Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

Background - Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). Results - Functional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. Conclusions - The production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields.

Pyrolysis of polyamide 6 under catalytic conditions and its application to reutilization of carpets

ε-caprolactam is a monomer of high value. Therefore, the chemical reutilization of polyamide 6 containing carpets for ε-caprolactam recovery offers some economic benefit and is performed on a technical scale with the help of the Zimmer-process. By this process polyamide 6 is depolymerized with steam and phosphoric acid. An alternative to this process is the thermal depolymerization - catalyzed or non-catalyzed. To investigate this alternative in more detail, the formal kinetic parameters of (i) the thermal depolymerization of polyamide 6, (ii) the thermal depolymerization in presence of sodium/potassium hydoxide, and (iii) the thermal depolymerization in presence of phosphoric acid are determined in this work. Based on the kinetics of the catalyzed or non-catalyzed depolymerization a stepwise pyrolysis procedure is designed by which the formation of ε-caprolactam from polyamide 6 can be separated from the formation of other pyrolysis products. © 2001 Elsevier Science B.V.

Impact in contact lenses and the anterior eye:challenges prevailing in 2015

As we settle into a new year, this second issue of Contact Lens and Anterior Eye allows us to reflect on how new research in this field impacts our understanding, but more importantly, how we use this evidence basis to enhance our day to day practice, to educate the next generation of students and to construct the research studies to deepen our knowledge still further. The end of 2014 saw the publication of the UK governments Research Exercise Framework (REF) which ranks Universities in terms of their outputs (which includes their paper, publications and research income), environment (infrastructure and staff support) and for the first time impact (defined as “any effect on, change or benefit to the economy, society, culture, public policy or services, health, the environment or quality of life, beyond academia” [8]). The REF is a process of expert review, carried out in 36 subject-based units of assessment, of which our field is typically submitted to the Allied Health, Dentistry, Nursing and Pharmacy panel. Universities that offer Optometry did very well with Cardiff, Manchester and Aston in the top 10% out of the 94 Universities that submitted to this panel (Grade point Average ranked order). While the format of the new exercise (probably in 2010) to allocate the more than £2 billion of UK government research funds is yet to be determined, it is already rumoured that impact will contribute an even larger proportion to the weighting. Hence it is even more important to reflect on the impact of our research. In this issue, Elisseef and colleagues [5] examine the intriguing potential of modifying a lens surface to allow it to bind to known wetting agents (in this case hyaluronic acid) to enhance water retention. Such a technique has the capacity to reduced friction between the lens surface and the eyelids/ocular surface, presumably leading to higher comfort and less reason for patients to discontinue with lens wear. Several papers in this issue report on the validity of new high precision, fast scanning imaging and quantification equipment, utilising techniques such as Scheimpflug, partial coherence interferometry, aberrometry and video allowing detailed assessment of anterior chamber biometry, corneal topography, corneal biomechanics, peripheral refraction, ocular aberrations and lens fit. The challenge is how to use this advanced instrumentation which is becoming increasingly available to create real impact. Many challenges in contact lenses and the anterior eye still prevail in 2015 such as: -While contact lens and refractive surgery complications are relatively rare, they are still too often devastating to the individual and their quality of life (such as the impact and prognosis of patients with Acanthmoeba Keratitis reported by Jhanji and colleagues in this issue [7]). How can we detect those patients who are going to be affected and what modifications do we need to make to contact lenses and patient management prevent this occurring? -Drop out from contact lenses still occurs at a rapid rate and symptoms of dry eye seem to be the leading cause driving this discontinuation of wear [1] and [2]. What design, coating, material and lubricant release mechanism will make a step change in end of day comfort in particular? -Presbyopia is a major challenge to hassle free quality vision and is one of the first signs of ageing noticed by many people. As an emmetrope approaching presbyopia, I have a vested interest in new medical devices that will give me high quality vision at all distances when my arms won’t stretch any further. Perhaps a new definition of presbyopia could be when you start to orientate your smartphone in the landscape direction to gain the small increase in print size needed to read! Effective accommodating intraocular lenses that truly mimic the pre-presbyopic crystalline lenses are still a way off [3] and hence simultaneous images achieved through contact lenses, intraocular lenses or refractive surgery still have a secure future. However, splitting light reaching the retina and requiring the brain to supress blurred images will always be a compromise on contrast sensitivity and is liable to cause dysphotopsia; so how will new designs account for differences in a patient's task demands and own optical aberrations to allow focused patient selection, optimising satisfaction? -Drug delivery from contact lenses offers much in terms of compliance and quality of life for patients with chronic ocular conditions such as glaucoma, dry eye and perhaps in the future, dry age-related macular degeneration; but scientific proof-of-concept publications (see EIShaer et al. [6]) have not yet led to commercial products. Part of this is presumably the regulatory complexity of combining a medical device (the contact lens) and a pharmaceutical agent. Will 2015 be the year when this innovation finally becomes a reality for patients, bringing them an enhanced quality of life through their eye care practitioners and allowing researchers to further validate the use of pharmaceutical contact lenses and propose enhancements as the technology matures? -Last, but no means least is the field of myopia control, the topic of the first day of the BCLA's Conference in Liverpool, June 6–9th 2015. The epidemic of myopia is a blight, particularly in Asia, with significant concerns over sight threatening pathology resulting from the elongated eye. This is a field where real impact is already being realised through new soft contact lens optics, orthokeratology and low dose pharmaceuticals [4], but we still need to be able to better predict which technique will work best for an individual and to develop new techniques to retard myopia progression in those who don’t respond to current treatments, without increasing their risk of complications or the treatment impacting their quality of life So what will your New Year's resolution be to make 2015 a year of real impact, whether by advancing science or applying the findings published in journals such as Contact Lens and Anterior Eye to make a real difference to your patients’ lives?

