A high performance liquid chromatography and electrospray ionization mass spectrometry method for the analysis of the natural medicine, Forsythia suspensa

Samples of Forsythia suspensa from raw (Laoqiao) and ripe (Qingqiao) fruit were analyzed with the use of HPLC-DAD and the EIS-MS techniques. Seventeen peaks were detected, and of these, twelve were identified. Most were related to the glucopyranoside molecular fragment. Samples collected from three geographical areas (Shanxi, Henan and Shandong Provinces), were discriminated with the use of hierarchical clustering analysis (HCA), discriminant analysis (DA), and principal component analysis (PCA) models, but only PCA was able to provide further information about the relationships between objects and loadings; eight peaks were related to the provinces of sample origin. The supervised classification models-K-nearest neighbor (KNN), least squares support vector machines (LS-SVM), and counter propagation artificial neural network (CP-ANN) methods, indicated successful classification but KNN produced 100% classification rate. Thus, the fruit were discriminated on the basis of their places of origin.

Versatility of polymethacrylate monoliths for chromatographic purification of biomolecules

Polymethacrylate monoliths, specifically poly(glycidyl methacrylate-co-ethylene dimethacrylate) or poly(GMA-co-EDMA) monoliths, are a new generation of chromatographic supports and are significantly different from conventional particle-based adsorbents, membranes, and other monolithic supports for biomolecule purification. Similar to other monoliths, polymethacrylate monoliths possess large pores which allow convective flow of mobile phase and result in high flow rates at reduced pressure drop, unlike particulate supports. The simplicity of the adsorbent synthesis, pH resistance, and the ease and flexibility of tailoring their pore size to that of the target biomolecule are the key properties which differentiate polymethacrylate monoliths from other monoliths. Polymethacrylate monoliths are endowed with reactive epoxy groups for easy functionalization (with anion-exchange, hydrophobic, and affinity ligands) and high ligand retention. In this review, the structure and performance of polymethacrylate monoliths for chromatographic purification of biomolecules are evaluated and compared to those of other supports. The development and use of polymethacrylate monoliths for research applications have grown rapidly in recent times and have enabled the achievement of high through-put biomolecule purification on semi-preparative and preparative scales.

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.

Large-volume methacrylate monolith for plasmid purification : process engineering approach to synthesis and application

The extent of exothermicity associated with the construction of large-volume methacrylate monolithic columns has somewhat obstructed the realisation of large-scale rapid biomolecule purification especially for plasmid-based products which have proven to herald future trends in biotechnology. A novel synthesis technique via a heat expulsion mechanism was employed to prepare a 40 mL methacrylate monolith with a homogeneous radial pore structure along its thickness. Radial temperature gradient was recorded to be only 1.8 °C. Maximum radial temperature recorded at the centre of the monolith was 62.3 °C, which was only 2.3 °C higher than the actual polymerisation temperature. Pore characterisation of the monolithic polymer showed unimodal pore size distributions at different radial positions with an identical modal pore size of 400 nm. Chromatographic characterisation of the polymer after functionalisation with amino groups displayed a persistent dynamic binding capacity of 15.5 mg of plasmid DNA/mL. The maximum pressure drop recorded was only 0.12 MPa at a flow rate of 10 mL/min. The polymer demonstrated rapid separation ability by fractionating Escherichia coli DH5α-pUC19 clarified lysate in only 3 min after loading. The plasmid sample collected after the fast purification process was tested to be a homogeneous supercoiled plasmid with DNA electrophoresis and restriction analysis.

