Protein loaded mesoporous silica spheres as a controlled delivery platform

Background The adsorption of bovine serum albumin (BSA) onto mesoporous silica spheres (MPS) synthesized from silica colloids was studied employing real time in situ measurements. The stabilities of the BSA at different pH values, their isoelectric points and zeta potentials were determined in order to probe the interactions between the protein and the mesoporous silica. Results The pore size of MPS was designed for protein, and this, coupled with an in depth understanding of the physico-chemical characteristics of the protein and MPS has yielded a better binding capacity and delivery profile. The adsorption isotherm at pH 4.2 fitted the Langmuir model and displayed the highest adsorption capacity (71.43 mg mL-1 MPS). Furthermore, the delivery rates of BSA from the MPS under physiological conditions were shown to be dependent on the ionic strength of the buffer and protein loading concentration. Conclusion Economics and scale-up considerations of mesoporous material synthesized via destabilization of colloids by electrolyte indicate the scaleability and commercial viability of this technology as a delivery platform for biopharmaceutical applications.

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.

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.

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.

A method to evaluate the response of the coronary circulation of perfused rat heart to adenosine

Exogenous adenosine causes a monophasic dilation of the coronary vessels in paced, perfused rat heart preparations. Because levels of endogenous adenosine in paced hearts may mask the presence of high potency adenosine receptors, we have developed a method to measure coronary vascular responses in a potassium-arrested heart. Hearts from adult male, Wistar rats were perfused at a constant flow rate of 10 mL/min in the nonrecirculating, Langendorff mode, using Krebs-Henseleit buffer. After 30 min, coronary perfusion pressure was 44 +/- 1 mmHg (mean +/- SEM). Hearts were then perfused with a modified Krebs-Henseleit buffer containing 35 mM potassium. Coronary perfusion pressure increased by 84 +/- 3 mmHg. Adenosine-induced reductions in coronary perfusion pressure were expressed as a percentage of the maximal increase in pressure produced by modified Krebs-Henseleit buffer from the equilibration level. A concentration-response curve for adenosine (n = 6) was biphasic and best described by the presence of two adenosine receptors, with negative log EC50 values of 8.8 +/- 0.3 and 4.3 +/- 0.1, representing 29 +/- 3 and 71 +/- 3%, respectively, of the observed response. Interstitial adenosine sampled by microdialysis during potassium arrest was 25% of the concentration found in paced hearts. Endogenous adenosine in nonarrested hearts may obscure the biphasic response of the coronary vessels to adenosine.

Bioaerosols associated with industrial green waste composting facilities and optimal microbiological indicators

This project developed and assessed a standard operating procedure for monitoring microbiological aerosol levels and dispersal from Australian industrial composting facilities. Development occurred via seasonal monitoring of such operations with evaluation of optimal microbial indicator organisms, sampling and analysis logistics. The resultant procedure allows practical end-user assessment of compost-associated bioaerosol levels, and potential health risks to proximal residential populations encroaching on such composting facilities and on-site industrial operations personnel.

The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse

Background The expression of biomass-degrading enzymes (such as cellobiohydrolases) in transgenic plants has the potential to reduce the costs of biomass saccharification by providing a source of enzymes to supplement commercial cellulase mixtures. Cellobiohydrolases are the main enzymes in commercial cellulase mixtures. In the present study, a cellobiohydrolase was expressed in transgenic corn stover leaf and assessed as an additive for two commercial cellulase mixtures for the saccharification of pretreated sugar cane bagasse obtained by different processes. Results Recombinant cellobiohydrolase in the senescent leaves of transgenic corn was extracted using a simple buffer with no concentration step. The extract significantly enhanced the performance of Celluclast 1.5 L (a commercial cellulase mixture) by up to fourfold on sugar cane bagasse pretreated at the pilot scale using a dilute sulfuric acid steam explosion process compared to the commercial cellulase mixture on its own. Also, the extracts were able to enhance the performance of Cellic CTec2 (a commercial cellulase mixture) up to fourfold on a range of residues from sugar cane bagasse pretreated at the laboratory (using acidified ethylene carbonate/ethylene glycol, 1-butyl-3-methylimidazolium chloride, and ball-milling) and pilot (dilute sodium hydroxide and glycerol/hydrochloric acid steam explosion) scales. We have demonstrated using tap water as a solvent (under conditions that mimic an industrial process) extraction of about 90% recombinant cellobiohydrolase from senescent, transgenic corn stover leaf that had minimal tissue disruption. Conclusions The accumulation of recombinant cellobiohydrolase in senescent, transgenic corn stover leaf is a viable strategy to reduce the saccharification cost associated with the production of fermentable sugars from pretreated biomass. We envisage an industrial-scale process in which transgenic plants provide both fibre and biomass-degrading enzymes for pretreatment and enzymatic hydrolysis, respectively.