Kinetic study of the pyrolysis of miscanthus and its acid hydrolysis residue by thermogravimetric analysis

The kinetic parameters of the pyrolysis of miscanthus and its acid hydrolysis residue (AHR) were determined using thermogravimetric analysis (TGA). The AHR was produced at the University of Limerick by treating miscanthus with 5 wt.% sulphuric acid at 175 °C as representative of a lignocellulosic acid hydrolysis product. For the TGA experiments, 3 to 6 g of sample, milled and sieved to a particle size below 250 μm, were placed in the TGA ceramic crucible. The experiments were carried out under non-isothermal conditions heating the samples from 50 to 900 °C at heating rates of 2.5, 5, 10, 17 and 25 °C/min. The activation energy (EA) of the decomposition process was determined from the TGA data by differential analysis (Friedman) and three isoconversional methods of integral analysis (Kissinger–Akahira–Sunose, Ozawa–Flynn–Wall, Vyazovkin). The activation energy ranged from 129 to 156 kJ/mol for miscanthus and from 200 to 376 kJ/mol for AHR increasing with increasing conversion. The reaction model was selected using the non-linear least squares method and the pre-exponential factor was calculated from the Arrhenius approximation. The results showed that the best fitting reaction model was the third order reaction for both feedstocks. The pre-exponential factor was in the range of 5.6 × 1010 to 3.9 × 10+ 13 min− 1 for miscanthus and 2.1 × 1016 to 7.7 × 1025 min− 1 for AHR.

Effect of oscillatory flow on nucleation kinetics of butyl paraben

More than 165 induction times of butyl paraben-ethanol solution in a batch moving fluid oscillation baffled crystallizer with various amplitudes (1-9 mm) and frequencies (1.0-9.0 Hz) have been determined to study the effect of COBR operating conditions on nucleation. The induction time decreases with increasing amplitude and frequency at power density below about 500 W/m3; however, a further increase of the frequency and amplitude leads to an increase of the induction time. The interfacial energies and pre-exponential factors in both homogeneous and heterogeneous nucleation are determined by classical nucleation theory at oscillatory frequency 2.0 Hz and amplitudes of 3 or 5 mm both with and without net flow. To capture the shear rate conditions in oscillatory flow crystallizers, a large eddy simulation approach in a computational fluid dynamics framework is applied. Under ideal conditions the shear rate distribution shows spatial and temporal periodicity and radial symmetry. The spatial distributions of the shear rate indicate an increase of average and maximum values of the shear rate with increasing amplitude and frequency. In continuous operation, net flow enhances the shear rate at most time points, promoting nucleation. The mechanism of the shear rate influence on nucleation is discussed.

Pre-existent expectancy effects in the relationship between caffeine and performance

The present study investigated the impact of pre-existent expectancy regarding the effects of the caffeine load of a drink and the perception of the caffeine content on subjective mood and vigilance performance. Caffeine deprived participants (N=25) were tested in four conditions (within subjects design), using a 2 × 2 design, with caffeine load and information regarding the caffeine content of the drink. In two sessions, they were given caffeinated coffee and in two were given decaffeinated coffee. Within these two conditions, on one occasion they were given accurate information about the drink and on the other they were given inaccurate information about the drink. Mood and vigilance performance were assessed post ingestion. Caffeine was found to enhance performance, but only when participants were accurately told they were receiving it. When decaffeinated coffee was given, performance was poorer, irrespective of expectancy. However, when caffeine was given, but participants were told it was decaffeinated coffee, performance was as poor as when no caffeine had been administered. There were no easily interpretable effects on mood. The pharmacological effects of caffeine appear to act synergistically with expectancy. © 2010.

Measurements of stress-optic coefficient and Young's modulus in PMMA fibers drawn under different conditions

We have systematically measured the differential stress-optic coefficient, ΔC, and Young's modulus, E, in a number of PMMA fibers drawn with different stress, ranging from 2 up to 27 MPa. Effect of temperature annealing on those parameters was also investigated. ΔC was determined in transverse illumination by measuring the dependence of birefringence on additional axial stress applied to the fiber. Our results show that ΔC in PMMA fibers has a negative sign and ranges from -4.5 to -1.5×10-12 Pa -1 depending on the drawing stress. Increase of the drawing stress results in greater initial fiber birefringence and lower ΔC. The dependence of ΔC and initial birefringence upon drawing stress is nonlinear and gradually saturates for higher drawing stress. Moreover, we find that ΔC is linearly proportional to initial fiber birefringence and that annealing the fiber has no impact on the slope of this dependence. On the other hand, no clear dependence was observed between the fiber drawing stress and the Young's modulus of the fibers as measured using microscopic digital image correlation with the fibers tensioned using an Instron tension tester. © 2010 SPIE.