylphosphonium methylsulphate ([P4441]MeSO4) are obtained using attenuated total reflectance-Fourier Transform Infra Red (FTIR). Absorption bands related to cellulose, hemicelluloses and lignin dissolution monitored in situ in biomass-IL mixtures indicate lignin dissolution in both ILs and some holocellulose dissolution in the hydrophilic [P4441]MeSO4. The kinetics of lignin dissolution reported here indicate that while dissolution in the hydrophobic IL [P66614]Cl appears to follow an accepted mechanism of acid catalysed β-aryl ether cleavage, dissolution in the hydrophilic IL [P4441]MeSO4 does not appear to follow this mechanism and may not be followed by condensation reactions (initiated by reactive ketones). The quantitative measurement of lignin dissolution in phosphonium ILs based on absorbance at 1510 cm-1 has demonstrated utility and greater precision than the conventional Klason lignin method. The cleavage of lignin β-aryl ether bonds in sugarcane bagasse by the ionic liquid [P66614]Cl, in the presence of catalytic amounts of mineral acid. (ca. 0.4 %). The delignification process of bagasse is studied over a range of temperatures (120 °C to 150 °C) by monitoring the production of β-ketones (indicative of cleavage of β-aryl ethers) using FTIR spectroscopy and by compositional analysis of the undissolved fractions. Maximum delignification is obtained at 150 °C, with 52 % of lignin removed from the original lignin content of bagasse. No delignification is observed in the absence of acid which suggests that the reaction is acid catalysed with the IL solubilising the lignin fragments. The rate of delignification was significantly higher at 150 °C, suggesting that crossing the glass transition temperature of lignin effects greater freedom of rotation about the propanoid carbon-carbon bonds and leads to increased cleavage of β-aryl ethers. An attempt has been made to propose a probable mechanism of delignifcation of bagasse with the phosphonuim IL. All polymeric components of bagasse, a lignocellulosic biomass, dissolve in the hydrophilic ionic liquid (IL) tributylmethylphosphonium methylsulfate ([P4441]MeSO4) with and without a catalytic amount of acid (H2SO4, ca. 0.4 %). The presence of acid significantly increases the extent of dissolution of bagasse in [P4441]MeSO4 (by ca. 2.5 times under conditions used here). The dissolved fractions can be partially recovered by the addition of an antisolvent (water) and are significantly enriched in lignin. Unlike acid catalysed dissolution in the hydrophobic IL tetradecyltrihexylphosphonium chloride there is little evidence of cleavage of β-aryl ether bonds of lignin dissolving in [P4441]MeSO4 (with and without acid), but this mechanism may play some role in the acid catalysed dissolution. The XRD of the undissolved fractions suggests that the IL may selectively dissolve the amorphous cellulose component, leaving behind crystalline material.

Dynamic covalent chemistry on surfaces employing highly reactive cyclopentadienyl moieties

Silicon substrates coated with a bromide-terminated silane are transformed into highly reactive, cyclopentadiene covered analogues. These surfaces undergo rapid cycloaddition reactions with various dienophile-capped polymers. Mild heating of the substrates causes the retro-Diels-Alder reaction to occur, thus reforming the reactive cyclopentadiene surface, generating an efficiently switchable surface.

Osteopenia in a teenage boy presenting with previously undiagnosed guanidinoacetate methyltransferase (GAMT) deficiency and response to creatine supplementation

A 16 y.o. fully ambulant boy born to consanguineous Indian parents, presented for assessment of a fragility femoral neck fracture sustained against a background of autism and moderately severe intellectual disability. He had a past history of infantile eczema, and epilepsy treated with anticonvulsants from 2 to 10 years of age, with no further seizures following cessation of anticonvulsants. He had a thin body habitus (see Table 1) with long fingers and a high arched palate. He had no speech and negligible social interaction, but physical examination was otherwise unremarkable. Positive investigations revealed an undetectable serum creatinine and a urinary metabolic screen which showed an elevated GUA:Phe of 160 (< 36) and a decreased creatinine of 0.3 mmol/l (1.2–29.5) consistent with the diagnosis of guanidinoacetate methyltransferase(GAMT) deficiency. He was commenced on oral creatine 5 g three times daily. Despite improvement in physical activity, height and bone density, there was no discernable improvement in his intellectual functioning. Proton and phosphorous brain and leg magnetic resonance spectroscopy(MRS) was performed at baseline and showed an increased inorganic phosphorus peak and decreased phosphocreatine synthesis in brain and decreased creatine concentration in muscle. Following creatine treatment total brain creatine(1H-MRS) and phosphocreatine/ATP ratio (31P-MRS) content increased to 30% and 60% of control values, respectively. Brain GUA returned to normal levels.

