662 resultados para Reproducing Transformation Method
Resumo:
We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.
Resumo:
We report the first successful Agrobacterium-mediated transformation of Australian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature embryos were used as target tissues. The binary vectors contained hph (encoding hygromycin resistance) or bar (encoding herbicide resistance) as the selectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivatives of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gene is interrupted by the castor bean catalase 1 intron, produced a 4-fold higher number of independent transgenic lines compared to that produced with the use of strain EHA101 carrying the binary vector pIG121-Hm wherein the CaMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold higher β-glucuronidase (GUS) activity (derivatives of binary vector pWBVec8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modified SCSV promoters produced GUS activity comparable to that produced by the Ubiquitin promoter. Progeny analysis (R1) for hygromycin resistance and GUS activity with selected lines showed both Mendelian and non-Mendelian segregation. Lines showing very high levels of GUS activity in T0 showed a reduced level of GUS activity in their T1 progeny, while lines with moderate levels of GUS activity showed increased levels in T1 progeny. Stable heritable green fluorescent protein (GFP) expression was also observed in few transgenic plants produced with the binary vector pTO134 which had the CaMV35S promoter-driven selectable marker gene bar and a modified CaMV35S promoter-driven reporter gene sgfpS65T.
Resumo:
In binary vectors, the antibiotic resistance gene used for selection of transformed plant cells is also usually expressed in the transforming Agrobacterium cells. This expression gives the bacterium antibiotic resistance, an unnecessary advantage on selective medium containing the antibiotic. Insertion of a castor bean catalase-1 (CAT-1) gene intron or a Parasponia andersonii haemoglobin gene intron into the coding region of the selectable marker gene, hph, completely abolished the expression of the gene in Agrobacterium, rendering it susceptible to hygromycin B. Use of these modified binary vectors minimized the overgrowth of Agrobacterium during plant transformation. Both of the introns were correctly spliced in plant cells and significantly enhanced hph gene expression in transformed rice tissue. The presence of these introns in the hph coding sequence not only maintained the selection efficiency of the hph gene, but with the CAT-1 intron also substantially increased the frequency of rice transformation. Transgenic lines with an intron-hph gene generally contained fewer gene copies and produced substantially more mRNA of the predicted size. Our results also indicate that transgenic plants with many copies of the transgene were more likely to show gene silencing than plants with 1-3 copies.
Resumo:
The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbil-gus plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs. Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T 1 generation.
Resumo:
Near-infrared spectroscopy (NIRS) calibrations were developed for the discrimination of Chinese hawthorn (Crataegus pinnatifida Bge. var. major) fruit from three geographical regions as well as for the estimation of the total sugar, total acid, total phenolic content, and total antioxidant activity. Principal component analysis (PCA) was used for the discrimination of the fruit on the basis of their geographical origin. Three pattern recognition methods, linear discriminant analysis, partial least-squares-discriminant analysis, and back-propagation artificial neural networks, were applied to classify and compare these samples. Furthermore, three multivariate calibration models based on the first derivative NIR spectroscopy, partial least-squares regression, back-propagation artificial neural networks, and least-squares-support vector machines, were constructed for quantitative analysis of the four analytes, total sugar, total acid, total phenolic content, and total antioxidant activity, and validated by prediction data sets.
Resumo:
Articular cartilage is the load-bearing tissue that consists of proteoglycan macromolecules entrapped between collagen fibrils in a three-dimensional architecture. To date, the drudgery of searching for mathematical models to represent the biomechanics of such a system continues without providing a fitting description of its functional response to load at micro-scale level. We believe that the major complication arose when cartilage was first envisaged as a multiphasic model with distinguishable components and that quantifying those and searching for the laws that govern their interaction is inadequate. To the thesis of this paper, cartilage as a bulk is as much continuum as is the response of its components to the external stimuli. For this reason, we framed the fundamental question as to what would be the mechano-structural functionality of such a system in the total absence of one of its key constituents-proteoglycans. To answer this, hydrated normal and proteoglycan depleted samples were tested under confined compression while finite element models were reproduced, for the first time, based on the structural microarchitecture of the cross-sectional profile of the matrices. These micro-porous in silico models served as virtual transducers to produce an internal noninvasive probing mechanism beyond experimental capabilities to render the matrices micromechanics and several others properties like permeability, orientation etc. The results demonstrated that load transfer was closely related to the microarchitecture of the hyperelastic models that represent solid skeleton stress and fluid response based on the state of the collagen network with and without the swollen proteoglycans. In other words, the stress gradient during deformation was a function of the structural pattern of the network and acted in concert with the position-dependent compositional state of the matrix. This reveals that the interaction between indistinguishable components in real cartilage is superimposed by its microarchitectural state which directly influences macromechanical behavior.
