Single step purification of plasmid DNA using peptide ligand affinity chromatography

Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide-monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels. © 2008 Elsevier B.V. All rights reserved.

Endotoxin removal : a critical step in vaccine production

A major drawback to the immunological potency of conventional vaccines, resulting in reduced level of immune responses, tissue injury, shock and high cytotoxicity, thus making their applications contraindicated in immunodeficiency diseases, is the presence of high contaminant concentrations in vaccine titers. Vaccine contamination arises from the simultaneous occurrence of competitive pathways resulting in the formation of other bio-products during cellular metabolism aside the pathways necessary for the production of vaccine molecules. One of such vaccine contaminating molecules is endotoxins which are mainly lipopolysaccharides (LPS) complexes found in the membrane of bacterial cell wall. The structural dynamics of these molecules make their removal from vaccine titers problematic, thus making vaccine endotoxin removal a major research endeavour. This presentation will discuss a novel technique for reducing the endotoxin level of vaccines. The technique commences with the disentanglement of endotoxin-vaccine molecular bonding and then capturing the vaccine molecules on an affinity monolith to separate the vaccine molecules from the endotoxins.

One step closer into the design of fall protective device for individuals fitted with a bone-anchored prosthesis

The consequences of falls are often dreadful for individuals with lower limb amputation using bone-anchored prosthesis.[1-5] Typically, the impact on the fixation is responsible for bending the intercutaneous piece that could lead to a complete breakage over time. .[3, 5-8] The surgical replacement of this piece is possible but complex and expensive. Clearly, there is a need for solid data enabling an evidence-based design of protective devices limiting impact forces and torsion applied during a fall. The impact on the fixation during an actual fall is obviously difficult to record during a scientific experiment.[6, 8-13] Consequently, Schwartze and colleagues opted for one of the next best options science has to offer: simulation with an able-bodied participant. They recorded body movements and knee impacts on the floor while mimicking several plausible falling scenarios. Then, they calculated the forces and moments that would be applied at four levels along the femur corresponding to amputation heights.[6, 8-11, 14-25] The overall forces applied during the falls were similar regardless of the amputation height indicating that the impact forces were simply translated along the femur. As expected, they showed that overall moments generally increased with amputation height due to changes in lever arm. This work demonstrates that devices preventing only against force overload do not require considering amputation height while those protecting against bending moments should. Another significant contribution is to provide, for the time, the magnitude of the impact load during different falls. This loading range is crucial to the overall design and, more precisely, the triggering threshold of protective devices. Unfortunately, the analysis of only a single able-bodied participant replicating falls limits greatly the generalisation of the findings. Nonetheless, this case study is an important milestone contributing to a better understanding of load impact during a fall. This new knowledge will improve the treatment, the safe ambulation and, ultimately, the quality of life of individuals fitted with bone-anchored prosthesis.

One-year clinical outcomes of a two-step surgical management for keratoconus-topography-guided photorefractive keratectomy/cross-linking after intrastromal corneal ring implantation

PURPOSE - To present the results of same-day topography-guided photorefractive keratectomy (TG-PRK) and corneal collagen cross-linking (CXL) after intrastromal corneal ring (ISCR) implantation in patients with keratoconus. METHODS - Thirty-three patients (41 eyes) aged between 19 and 45 years were included in this prospective study. All patients underwent a femtosecond laser-enabled (Intralase FS; Abbott Medical Optics, Inc.) placement of intracorneal ring segments (Kerarings; Mediphacos, Brazil). Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), and keratometry readings remained stable for 6 months. Same-day PRK and CXL was subsequently performed in all patients. RESULTS - After 12 months of completion of the procedure, mean UDVA in log of minimal angle of resolution was significantly improved (0.74±0.54-0.10±0.16); CDVA did not improve significantly but 85% of eyes maintained or gained multiple lines of CDVA; mean refraction spherical equivalent improved (from -3.03±1.98 to -0.04±0.99 D), all keratometry readings were significantly reduced, from preoperative values, but coma did not vary significantly from preoperative values. Central corneal thickness and corneal thickness at the thinnest point were significantly (P<0.0001) reduced from 519.76±29.33 and 501.87±31.50 preoperatively to 464.71±36.79 and 436.55±47.42 postoperatively, respectively. Safety and efficacy indices were 0.97 and 0.88, respectively. From 6 months up until more than 1 year of follow-up, further significant improvement was observed only for UDVA (P<0.0001). CONCLUSIONS - Same-day combined TG-PRK and CXL after ISCR implantation is a safe and effective option for improving visual acuity and visual function, and it halts the progression of the keratoconus. The improvements recorded after 6 months of follow-up were maintained or improved upon 1 year after the procedure.

