Towards the biofortification of banana fruit for enhanced micronutrient content

In Uganda, vitamin A deficiency (VAD) and iron deficiency anaemia (IDA) are major public health problems with between 15-32% of children under 5 years of age showing VAD and 73% being anaemic. This is largely due to the fact that the staple food crop of the country, banana, is low in pro-vitamin A and iron, therefore leading to dietary deficiencies. Although worldwide progress has been made to control VAD and IDA through supplementation, food fortification and diet diversification, their long term sustainability and impact in developing countries such as Uganda is limited. The approach taken by researchers at Queensland University of Technology (QUT), Australia, in collaboration with the National Agricultural Research Organization (NARO), Uganda, to address this problem, is to generate consumer acceptable banana varieties with significantly increased levels of pro-vitamin A and iron in the fruit using genetic engineering techniques. Such an approach requires the use of suitable, well characterised genes and promoters for targeted transgene expression. Recently, a new banana phytoene synthase gene (APsy2a) involved in the synthesis of pro-vitamin A (pVA) carotenoids was isolated from a high â-carotene banana (F’ei cv Asupina). In addition, sequences of banana ferritin, an iron storage protein, have been isolated from Cavendish banana. The aim of the research described in this thesis was to evaluate the function of these genes to assess their suitability for the biofortification of banana fruit. In addition, a range of banana-derived promoters were characterised to determine their suitability for controlling the expression of transgenes in banana fruit. Due to the time constraints involved with generating transgenic banana fruit, rice was used as the model crop to investigate the functionality of the banana-derived APsy2a and ferritin genes. Using Agrobacterium-mediated transformation, rice callus was transformed with APsy2a +/- the bacterial-derived carotene desaturase gene (CrtI) each under the control of the constitutive maize poly-ubiquitin promoter (ZmUbi) or seed-specific rice glutelin1 (Gt1) promoter. The maize phytoene synthase (ZmPsy1) gene was included as a control. On selective media, with the exception of ZmUbi-CrtI-transgenic callus, all antibiotic resistant callus displayed a yellow-orange colour from which the presence of â-carotene was demonstrated using Raman spectroscopy. Although the regeneration of plants from yellow-orange callus was difficult, 16 transgenic plants were obtained and characterised from callus transformed with ZmUbi-APys2a alone. At least 50% of the T1 seeds developed a yellow-orange coloured callus which was found to contain levels of â-carotene ranging from 4.6-fold to 72-fold higher than that in non-transgenic rice callus. Using the seed-specific Gt1 promoter, 38 transgenic rice plants were generated from APsy2a-CrtI-transformed callus while 32 plants were regenerated from ZmPsy1-CrtI-transformed callus. However, when analysed for presence of transgene by PCR, all transgenic plants contained the APsy2a, ZmPsy1 or CrtI transgene, with none of the plants found to be co-transformed. Using Raman spectroscopy, no â-carotene was detected in-situ in representative T1 seeds. To investigate the potential of the banana-derived ferritin gene (BanFer1) to enhance iron content, rice callus was transformed with constitutively expressed BanFer1 using the soybean ferritin gene (SoyFer) as a control. A total of 12 and 11 callus lines independently transformed with BanFer1 and SoyFer, respectively, were multiplied and transgene expression was verified by RT-PCR. Pearl’s Prussian blue staining for in-situ detection of ferric iron showed a stronger blue colour in rice callus transformed with BanFer1 compared to SoyFer. Using flame atomic absorption spectrometry, the highest mean amount of iron quantified in callus transformed with BanFer1 was 30-fold while that obtained using the SoyFer was 14-fold higher than the controls. In addition, ~78% of BanFer1-transgenic callus lines and ~27% of SoyFer-transgenic callus lines had significantly higher iron content than the non-transformed controls. Since the genes used for enhancing micronutrient content need to be expressed in banana fruit, the activity of a range of banana-derived, potentially fruit-active promoters in banana was investigated. Using uidA (GUS) as a reporter gene, the function of the Expansin1 (MaExp1), Expansin1 containing the rice actin intron (MaExp1a), Expansin4 (MaExp4), Extensin (MaExt), ACS (MaACS), ACO (MaACO), Metallothionein (MaMT2a) and phytoene synthase (APsy2a) promoters were transiently analysed in intact banana fruit using two transformation methods, particle bombardment and Agrobacterium-mediated infiltration (agro-infiltration). Although a considerable amount of variation in promoter activity was observed both within and between experiments, similar trends were obtained using both transformation methods. The MaExp1 and MaExp1a directed high levels of GUS expression in banana fruit which were comparable to those observed from the ZmUbi and Banana bunchy top virus-derived BT4 promoters that were included as positive controls. Lower levels of promoter activity were obtained in both methods using the MaACO and MaExt promoters while the MaExp4, MaACS, and APsy2a promoters directed the lowest GUS activity in banana fruit. An attempt was subsequently made to use agro-infiltration to assess the expression of pVA biosynthesis genes in banana fruit by infiltrating fruit with constructs in which the ZmUbi promoter controlled the expression of APsy2a +/- CrtI, and with the maize phytoene synthase gene (ZmPsy1) included as a control. Unfortunately, the large amount of variation and inconsistency observed within and between experiments precluded any meaningful conclusions to be drawn. The final component of this research was to assess the level of promoter activity and specificity in non-target tissue. These analyses were done on leaves obtained from glasshouse-grown banana plants stably transformed with MaExp1, MaACO, APsy2a, BT4 and ZmUbi promoters driving the expression of the GUS gene in addition to leaves from a selection of the same transgenic plants which were growing in a field trial in North Queensland. The results from both histochemical and fluorometric GUS assays showed that the MaExp1 and MaACO promoters directed very low GUS activities in leaves of stably transformed banana plants compared to the constitutive ZmUbi and BT4 promoters. In summary, the results from this research provide evidence that the banana phytoene synthase gene (APsy2a) and the banana ferritin gene (BanFer1) are functional, since the constitutive over-expression of each of these transgenes led to increased levels of pVA carotenoids (for APsy2a) and iron content (for BanFer1) in transgenic rice callus. Further work is now required to determine the functionality of these genes in stably-transformed banana fruit. This research also demonstrated that the MaExp1 and MaACO promoters are fruit-active but have low activity in non-target tissue (leaves), characteristics that make them potentially useful for the biofortification of banana fruit. Ultimately, however, analysis of fruit from field-grown transgenic plants will be required to fully evaluate the suitability of pVA biosynthesis genes and the fruit-active promoters for fruit biofortification.