Resumo:
In a people-to-people matching systems, filtering is widely applied to find the most suitable matches. The results returned are either too many or only a few when the search is generic or specific respectively. The use of a sophisticated recommendation approach becomes necessary. Traditionally, the object of recommendation is the item which is inanimate. In online dating systems, reciprocal recommendation is required to suggest a partner only when the user and the recommended candidate both are satisfied. In this paper, an innovative reciprocal collaborative method is developed based on the idea of similarity and common neighbors, utilizing the information of relevance feedback and feature importance. Extensive experiments are carried out using data gathered from a real online dating service. Compared to benchmarking methods, our results show the proposed method can achieve noticeable better performance.
Resumo:
This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between the drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from a dry powder inhaler (DPI) formulation. Initially model silica probes of approximately 4 μm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-Ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres preattached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.
Resumo:
Robust descriptor matching across varying lighting conditions is important for vision-based robotics. We present a novel strategy for quantifying the lighting variance of descriptors. The strategy works by utilising recovered low dimensional mappings from Isomap and our measure of the lighting variance of each of these mappings. The resultant metric allows different descriptors to be compared given a dataset and a set of keypoints. We demonstrate that the SIFT descriptor typically has lower lighting variance than other descriptors, although the result depends on semantic class and lighting conditions.
Resumo:
A novel method of matching stiffness and continuous variable damping of an ECAS (electronically controlled air suspension) based on LQG (linear quadratic Gaussian) control was proposed to simultaneously improve the road-friendliness and ride comfort of a two-axle school bus. Taking account of the suspension nonlinearities and target-height-dependent variation in suspension characteristics, a stiffness model of the ECAS mounted on the drive axle of the bus was developed based on thermodynamics and the key parameters were obtained through field tests. By determining the proper range of the target height for the ECAS of the fully-loaded bus based on the design requirements of vehicle body bounce frequency, the control algorithm of the target suspension height (i.e., stiffness) was derived according to driving speed and road roughness. Taking account of the nonlinearities of a continuous variable semi-active damper, the damping force was obtained through the subtraction of the air spring force from the optimum integrated suspension force, which was calculated based on LQG control. Finally, a GA (genetic algorithm)-based matching method between stepped variable damping and stiffness was employed as a benchmark to evaluate the effectiveness of the LQG-based matching method. Simulation results indicate that compared with the GA-based matching method, both dynamic tire force and vehicle body vertical acceleration responses are markedly reduced around the vehicle body bounce frequency employing the LQG-based matching method, with peak values of the dynamic tire force PSD (power spectral density) decreased by 73.6%, 60.8% and 71.9% in the three cases, and corresponding reduction are 71.3%, 59.4% and 68.2% for the vehicle body vertical acceleration. A strong robustness to variation of driving speed and road roughness is also observed for the LQG-based matching method.
Resumo:
Cognitive impairment and physical disability are common in Parkinson’s disease (PD). As a result diet can be difficult to measure. This study aimed to evaluate the use of a photographic dietary record (PhDR) in people with PD. During a 12-week nutrition intervention study, 19 individuals with PD kept 3-day PhDRs on three occasions using point-and-shoot digital cameras. Details on food items present in the PhDRs and those not photographed were collected retrospectively during an interview. Following the first use of the PhDR method, the photographer completed a questionnaire (n=18). In addition, the quality of the PhDRs was evaluated at each time point. The person with PD was the sole photographer in 56% of the cases, with the remainder by the carer or combination of person with PD and the carer. The camera was rated as easy to use by 89%, keeping a PhDR was considered acceptable by 94% and none would rather use a “pen and paper” method. Eighty-three percent felt confident to use the camera again to record intake. Of the photos captured (n=730), 89% were of adequate quality (items visible, in-focus), while only 21% could be used alone (without interview information) to assess intake. Over the study, 22% of eating/drinking occasions were not photographed. PhDRs were considered an easy and acceptable method to measure intake among individuals with PD and their carers. The majority of PhDRs were of adequate quality, however in order to quantify intake the interview was necessary to obtain sufficient detail and capture missing items.
Resumo:
Access to dietetic care is important in chronic disease management and innovative technologies assists in this purpose. Photographic dietary records (PhDR) using mobile phones or cameras are valid and convenient for patients. Innovations in providing dietary interventions via telephone and computer can also inform dietetic practice. Three studies are presented. A mobile phone method was validated by comparing energy intake (EI) to a weighed food record and a measure of energy expenditure (EE) obtained using the doubly labelled water technique in 10 adults with T2 diabetes. The level of agreement between mean (±sd) energy intake mobile phone (8.2±1.7 MJ) and weighed record (8.5±1.6 MJ) was high (p=0.392), however EI/EE for both methods gave similar levels of under-reporting (0.69 and 0.72). All subjects preferred using the mobile phone vs. weighed record. Nineteen individuals with Parkinsons disease kept 3-day PhDRs on three occasions using point-and-shoot digital cameras over a 12 week period. The camera was rated as easy to use by 89%, keeping a PhDR was considered acceptable by 94% and none would rather use a “pen and paper” method. Eighty-three percent felt confident to use the camera again to record intake. An interactive, automated telephone system designed to coach people with T2 diabetes to adopt and maintain diabetes self-care behaviours, including nutrition, showed trends for improvements in total fat, saturated fat and vegetable intake of the intervention group compared to control participants over 6 months. Innovative technologies are acceptable to patients with chronic conditions and can be incorporated into dietetic care.