Independent distractor frequency and age-of-acquisition dffects in picture-word interference: FMRI evidence for post-lexical and lexical accounts according to distractor type

In two fMRI experiments, participants named pictures with superimposed distractors that were high or low in frequency or varied in terms of age of acquisition. Pictures superimposed with low-frequency words were named more slowly than those superimposed with high-frequency words, and late-acquired words interfered with picture naming to a greater extent than early-acquired words. The distractor frequency effect (Experiment 1) was associated with increased activity in left premotor and posterior superior temporal cortices, consistent with the operation of an articulatory response buffer and verbal selfmonitoring system. Conversely, the distractor age-of-acquisition effect (Experiment 2) was associated with increased activity in the left middle and posterior middle temporal cortex, consistent with the operation of lexical level processes such as lemma and phonological word form retrieval. The spatially dissociated patterns of activity across the two experiments indicate that distractor effects in picture-word interference may occur at lexical or postlexical levels of processing in speech production.

Progressive dysgraphia in a case of posterior cortical atrophy

Dysgraphia (agraphia) is a common feature of posterior cortical atrophy (PCA). However, detailed analyses of these spelling and writing impairments are infrequently conducted. LM is a 59-year-old woman with dysgraphia associated with PCA. She presented with a two-year history of decline in her writing and dressmaking skills. A 3D T1-weighted MRI scan confirmed selective bi-parietal atrophy, with relative sparing of the hippocampi and other cortical regions. Analyses of LM's preserved and impaired spelling abilities indicated mild physical letter distortions and a significant spelling deficit characterised by letter substitutions, insertions, omissions, and transpositions that was systematically sensitive to word length while insensitive to real word versus nonword category, word frequency, regularity, imagery, grammatical class and ambiguity. Our findings suggest a primary graphemic buffer disorder underlies LM's spelling errors, possibly originating from disruption to the operation of a fronto-parietal network implicated in verbal working memory.

An experimental study of the effects of work stress, work control, and task information on adjustment

The present study was designed to examine the main and interactive effects of task demands, work control, and task information on levels of adjustment. Task demands, work control, and task information were manipulated in an experimental setting where participants completed a letter-sorting activity (N= 128). Indicators of adjustment included measures of positive mood, participants' perceptions of task performance, and task satisfaction. Results of the present study provided some support for the main effects of objective task demands, work control, and task information on levels of adjustment. At the subjective level of analysis, there was some evidence to suggest that work control and task information interacted in their effects on levels of adjustment. There was minimal support for the proposal that work control and task information would buffer the negative effects of task demands on adjustment. There was, however, some evidence to suggest that the stress-buffering role of subjective work control was more marked at high, rather than low, levels of subjective task information.

The effects of prediction, understanding, and control: A test of the stress antidote model

A study was undertaken to examine further the effects of perceived work control on employee adjustment. On the basis of the stress antidote model, it was proposed that high levels of prediction, understanding, and control of work-related events would have direct, indirect, and interactive effects on levels of employee adjustment. These hypotheses were tested in a short-term longitudinal study of 137 employees of a large retail organization. The stress antidote measures appeared to be indirectly related to employee adjustment, via their effects on perceptions of work stress. There was weak evidence for the proposal that prediction, understanding, and control would buffer the negative effects of work stress. Additional analyses indicated that the observed effects of prediction, understanding, and control were independent of employees' generalized control beliefs. However, there was no support for the proposal that the effects of the stress antidote measures would be dependent on employees' generalized control beliefs.

Optimized multilayer indium-free electrodes for organic photovoltaics

Flexible multilayer electrodes that combine high transparency, high conductivity, and efficient charge extraction have been deposited, characterised and used as the anode in organic solar cells. The anode consists of an AZO/Ag/AZO stack plus a very thin oxide interlayer whose ionization potential is fine-tuned by manipulating its gap state density to optimise charge transfer with the bulk heterojunction active layer consisting of poly(n-3- hexylthiophene-2,5-diyl) and phenyl-C61-butyric acid methyl ester (P3HT:BC61BM). The deposition method for the stack was compatible with the low temperatures required for polymer substrates. Optimisation of the electrode stack was achieved by modelling the optical and electrical properties of the device and a power conversion efficiency of 2.9% under AM1.5 illumination compared to 3.0% with an ITO-only anode and 3.5% for an ITO:PEDOT electrode. Dark I-V reverse bias characteristics indicate very low densities of occupied buffer states close to the HOMO level of the hole conductor, despite observed ionization potential being high enough. Their elimination should raise efficiency to that with ITO:PEDOT.