Enhancing MRI of prostate cancer using PSMA-targeting iron oxide magnetic nanoparticles

Introduction Novel imaging techniques for prostate cancer (PCa) are required to improve staging and real-time assessment of therapeutic response. We performed preclinical evaluation of newly-developed, biocompatible magnetic nanoparticles (MNPs) conjugated with J591, an antibody specific for prostate specific membrane antigen (PSMA), to enhance magnetic resonance imaging (MRI) of PCa. PSMA is expressed on ∼90% of PCa, including those that are castrate-resistant, rendering it as a rational target for PCa imaging. Materials and Methods The specificity of J591 for PSMA was confirmed by flow cytometric analysis of several PCa cell lines of known PSMA status. MNPs were prepared, engineered to the appropriate size, labeled with DiR fluorophore, and their toxicity to a panel of PC cells was assessed by in vitro Alamar Blue assay. Immunohistochemistry, fluorescence microscopy and Prussian Blue staining (iron uptake) were used to evaluate PSMA specificity of J591-MNP conjugates. In vivo MRI studies (16.4T MRI system) were performed using live immunodeficient mice bearing orthotopic LNCaP xenografts and injected intravenously with J591-MNPs or MNPs alone. Results MNPs were non-toxic to PCa cells. J591-MNP conjugates showed no compromise in specificity of binding to PSMA+ cells and showed enhanced iron uptake compared with MNPs alone. In vivo, tumour targeting (significant MR image contrast) was evident in mice injected with J591-MNPs, but not MNPs alone. Resected tumours from targeted mice had an accumulation of MNPs, not seen in normal control prostate. Conclusions Application of PSMA-targeting MNPs into conventional MRI has potential to enhance PCa detection and localization in real-time, improving patient management.