Resumo:
Suspension bridges meet the steadily growing demand for lighter and longer bridges in today’s infrastructure systems. These bridges are designed to have long life spans, but with age, their main cables and hangers could suffer from corrosion and fatigue. There is a need for a simple and reliable procedure to detect and locate such damage, so that appropriate retrofitting can be carried out to prevent bridge failure. Damage in a structure causes changes in its properties (mass, damping and stiffness) which in turn will cause changes in its vibration characteristics (natural frequencies, modal damping and mode shapes). Methods based on modal flexibility, which depends on both the natural frequencies and mode shapes, have the potential for damage detection. They have been applied successfully to beam and plate elements, trusses and simple structures in reinforced concrete and steel. However very limited applications for damage detection in suspension bridges have been identified to date. This paper examines the potential of modal flexibility methods for damage detection and localization of a suspension bridge under different damage scenarios in the main cables and hangers using numerical simulation techniques. Validated finite element model (FEM) of a suspension bridge is used to acquire mass normalized mode shape vectors and natural frequencies at intact and damaged states. Damage scenarios will be simulated in the validated FE models by varying stiffness of the damaged structural members. The capability of damage index based on modal flexibility to detect and locate damage is evaluated. Results confirm that modal flexibility based methods have the ability to successfully identify damage in suspension bridge main cables and hangers.
Resumo:
Community-based arts and media movements have been intsrumental in building population-wide creative capacity for cultural development, social participation and social transformation in many parts of the world. Digital storytelling is a form of media practice that was pioneered in the United States at the intersection of these movements. It is described here as a ‘co-creative’ media production method. This description aims to differentiate the approaches to collaborative content creation that are used in community cultural development (CCD) and community media movements from those valued in professional and consumer modes of media production. Yet, the products of co-creative practices, such as digital stories, do not circulate widely through existing media networks or through the newer social media networks that Australian CCD and community media movements anticipated by at least twenty years. The complex politics of story ownership are one of a number of factors that often render ‘publication’ a secondary consideration in the making of digital stories. The possibility of ‘downstream’ use and re-use of stories in other networks is not usually considered in initial planning and development processes. As landmark projects such as Capture Wales indicate, even where stories are made for broadcast outcomes, television can be a problematic window for exhibiting digital stories. Scepticism about the brave new world of reality television and user generated content also circulates in digital storytelling networks, especially when it comes to ethical concerns for managing the risks of harm associated with widespread distribution of digital stories to indiscriminate publics. This publication reports on a collaborative action research project that took a closer look at some of the constraints relating to the problems of re-purposing digital stories for television. It focussed on ‘best practice’ for managing the risks of harm to storytellers in the process of re-purposing digital stories for broadcast on community television.
Resumo:
Composite steel-concrete structures experience non-linear effects which arise from both instability-related geometric non-linearity and from material non-linearity in all of their component members. Because of this, conventional design procedures cannot capture the true behaviour of a composite frame throughout its full loading range, and so a procedure to account for those non-linearities is much needed. This paper therefore presents a numerical procedure capable of addressing geometric and material non-linearities at the strength limit state based on the refined plastic hinge method. Different material non-linearity for different composite structural components such as T-beams, concrete-filled tubular (CFT) and steel-encased reinforced concrete (SRC) sections can be treated using a routine numerical procedure for their section properties in this plastic hinge approach. Simple and conservative initial and full yield surfaces for general composite sections are proposed in this paper. The refined plastic hinge approach models springs at the ends of the element which are activated when the surface defining the interaction of bending and axial force at first yield is reached; a transition from the first yield interaction surface to the fully plastic interaction surface is postulated based on a proposed refined spring stiffness, which formulates the load-displacement relation for material non-linearity under the interaction of bending and axial actions. This produces a benign method for a beam-column composite element under general loading cases. Another main feature of this paper is that, for members containing a point of contraflexure, its location is determined with a simple application of the method herein and a node is then located at this position to reproduce the real flexural behaviour and associated material non-linearity of the member. Recourse is made to an updated Lagrangian formulation to consider geometric non-linear behaviour and to develop a non-linear solution strategy. The formulation with the refined plastic hinge approach is efficacious and robust, and so a full frame analysis incorporating geometric and material non-linearity is tractable. By way of contrast, the plastic zone approach possesses the drawback of strain-based procedures which rely on determining plastic zones within a cross-section and which require lengthwise integration. Following development of the theory, its application is illustrated with a number of varied examples.