Performance of R-N(R′)-R″ functionalised poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolithic sorbent for plasmid DNA adsorption

High-throughput plasmid DNA (pDNA) manufacture is obstructed predominantly by the performance of conventional stationary phases. For this reason, the search for new materials for fast chromatographic separation of pDNA is ongoing. A poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (GMA-EGDMA) monolithic material was synthesised via a thermal-free radical reaction, functionalised with different amino groups from urea, 2-chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) and ammonia in order to investigate their plasmid adsorption capacities. Physical characterisation of the monolithic polymer showed a macroporous polymer having a unimodal pore size distribution pivoted at 600 nm. Chromatographic characterisation of the functionalised polymers using pUC19 plasmid isolated from E. coli DH5α-pUC19 showed a maximum plasmid adsorption capacity of 18.73 mg pDNA/mL with a dissociation constant (KD) of 0.11 mg/mL for GMA-EGDMA/DEAE-Cl polymer. Studies on ligand leaching and degradation demonstrated the stability of GMA-EGDMA/DEAE-Cl after the functionalised polymers were contacted with 1.0 M NaOH, which is a model reagent for most 'cleaning in place' (CIP) systems. However, it is the economic advantage of an adsorbent material that makes it so attractive for commercial purification purposes. Economic evaluation of the performance of the functionalised polymers on the grounds of polymer cost (PC)/mg pDNA retained endorsed the suitability of GMA-EGDMA/DEAE-Cl polymer.

Enhancing methacrylate-monolith-based downstream processes to champion plasmid DNA production

Increasing numbers of preclinical and clinical studies are utilizing pDNA (plasmid DNA) as the vector. In addition, there has been a growing trend towards larger and larger doses of pDNA utilized in human trials. The growing demand on pDNA manufacture leads to pressure to make more in less time. A key intervention has been the use of monoliths as stationary phases in liquid chromatography. Monolithic stationary phases offer fast separation to pDNA owing to their large pore size, making pDNA in the size range from 100 nm to over 300 nm easily accessible. However, the convective transport mechanism of monoliths does not guarantee plasmid purity. The recovery of pure pDNA hinges on a proper balance in the properties of the adsorbent phase, the mobile phase and the feedstock. The effects of pH and ionic strength of binding buffer, temperature of feedstock, active group density and the pore size of the stationary phase were considered as avenues to improve the recovery and purity of pDNA using a methacrylate-based monolithic adsorbent and Escherichia coli DH5α-pUC19 clarified lysate as feedstock. pDNA recovery was found to be critically dependent on the pH and ionic strength of the mobile phase. Up to a maximum of approx. 92% recovery was obtained under optimum conditions of pH and ionic strength. Increasing the feedstock temperature to 80°C increased the purity of pDNA owing to the extra thermal stability associated with pDNA over contaminants such as proteins. Results from toxicological studies of the plasmid samples using endotoxin standard (E. coli 0.55:B5 lipopolysaccharide) show that endotoxin level decreases with increasing salt concentration. It was obvious that large quantities of pure pDNA can be obtained with minimal extra effort simply by optimizing process parameters and conditions for pDNA purification.

Investigating the link between carbon dioxide emissions and transport-related social exclusion in rural Northern Ireland

Carbon dioxide (CO2) is considered the most harmful of the greenhouse gases. Despite policy efforts, transport is the only sector experiencing an increase in the level of CO2 emissions and thereby possesses a major threat to sustainable development. In contrast, a reduced level of mobility has been associated with an increasing risk of being socially excluded. However, despite being the two key elements in transport policy, little effort has so far been made to investigate the links between CO2 emissions and social exclusion. This research contributes to this gap by analysing data from 157 weekly activity-travel diaries collected in rural Northern Ireland. CO2 emission levels were calculated using average speed models for different modes of transport. Regression analyses were then conducted to identify the socio-spatial patterns associated with these CO2 emissions, mode choice behaviour, and patterns of participation in activities. This research found that despite emitting a higher level of carbon dioxide, groups in rural areas possess the risk of being socially excluded due to their higher levels of mobility.