Comparison of performance parameters of poly(3,4 ethylenedioxythiophene) (PEDOT) based electrochromic device on glass with and without counter electrode

Conjugated polymers are promising materials for electrochromic device technology. Aqueous dispersions of poly(3,4-ethylenedioxythiophene)-(PEDOT) were spin coated onto transparent conducting oxide (TCO) coated glass substrates. A seven-layer electrochromic device was fabricated with the following configuration: glass/transparent conducting oxide (TCO)/PEDOT (main electrochromic layer)/gel electrolyte/prussian blue (counter electrode)/TCO/glass. The device fabricated with counter electrode (Prussian blue) showed a contrast of 18% and without counter electrode showed visible contrast of 5% at 632 nm at a voltage of 1.9 V. The comparison of the device is done in terms of the colouration efficiency of the devices with and without counter electrode.

Studies on Carbon Mediated Paste Screen Printed Sensors for Blood Glucose Sensing Application

Ready-to-use screen printed glucose sensors are fabricated using Prussian Blue (PB) and Cobalt Phthalocyanine (CoPC) mediated carbon inks as working electrodes. The reference and counter electrodes are screen printed using silver/silver chloride and graphitic carbon paste respectively. The screen printed reference electrodes (internal reference electrode (IRE)) are found to be stable for more than 60 minutes when examined with saturated calomel electrode. Optimal operating voltage for PB and CoPC screen printed sensors are determined by hydrodynamic voltammetric technique. Glucose oxidase is immobilized on the working electrodes by cross-linking method. PB mediated glucose sensor exhibits a sensitivity of 5.60 mA cm(-2)/mM for the range, 10 to 1000 mu M. Sensitivity of CoPC mediated glucose sensor is found to be 5.224 mu A cm(-2)/mM and amperometeric response is linear for the range, 100 to 1500 mu M. Interference studies on the fabricated glucose sensors are conducted with species like uric acid and ascorbic acid. PB mediated sensors showed a completely interference-free behavior. The sensing characteristics of PB mediated glucose sensors are also studied in diluted human serum samples and the results are compared with the values obtained through standard clinical method. The co-efficient of variation is found to be less than 5%. (C) 2015 The Electrochemical Society. All rights reserved.

Assembly process of CuHCF/MPA multilayers on gold nanoparticles modified electrode and characterization by electrochemical SPR

Gold nanoparticles were deposited onto 2-mercaptoethylamine (MEA)-assembled planar gold thin film to construct gold nanoparticles modified electrode by virtue of a solution-based self-assembly strategy. Subsequently, 3-mercaptopropionic acid (MPA)-bridged copper hexacyanoferrate (CuHCF) multilayers were constructed on the as-prepared gold nanoparticles modified electrode. The resulted multilayer nanostructures were investigated by electrochemical surface plasmon resonance (EC-SPR) and atomic force microscopy (AFM) with primary emphasis upon the effect of the gold nanoparticles on the MPA/CuHCF multilayers growth and their surface morphology. Compared with the multilayer system on a planar gold electrode, the different electrochemical and optical properties might result from higher curvature effect and extraordinary surface-to-volume ratio characteristic of gold nanoparticles and the nanoparticle-selective growth of CuHCF. A dendrimer-like assembly process was proposed to explain the experiment results. This new motif of multilayer on the gold nanoparticles modified electrode was different from that of on a planar gold electrode, indicating a potential application of EC-SPR technique in the study of nanocomposite materials.

Nanotubes of mixed-valence, transition metal compounds synthesized by solution phase approach

A solution-phase approach to synthesize four kinds of mixed-valence, transition metal compounds nanotube is described. The approach is based on the self-assembly of siloxane sol. The resulted production of mixed-valence, transition metal compounds share a common structural characteristic of tubular geometrical morphology, at least for the ones we studied. The results demonstrate that the synthesis strategy can be a general route for preparation of compound nanotubes. In addition, the size control of nanotubular materials can be easily achieved through varying the ionic strength of solution. Based on the strategy, the diameters of ultrathin Ru-Fe nanotubes can be easily tuned between 100 nm and 800 nm.