Implications of lipid biology for the pathogenesis of schizophrenia

Objective: Preclinical and clinical data suggest that lipid biology is integral to brain development and neurodegeneration. Both aspects are proposed as being important in the pathogenesis of schizophrenia. The purpose of this paper is to examine the implications of lipid biology, in particular the role of essential fatty acids (EFA), for schizophrenia. Methods: Medline databases were searched from 1966 to 2001 followed by the crosschecking of references. Results: Most studies investigating lipids in schizophrenia described reduced EFA, altered glycerophospholipids and an increased activity of a calcium-independent phospholipase A2 in blood cells and in post-mortem brain tissue. Additionally, in vivo brain phosphorus-31 Magnetic Resonance Spectroscopy (31P-MRS) demonstrated lower phosphomonoesters (implying reduced membrane precursors) in first- and multi-episode patients. In contrast, phosphodiesters were elevated mainly in first-episode patients (implying increased membrane breakdown products), whereas inconclusive results were found in chronic patients. EFA supplementation trials in chronic patient populations with residual symptoms have demonstrated conflicting results. More consistent results were observed in the early and symptomatic stages of illness, especially if EFA with a high proportion of eicosapentaenoic acid was used. Conclusion: Peripheral blood cell, brain necropsy and 31P-MRS analysis reveal a disturbed lipid biology, suggesting generalized membrane alterations in schizophrenia. 31P-MRS data suggest increased membrane turnover at illness onset and persisting membrane abnormalities in established schizophrenia. Cellular processes regulating membrane lipid metabolism are potential new targets for antipsychotic drugs and might explain the mechanism of action of treatments such as eicosapentaenoic acid.

Advanced glycation endproducts in horses with insulin-induced laminitis

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6 h, 12 h and 24 h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.

A mass spectrometric assay for the quantification of neuropeptide PYY in plasma

A multiple reaction monitoring mass spectrometric assay for the quantification of PYY in human plasma has been developed. A two stage sample preparation protocol was employed in which plasma containing the full length neuropeptide was first digested using trypsin, followed by solid-phase extraction to extract the digested peptide from the complex plasma matrix. The peptide extracts were analysed by LC-MS using multiple reaction monitoring to detect and quantify PYY. The method has been validated for plasma samples, yielding linear responses over the range 5–1,000 ng mL−1. The method is rapid, robust and specific for plasma PYY detection.

Eliciting metacognitive experiences and reflection in a year 11 chemistry classroom : an activity theory perspective

Concerns regarding students' learning and reasoning in chemistry classrooms are well documented. Students' reasoning in chemistry should be characterized by conscious consideration of chemical phenomenon from laboratory work at macroscopic, molecular/sub-micro and symbolic levels. Further, students should develop metacognition in relation to such ways of reasoning about chemistry phenomena. Classroom change eliciting metacognitive experiences and metacognitive reflection is necessary to shift entrenched views of teaching and learning in students. In this study, Activity Theory is used as the framework for intepreting changes to the rules/customs and tools of the activity systems of two different classes of students taught by the same teacher, Frances, who was teaching chemical equilibrium to those classes in consecutive years. An interpretive methodolgy involving multiple data sources was employed. Frances explicitly changed her pedagogy in the second year to direct students attention to increasingly consider chemical phenomena at the molecular/sub-micro level. Additonally, she asked students not to use the textbook until toward the end of the equilibrium unit and sought to engage them in using their prior knowledge of chemistry to understand their observations from experiments. Frances' changed pedagogy elicited metacognitive experiences and reflection in students and challenged them to reconsider their metacognitive beliefs about learning chemistry and how it might be achieved. While teacher change is essential for science education reform, students are not passive players in the change efforts and they need to be convinced of the viability of teacher pedagogical change in the context of their goals, intentions, and beliefs.

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 microM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and > 50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and > 37.5 microM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2'-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5'-oxygen in the transition state. We suggest structural reasons why the Mg(2+)-La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.

Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism

Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the influence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium isotope effect has been taken as evidence against a proton transfer in the rate-limiting step of the cleavage reaction. We propose that these data are more easily explained by a two-metal-ion mechanism that does not involve a metal hydroxide, but does involve a proton transfer in the rate-limiting step.

The effect of Tenofovir on vitamin D metabolism in HIV-infected adults is dependent on sex and ethnicity

Background: Tenofovir has been associated with renal phosphate wasting, reduced bone mineral density, and higher parathyroid hormone levels. The aim of this study was to carry out a detailed comparison of the effects of tenofovir versus non-tenofovir use on calcium, phosphate and, vitamin D, parathyroid hormone (PTH), and bone mineral density. Methods: A cohort study of 56 HIV-1 infected adults at a single centre in the UK on stable antiretroviral regimes comparing biochemical and bone mineral density parameters between patients receiving either tenofovir or another nucleoside reverse transcriptase inhibitor. Principal Findings: In the unadjusted analysis, there was no significant difference between the two groups in PTH levels (tenofovir mean 5.9 pmol/L, 95% confidence intervals 5.0 to 6.8, versus non-tenofovir; 5.9, 4.9 to 6.9; p = 0.98). Patients on tenofovir had significantly reduced urinary calcium excretion (median 3.01 mmol/24 hours) compared to non-tenofovir users (4.56; p,0.0001). Stratification of the analysis by age and ethnicity revealed that non-white men but not women, on tenofovir had higher PTH levels than non-white men not on tenofovir (mean difference 3.1 pmol/L, 95% CI 5.3 to 0.9; p = 0.007). Those patients with optimal 25-hydroxyvitamin D (.75 nmol/L) on tenofovir had higher 1,25-dihydroxyvitamin D [1,25(OH)2D] (median 48 pg/mL versus 31; p = 0.012), fractional excretion of phosphate (median 26.1%, versus 14.6;p = 0.025) and lower serum phosphate (median 0.79 mmol/L versus 1.02; p = 0.040) than those not taking tenofovir. Conclusions: The effects of tenofovir on PTH levels were modified by sex and ethnicity in this cohort. Vitamin D status also modified the effects of tenofovir on serum concentrations of 1,25(OH)2D and phosphate.