Towards strongly consistent online HMM parameter estimation using one-step Kerridge inaccuracy

In this paper, we propose a novel online hidden Markov model (HMM) parameter estimator based on the new information-theoretic concept of one-step Kerridge inaccuracy (OKI). Under several regulatory conditions, we establish a convergence result (and some limited strong consistency results) for our proposed online OKI-based parameter estimator. In simulation studies, we illustrate the global convergence behaviour of our proposed estimator and provide a counter-example illustrating the local convergence of other popular HMM parameter estimators.

In-situ carbon control in the preparation of precursors to boron carbide by a non-aqueous solution technique

Synthesis of high quality boron carbide (B4C) powders is achieved by carbothermal reduction of boron oxide (B2O3) from a condensed boric acid (H3BO3)/polyvinyl acetate (PVAc) product. Precursor solutions are prepared via free radical polymerisation of vinyl acetate (VA) monomer in methanol in the presence of dissolved H3BO3. A condensed product is then formed by flash evaporation under vacuum. As excess VA monomer is removed at the evaporation step, the polymerisation time is used to manage availability of carbon for reaction. This control of carbon facilitates dispersion of H3BO3 in solution due to the presence of residual VA monomer. B4C powders with very low residual carbon are formed at temperatures as low as 1,250 °C with a 4 hour residence time.

Structural identification of a laboratory-based steel through truss cantilevered bridge with suspended span using a layered genetic algorithm with patternsearch step (GAPS)

Structural identification (St-Id) can be considered as the process of updating a finite element (FE) model of a structural system to match the measured response of the structure. This paper presents the St-Id of a laboratory-based steel through-truss cantilevered bridge with suspended span. There are a total of 600 degrees of freedom (DOFs) in the superstructure plus additional DOFs in the substructure. The St-Id of the bridge model used the modal parameters from a preliminary modal test in the objective function of a global optimisation technique using a layered genetic algorithm with patternsearch step (GAPS). Each layer of the St-Id process involved grouping of the structural parameters into a number of updating parameters and running parallel optimisations. The number of updating parameters was increased at each layer of the process. In order to accelerate the optimisation and ensure improved diversity within the population, a patternsearch step was applied to the fittest individuals at the end of each generation of the GA. The GAPS process was able to replicate the mode shapes for the first two lateral sway modes and the first vertical bending mode to a high degree of accuracy and, to a lesser degree, the mode shape of the first lateral bending mode. The mode shape and frequency of the torsional mode did not match very well. The frequencies of the first lateral bending mode, the first longitudinal mode and the first vertical mode matched very well. The frequency of the first sway mode was lower and that of the second sway mode was higher than the true values, indicating a possible problem with the FE model. Improvements to the model and the St-Id process will be presented at the upcoming conference and compared to the results presented in this paper. These improvements will include the use of multiple FE models in a multi-layered, multi-solution, GAPS St-Id approach.

Epitaxial graphene growth on 3C SiC by Si sublimation in UHV

This thesis is a step forward in understanding the growth of graphene, a single layer of carbon atoms, by annealing Silicon Carbide (SiC) thin films in Ultra High Vacuum. The research lead to the discovery that the details of the transition from SiC to graphene, providing, for the first time, atomic resolution images of the different stages of the transformation and a model of the growth. The epitaxial growth of graphene developed by this study is a cost effective procedure to obtain this material directly on Si chips, a breakthrough for the future electronic industry.