Effects of a surfactant on the electrocatalytic activity of cobalt hexacyanoferrate modified glassy carbon electrode towards the oxidation of dopamine

The cobalt hexacyanoferrate film (CoHCF) was deposited on the surface of a glassy carbon (GC) electrode with a potential cycling procedure in the presence and absence of the cationic surfactant, cetyl trimethylammonium bromide (CTAB), to form CoHCF modified GC (CoHCF/GC) electrode. It was found that CTAB would affect the growth of the CoHCF film, the electrochemical behavior of the CoHCF film and the electrocatalytic activity of the CoHCF/GC electrode towards the electrochemical oxidation of dopamine (DA). The reasons of the electrochemical behavior of CoHCF/GC electrode influenced by CTAB were investigated using FTIR and scanning electron microscope (SEM) techniques. The apparent rate constant of electrocatalytic oxidation of DA catalyzed by CoHCF was determined using the rotating disk electrode measurements.

Large-scale synthesis of Berlin green Fe[Fe(CN)(6)] microcubic crystals

Berlin green FeFe(CN)(6) microcubic crystals have been successfully prepared by a simple hydrothermal process between K-3[Fe(CN)(6)] with Na2S2O3 aqueous solution, free of any surfactant or template. The experimental results clearly show that the molar ratio of K-3[Fe(CN)(6)] to Na2S2O3 and their concentrations are the dominant processing factors in controlling the size, morphology, and composition of the resulting products.

Copper hexacyanoferrate multilayer films on glassy carbon electrode modified with 4-aminobenzoic acid in aqueous solution

4-Aminobenzoic acid (4-ABA) was covalently grafted on a glassy carbon electrode (GCE) by amine cation radical formation during the electrooxidation process in 0.1 M KCl aqueous Solution. X-ray photoelectron spectroscopy (XPS) measurement proves the presence of 4-carboxylphenylamine on the GCE. Electron transfer processes of Fe(CN)(6)(3-) in solutions of various pHs at the modified electrode are studied by both cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Changing the solution pH would result in the variation of the terminal group's charge state, based on which the surface pK(a) values were estimated. The copper hexacyanoferrate (CuHCF) multilayer films were formed on 4-ABA/GCE prepared in aqueous solution, and which exhibit good electrochemical behavior with high stability.

Preparation of cobalt hexacyanoferrate nanowires using carbon nanotubes as templates

A simple and convenient method for preparation of cobalt hexacyanoferrate (CoHCF) nanowires by electrodeposition was reported. Multiwall carbon nanotubes (MWNTs) were used as templates to fabricate CoHCF nanowires. MWNTs could affect the size of CoHCF nanoparticles and made them grow on the sidewalls of carbon nanotubes during the process of electrodeposition. Thus CoHCF nanowires could be obtained by this method. Field-emission scanning electron microscopy, UV-vis spectroscopy, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were used to characterize these nanowires. These results showed the CoHCF nanowires could be easily and successfully obtained and it gave a novel approach to prepare inorganic nanowires.

Site-selective self-assembly of MPA-bridged CuHCF multilayers on APTMS-supported gold colloid electrodes

(3-Aminopropyl)trimethoxysilane (APTMS)-supported gold colloid electrode was constructed by virtue of a recently developed solution-based self-assembly strategy. The preparing procedure of 3-mercaptopropionic acid (MPA)-bridged copper hexacyanoferrate (CuHCF) multilayers on a planar macroelectrode (Bharathi et al. Langmuir 2001, 17, 7468) was copied to the as-prepared colloid electrode. The optical spectra, atomic force microscopy, and electrochemistry demonstrate successful copy of the multilayer system on a macroelectrode to the as-prepared colloid electrode. Remarkably, it was found that multilayer growth is highly selective to the nanoscale sites where gold nanoparticles are immobilized, and multilayer growth does not take place on the sites without nanoparticles. Interestingly, a preliminary electrochemical investigation indicates that electrochemical properties of multilayers systems on the colloid electrode are different from their counterparts on a planar macroelectrode, which might be due to high curvature effects of the gold nanoparticles. This indicates a different motif of multilayers on the colloid electrode from that on a planar macroelectrode.