The effect of β-alanine and NaHCO3 co-ingestion on buffering capacity and exercise performance with high-intensity exercise in healthy males

Introduction β-alanine (BAl) and NaHCO3 (SB) ingestion may provide performance benefits by enhancing concentrations of their respective physiochemical buffer counterparts, muscle carnosine and blood bicarbonate, counteracting acidosis during intense exercise. This study examined the effect of BAl and SB co-supplementation as an ergogenic strategy during high-intensity exercise. Methods Eight healthy males ingested either BAl (4.8 g day−1 for 4 weeks, increased to 6.4 g day−1 for 2 weeks) or placebo (Pl) (CaCO3) for 6 weeks, in a crossover design (6-week washout between supplements). After each chronic supplementation period participants performed two trials, each consisting of two intense exercise tests performed over consecutive days. Trials were separated by 1 week and consisted of a repeated sprint ability (RSA) test and cycling capacity test at 110 % Wmax (CCT110 %). Placebo (Pl) or SB (300 mg kgbw−1) was ingested prior to exercise in a crossover design to creating four supplement conditions (BAl-Pl, BAl-SB, Pl–Pl, Pl-SB). Results Carnosine increased in the gastrocnemius (n = 5) (p = 0.03) and soleus (n = 5) (p = 0.02) following BAl supplementation, and Pl-SB and BAl-SB ingestion elevated blood HCO3 − concentrations (p < 0.01). Although buffering capacity was elevated following both BAl and SB ingestion, performance improvement was only observed with BAl-Pl and BAl-SB increasing time to exhaustion of the CCT110 % test 14 and 16 %, respectively, compared to Pl–Pl (p < 0.01). Conclusion Supplementation of BAl and SB elevated buffering potential by increasing muscle carnosine and blood bicarbonate levels, respectively. BAl ingestion improved performance during the CCT110 %, with no aggregating effect of SB supplementation (p > 0.05). Performance was not different between treatments during the RSA test.

Threat of resource loss: The role of self-regulation in adaptive task performance

Changes at work are often accompanied with the threat of, or actual, resource loss. Through an experiment, we investigated the detrimental effect of the threat of resource loss on adaptive task performance. Self-regulation (i.e., task focus and emotion control) was hypothesized to buffer the negative relationship between the threat of resource loss and adaptive task performance. Adaptation was conceptualized as relearning after a change in task execution rules. Threat of resource loss was manipulated for 100 participants undertaking an air traffic control task. Using discontinuous growth curve modeling, 2 kinds of adaptation—transition adaptation and reacquisition adaptation—were differentiated. The results showed that individuals who experienced the threat of resource loss had a stronger drop in performance (less transition adaptation) and a subsequent slower recovery (less reacquisition adaptation) compared with the control group who experienced no threat. Emotion control (but not task focus) moderated the relationship between the threat of resource loss and transition adaptation. In this respect, individuals who felt threatened but regulated their emotions performed better immediately after the task change (but not later on) compared with those individuals who felt threatened and did not regulate their emotions as well. However, later on, relearning (reacquisition adaptation) under the threat of resource loss was facilitated when individuals concentrated on the task at hand.

Fate of swallowed interleukin 8 and TNFα, and effect on the Wistar rat gut

To evaluate the passage of cytokines through the gastrointestinal tract, we investigated the digestion of interleukin-8 (IL-8) and tumour necrosis factor α (TNFα), in vitro and in vivo, and their propensity to induce intestinal inflammation. We serially immuno-assayed IL-8 and TNFα solutions co-incubated with each of three pancreatin preparations at pH 4.5 and pH 8. We gavaged IL-8, TNFα and marker into 15 Wistar rats, and measured their faecal cytokine concentrations by ELISA and histologically examined their guts. IL-8 immunoreactivity was extinguished by all pancreatin preparations after 1 h of incubation at 37 °C. TNFα concentration progressively fell from 1 to 4 h with all enzyme preparations. Buffer control samples maintained their cytokine concentrations throughout incubation. No IL-8 or TNFα was detected in any rat faecal pellets. There was no significant proinflammatory effect of the gavaged cytokines on rat intestine. IL-8 and TNFα in aqueous solution could well be fully digested in the CF gut when transit time is normal and exogenous enzymes are provided, although cytokines swallowed in viscous sputum may be protected from such digestion