Barriers to good occupational health and safety (OHS) practices by small construction firms

The construction industry has long been considered to have unacceptably high injury and fatality rates. Previous research has shown that small construction companies sustain higher injury rates than large companies. However, despite the industry being dominated by a very large number of such small companies, little is known of their occupational health and safety (OHS) needs, practices and constraints. This paper takes a first step in aiming to identify the principal barriers that affect good OHS performance of small construction companies so that effective OHS practices can be developed to improve this in future. The contents of the literature are first summarised, in which three critical barriers to good OHS practice in small construction firms are proposed. They are : cost, time, lack of safety awareness and concern. The results of a questionnaire survey carried out with South East Queensland construction personnel are presented, which largely confirm what is suggested by the literature research and also succeed in providing an indication of their ranking in terms of importance and suggestions for overcoming these barriers. The research results provide a better understanding of the issues that restrict good OHS practice in small construction companies and potential measures for improvement.

Enhanced earth grid performance by improved conductor properties

Encroaching built environment with increased fault current levels is demanding a robust design approach and prolonged improved performance of the earth grid. With this in mind, the aim of the project was to perform a sensitivity analysis of the earth grid and an earthing performance evaluation with graphene coated conductors. Subsequent to these, a conceptual design to continuously monitor the performance of the earth grid was developed. In this study, earth grid design standards were compared to evaluate their appropriate use in determining the safety condition. A process to grow a thin film of graphene on the surface of cylindrical copper rods was developed to evaluate earthing performance in terms of conductivity and corrosion susceptibility.

A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens

Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 μg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

Directional synthesis of tin oxide@graphene nanocomposites via a one-step up-scalable wet-mechanochemical route for lithium ion batteries

Directional synthesis of SnO2@graphene nanocomposites via a one-step, low-cost, and up-scalable wetmechanochemical method is achieved using graphene oxide and SnCl2 as precursors. The graphene oxides are reduced to graphene while the SnCl2 is oxidized to SnO2 nanoparticles that are in situ anchored onto the graphene sheets evenly and densely, resulting in uniform SnO2@graphene nanocomposites. The prepared nanocomposites possess excellent electrochemical performance and outstanding cycling in Li-ion batteries.

A two-stage SVM method to predict membrane protein types by incorporating amino acid classifications and physicochemical properties into a general form of Chou's PseAAC

Membrane proteins play important roles in many biochemical processes and are also attractive targets of drug discovery for various diseases. The elucidation of membrane protein types provides clues for understanding the structure and function of proteins. Recently we developed a novel system for predicting protein subnuclear localizations. In this paper, we propose a simplified version of our system for predicting membrane protein types directly from primary protein structures, which incorporates amino acid classifications and physicochemical properties into a general form of pseudo-amino acid composition. In this simplified system, we will design a two-stage multi-class support vector machine combined with a two-step optimal feature selection process, which proves very effective in our experiments. The performance of the present method is evaluated on two benchmark datasets consisting of five types of membrane proteins. The overall accuracies of prediction for five types are 93.25% and 96.61% via the jackknife test and independent dataset test, respectively. These results indicate that our method is effective and valuable for predicting membrane protein types. A web server for the proposed method is available at http://www.juemengt.com/jcc/memty_page.php

Step based physical activity guidelines for preschool-aged children

OBJECTIVE Public health organizations recommend that preschool-aged children accumulate at least 3h of physical activity (PA) daily. Objective monitoring using pedometers offers an opportunity to measure preschooler's PA and assess compliance with this recommendation. The purpose of this study was to derive step-based recommendations consistent with the 3h PA recommendation for preschool-aged children. METHOD The study sample comprised 916 preschool-aged children, aged 3 to 6years (mean age=5.0+/-0.8years). Children were recruited from kindergartens located in Portugal, between 2009 and 2013. Children wore an ActiGraph GT1M accelerometer that measured PA intensity and steps per day simultaneously over a 7-day monitoring period. Receiver operating characteristic (ROC) curve analysis was used to identify the daily step count threshold associated with meeting the daily 3hour PA recommendation. RESULTS A significant correlation was observed between minutes of total PA and steps per day (r=0.76, p<0.001). The optimal step count for >/=3h of total PA was 9099 steps per day (sensitivity (90%) and specificity (66%)) with area under the ROC curve=0.86 (95% CI: 0.84 to 0.88). CONCLUSION Preschool-aged children who accumulate less than 9000 steps per day may be considered Insufficiently Active.