Electrocatalytic oxidation of dopamine at a cobalt hexacyanoferrate modified glassy carbon electrode prepared by a new method

A stable electroactive thin film of cobalt hexacyanoferrate (CoHCF) was electrochemically deposited on the surface of a glassy carbon (GC) electrode with a new and simple method. The cyclic voltammograms of the CoHCF Film modified GC (CoHCF/GC) electrode prepared by this method exhibit two pairs of well-defined redox peaks, at scan rates up to 200 mV s(-1). The advantage of this method is that it is easy to manipulate and to control the surface coverage of CoHCF on the electrode surface. The modified electrode shows good electrocatalytic activity towards the electrochemical reaction of dopamine (DA) in a 0.1 mol dm (3) KNO3 + phosphate buffer solution (pH 7.0). The rate constant of the electrocatalytic oxidation of DA at the CoHCF/GC electrode is determined by employing rotating disk electrode measurements.

Cupric hexacyanoferrate nanoparticle modified carbon ceramic composite electrodes

Graphite powder-supported cupric hexacyanoferrate (CuHCF) nanoparticles were dispersed into methyltrimethoxysilane based gels to produce a conducting carbon ceramic Composite, which was used as electrode material to fabricate surface- renewable CuHCF-modified electrodes. Electrochemical behavior of the CuHCF-modifled carbon ceramic composite electrodes was characterized using cyclic and square-wave voltammetry. Cyclic voltammograms at various scan rates indicated that peak currents were surface-confined at low scan rates. In the presence of glutathione, a clear electrocatalytic response was observed at the CuHCF-modified composite electrodes. In addition, the electrodes exhibited a distinct advantage of reproducible surface-renewal by simple mechanical polishing on emery paper, as well as ease of preparation, and good chemical and mechanical stability in a flowing stream.

Surface renewable graphite organosilicate composite electrode containing indium(III) hexacyanoferrate(II/III)

Indium(III) hexacyanoferrate(II/III) (InHCF) supported on graphite powder was prepared using the in situ chemical deposition procedure and subsequently dispersed into methyltrimethoxysilane-derived gels to yield a conductive graphite organosilicate composite. The composite was used as the electrode material to fabricate a three-dimensional InHCF-modified electrode. InHCF acts as a catalyst, graphite powder ensures conductivity by percolation, the silicate provides a rigid porous backbone and the methyl groups endow hydrophobicity and thus limit the wetting section of the modified electrode. The chemically modified electrode can electrocatalyze the oxidation of thiosulfate, and exhibits a good repeatability of surface-renewal by simple mechanical polishing, as well as simple preparation, good chemical and mechanical stability.

Sol-gel-derived carbon ceramic electrode containing 9,10-phenanthrenequinone, and its electrocatalytic activity toward iodate

9,10-Phenanthrenequinone (PQ) supported on graphite powder by adsorption was dispersed in propyltrimethoxysilane-derived gels to yield a conductive composite which was used as electrode material to fabricate a PQ-modified carbon ceramic electrode. In this configuration, PQ acts as a catalyst, graphite powder guarantees conductivity by percolation, the silicate provides a rigid porous backbone, and the propyl groups endow hydrophobicity and thus limit the wetting region of the modified electrode. Square-wave voltammetry was exploited to investigate the pH-dependent electrochemical behavior of the composite electrode and an almost Nernstian response was obtained from pH 0.42 to 6.84. Because the chemically modified electrode can electrocatalyze the reduction of iodate in acidic aqueous solution (pH 2.45), it was used as an amperometric sensor for the determination of iodate in table salt. The advantages of the electrode are that it can be polished in the event of surface fouling, it is simple to prepare, has excellent chemical and mechanical stability, and the reproducibility of surface-renewal is good.