Assessment and application of clustering techniques to atmospheric particle number size distribution for the purpose of source apportionment

Long-term measurements of particle number size distribution (PNSD) produce a very large number of observations and their analysis requires an efficient approach in order to produce results in the least possible time and with maximum accuracy. Clustering techniques are a family of sophisticated methods which have been recently employed to analyse PNSD data, however, very little information is available comparing the performance of different clustering techniques on PNSD data. This study aims to apply several clustering techniques (i.e. K-means, PAM, CLARA and SOM) to PNSD data, in order to identify and apply the optimum technique to PNSD data measured at 25 sites across Brisbane, Australia. A new method, based on the Generalised Additive Model (GAM) with a basis of penalised B-splines, was proposed to parameterise the PNSD data and the temporal weight of each cluster was also estimated using the GAM. In addition, each cluster was associated with its possible source based on the results of this parameterisation, together with the characteristics of each cluster. The performances of four clustering techniques were compared using the Dunn index and Silhouette width validation values and the K-means technique was found to have the highest performance, with five clusters being the optimum. Therefore, five clusters were found within the data using the K-means technique. The diurnal occurrence of each cluster was used together with other air quality parameters, temporal trends and the physical properties of each cluster, in order to attribute each cluster to its source and origin. The five clusters were attributed to three major sources and origins, including regional background particles, photochemically induced nucleated particles and vehicle generated particles. Overall, clustering was found to be an effective technique for attributing each particle size spectra to its source and the GAM was suitable to parameterise the PNSD data. These two techniques can help researchers immensely in analysing PNSD data for characterisation and source apportionment purposes.

Children, violence, and social exclusion: Negotiation of everyday insecurity in a Colombian barrio

Discourses on in/security are often concerned with structures and meta-narratives of the state and other institutions; however, such attention misses the complexities of the everyday consequences of insecurity. In Colombia’s protracted conflict, children are disproportionately affected yet rarely consulted, rendering it difficult to account for their experiences in meaningful ways. This article draws on fieldwork conducted with conflict-affected children in an informal barrio community on the periphery of Colombia’s capital, Bogotá, to explore how children articulate experiences of insecurity. It examines how stereotypes of violence and delinquency reinforce insecurity; how multiple violences impact young people’s lives; and how children themselves conceive of responses to these negative experiences. These discussions are underpinned by a feminist commitment of attention to the margins and engage with those for whom insecurity is a daily phenomenon. The effects of deeply embedded insecurity, violence, and fear for young people in Colombia require a more nuanced theoretical engagement with notions of insecurity, as well as the complexities of connections and dissonances within everyday life.

Specificity and context in post-exercise recovery: It is not a one-size-fits-all approach

The concept of specificity of exercise prescription and training is a longstanding and widely accepted foundation of the exercise sciences. Simply, the principle holds that training adaptations are achieved relative to the stimulus applied. That is, the manipulation of training variables (e.g. intensity or loading, mode, volume and frequency) directly influences the acute training stimulus, and so the long-term adaptive response (Young et al., 2001; Bird et al., 2005). Translating this concept to practice then recommends that exercise be prescribed specific to the desired outcomes, and the more closely this is achieved, the greater the performance gain is likely to be. However, the cardiovascular and metabolic adaptations traditionally associated with long, slow distance training types, similarly achieved using high-intensity training methods (for a review see Gibala et al., 2012), highlights understanding of underlying physiology as paramount for effective training program design. Various other factors including illness, sleep and psychology also impact on the training stimulus (Halson, 2014) and must be managed collectively with appropriate post-exercise recovery to continue performance improvements and reduce overtraining and injury risks (Kenttä and Hassmén, 1998).

A laboratory assessment of the choice of vessel size under individual transferable quota regimes

This paper examines the effect of individual transferable quota regimes on technology choice, such as choice of vessel size, by using the laboratory experiment method. We find that even if vessel sizes change over time, the quota price can converge to the fundamental value conditioned on the vessels chosen. We also find that subjects choose their vessel type to maximise their profits based on the quota trading prices in the previous period. This result implies that the efficiency of quota markets in the beginning period is important because any inefficiency in quota markets may affect vessel sizes in ensuing periods. Moreover, we find that the initial allocations may significantly influence vessel sizes through two channels: first, a higher initial allocation to a subject increases the likelihood that the subject invests in a large-sized vessel; second, the quota price may be higher and more unstable under unequal allocation than under equal allocation; thus, whether the allocation is equal influences subjects' choice of vessel type. © 2014 Australian Agricultural and Resource Economics Society Inc.

Size-resolved particle distribution and gaseous concentrations by real-world road tunnel measurement

Measurements of aerosol particle number size distributions (15-700 nm), CO and NOx were performed in a bus tunnel, Australia. Daily mean particle size distributions of mixed diesel/CNG (Compressed Natural Gas) buses traffic flow were determined in 4 consecutive measurement days. EFs (Emission Factors) of Particle size distribution of diesel buses and CNG buses were obtained by MLR (Multiple Linear Regression) methods, particle distributions of diesel buses and CNG buses were observed as single accumulation mode and nuclei-mode separately. Particle size distributions of mixed traffic flow were decomposed by two log-normal fitting curves for each 30 minutes interval mean scans, all the mix fleet PSD emission can be well fitted by the summation of two log-normal distribution curves, and these were composed of nuclei mode curve and accumulation curve, which were affirmed as the CNG buses and diesel buses PN emission curves respectively. Finally, particle size distributions of diesel buses and CNG buses were quantified by statistical whisker-box charts. For log-normal particle size distribution of diesel buses, accumulation mode diameters were 74.5～87.5nm, geometric standard deviations were 1.89～1.98. As to log-normal particle size distribution of CNG buses, nuclei-mode diameters were 21～24 nm, geometric standard deviations were 1.27～1.31.

Field size consistency of nominally matched linacs

Given that there is increasing recognition of the effect that submillimetre changes in collimator position can have on radiotherapy beam dosimetry, this study aimed to evaluate the potential variability in small field collimation that may exist between otherwise matched linacs. Field sizes and field output factors were measured using radiochromic film and an electron diode, for jaw- and MLC-collimated fields produced by eight dosimetrically matched Varian iX linacs (Varian Medical Systems, Palo Alto, USA). This study used nominal sizes from 0.6×0.6 to 10×10 cm215 , for jaw-collimated fields,and from 1×1 to 10×10 cm216 , for MLC-collimated fields, delivered from a zero (head up, beam directed vertically downward) gantry angle. Differences between the field sizes measured for the eight linacs exceeded the uncertainty of the film measurements and the repositioning uncertainty of the jaws and MLCs on one linac. The dimensions of fields defined by MLC leaves were more consistent between linacs, while also differing more from their nominal values than fields defined by orthogonal jaws. The field output factors measured for the different linacs generally increased with increasing measured field size for the nominal 0.6×0.6 and 1×1 cm2 fields, and became consistent between linacs for nominal field sizes of 2×2 cm2 25 and larger. The inclusion in radiotherapy treatment planning system beam data of small field output factors acquired in fields collimated by jaws (rather than the more-reproducible MLCs), associated with either the nominal or the measured field sizes, should be viewed with caution. The size and reproducibility of the fields (especially the small fields) used to acquire treatment planning data should be investigated thoroughly as part of the linac or planning system commissioning process. Further investigation of these issues, using different linac models, collimation systems and beam orientations, is recommended.

Effect of carrier size on the dispersion of salmeterol xinafoate from interactive mixtures

The objective of this study was to determine the influence of lactose carrier size on drug dispersion of salmeterol xinafoate (SX) from interactive mixtures. SX dispersion was measured by using the fine particle fractions determined by a twin stage impinger attached to a Rotahaler1. The particle size of the lactose carrier in the SX interactive mixtures was varied using a range of commercial inhalation-grade lactoses. In addition, differing size fractions of individual lactose samples were achieved by dry sieving. The dispersion ofSXappeared to increase as the particle size of the lactose carrier decreased for the mixtures prepared from different particle size commercial samples of lactose and from different sieve fractions of the same lactose. Fine particles of lactose (<5 mm) associated with the lactose carrier were removed from the carrier surface by a wet decantation process to produce lactose samples with low but similar concentrations of fine lactose particles. The fine particle fractions of SX in mixtures prepared with the decanted lactose decreased significantly (analysis of variance, p<0.001) and the degree of dispersion became independent of the volume mean diameter of the carriers (analysis of variance, p<0.05). The dispersion behavior is therefore associated with the presence of fine adhered particles associated with the carriers and the inherent size of the carrier itself has little influence on dispersion.