High throughput approaches to mammalian cell culture process development

Commercial pressures to reduce the costs and timelines involved in bringing new medicines to market are driving investment in methods for high throughput bioprocess development. The establishment of mammalian cell culture processes for therapeutic antibody production is an area of particular interest. This is an experimentally intensive procedure initially involving evaluation of a large number of clones followed by optimisation and scale-up of cell culture conditions. In this thesis a high throughput microwell platform is established for use in early stage cell line selection and cell culture process development. It was first demonstrated that shaken 24 SRW microwells were suitable for batch suspension culture of a commercially available CHO cell line, provided that appropriate culture conditions were selected. However, a high rate of evaporation from the microwells was identified as a potential limiting factor. Further work with an industrial GS-CHO cell line led to the development of a fed-batch method. This used a combination of diluted liquid feeds and a ‘sandwich lid’ to counteract microwell liquid losses by water replacement and reduction of evaporation to negligible levels. This led to comparable cell growth and product formation kinetics as well as similar metabolite utilization kinetics in 24 SRW plates and conventional shake flasks. Engineering characterisation of 24 SRW, shake flask and laboratory scale (5L) stirred tank systems was subsequently performed, encompassing the oxygen mass transfer coefficient (kLa), mean energy dissipation rate (P/V), and liquid phase mixing time (tm). Mixing times in particular showed a strong dependence on the speed and diameter of orbital shaking while in general kLa values were sufficient (>1h-1) for oxygen transfer not to be rate limiting. Consequently it was suggested that matched liquid phase mixing times could be a suitable scale translation parameter for the types of small scale bioreactor formats used in early stage cell culture process development. It was subsequently demonstrated that at mixing times that promoted a homogeneous culture environment at each scale (tm = 5s) similar culture performance in all three bioreactor formats could be achieved using this engineering basis for two distinct cell culture processes involving different GS-CHO cell lines and alternative feeding methodologies. Peak viable cell densities achieved in stirred tank, shake flask and 24 SRW formats were 5.9, 6.7 and 6.4 x 106 cells mL-1 respectively, while antibody titres were 1.23, 0.81 and 0.88 g L-1. The higher IgG concentration in the stirred tank was attributed to tighter on-line pH control. The utilization rates for key metabolites were also closely matched. Finally the industrial relevance of this methodology was demonstrated through the successful parallel fed-batch cultivation of more than 50 GS-CHO cell lines. The rank order of cells based on product titre agreed closely with existing development procedures using shake flasks and static microwell plates. The microwell cell culture process presented here thus offers the potential for a considerable increase in throughput during the early stages of biopharmaceutical product development

Monster black holes

A combination of ground-based and spacecraft observations has uncovered two black holes of 10 billion solar masses in the nearby Universe. The finding sheds light on how these cosmic monsters co-evolve with galaxies.Comment: 2 pages, 1 figure, LaTeX. Published in Nature "News & Views

Bridging the gap between energy and the environment

Meeting the world’s energy demand is a major challenge for society over the coming century. To identify the most sustainable energy pathways to meet this demand, analysis of energy systems on which policy is based must move beyond the current primary focus on carbon to include a broad range of ecosystem services on which human well-being depends. Incorporation of a broad set of ecosystem services into the design of energy policy will differentiates between energy technology options to identify policy options that reconcile national and international obligations to address climate change and the loss of biodiversity and ecosystem services. In this paper we consider our current understanding of the implications of energy systems for ecosystem services and identify key elements of an assessment. Analysis must consider the full life cycle of energy systems, the territorial and international footprint, use a consistent ecosystem service framework that incorporates the value of both market and non-market goods, and consider the spatial and temporal dynamics of both the energy and environmental system. While significant methodological challenges exist, the approach we detail can provide the holistic view of energy and ecosystem services interactions required to inform the future of global energy policy

Disorder-specific functional abnormalities during sustained attention in youth with Attention Deficit Hyperactivity Disorder (ADHD) and with Autism

Attention Deficit Hyperactivity Disorder (ADHD) and Autism Spectrum Disorder (ASD) are often comorbid and share behavioural-cognitive abnormalities in sustained attention. A key question is whether this shared cognitive phenotype is based on common or different underlying pathophysiologies. To elucidate this question, we compared 20 boys with ADHD to 20 age and IQ matched ASD and 20 healthy boys using functional magnetic resonance imaging (fMRI) during a parametrically modulated vigilance task with a progressively increasing load of sustained attention. ADHD and ASD boys had significantly reduced activation relative to controls in bilateral striato–thalamic regions, left dorsolateral prefrontal cortex (DLPFC) and superior parietal cortex. Both groups also displayed significantly increased precuneus activation relative to controls. Precuneus was negatively correlated with the DLPFC activation, and progressively more deactivated with increasing attention load in controls, but not patients, suggesting problems with deactivation of a task-related default mode network in both disorders. However, left DLPFC underactivation was significantly more pronounced in ADHD relative to ASD boys, which furthermore was associated with sustained performance measures that were only impaired in ADHD patients. ASD boys, on the other hand, had disorder-specific enhanced cerebellar activation relative to both ADHD and control boys, presumably reflecting compensation. The findings show that ADHD and ASD boys have both shared and disorder-specific abnormalities in brain function during sustained attention. Shared deficits were in fronto–striato–parietal activation and default mode suppression. Differences were a more severe DLPFC dysfunction in ADHD and a disorder-specific fronto–striato–cerebellar dysregulation in ASD

Nucleotide supplementation: a randomised double-blind placebo controlled trial of IntestAidIB in people with Irritable Bowel Syndrome [ISRCTN67764449]

BACKGROUND: Dietary nucleotide supplementation has been shown to have important effects on the growth and development of cells which have a rapid turnover such as those in the immune system and the gastrointestinal tract. Work with infants has shown that the incidence and duration of diarrhoea is lower when nucleotide supplementation is given, and animal work shows that villi height and crypt depth in the intestine is increased as a result of dietary nucleotides. Dietary nucleotides may be semi-essential under conditions of ill-health, poor diet or stress. Since people with Irritable Bowel Syndrome tend to fulfil these conditions, we tested the hypothesis that symptoms would be improved with dietary nucleotide supplementation. METHODS: Thirty-seven people with a diagnosis of Irritable Bowel gave daily symptom severity ratings for abdominal pain, diarrhoea, urgency to have a bowel movement, incomplete feeling of evacuation after a bowel movement, bloating, flatulence and constipation for 28 days (baseline). They were then assigned to either placebo (56 days) followed by experimental (56 days) or the reverse. There was a four week washout period before crossover. During the placebo and experimental conditions participants took one 500 mg capsule three times a day; in the experimental condition the capsule contained the nutroceutical substances. Symptom severity ratings and psychological measures (anxiety, depression, illness intrusiveness and general health) were obtained and analysed by repeated measures ANOVAs. RESULTS: Symptom severity for all symptoms (except constipation) were in the expected direction of baseline>placebo>experimental condition. Symptom improvement was in the range 4 – 6%. A feeling of incomplete evacuation and abdominal pain showed the most improvement. The differences between conditions for diarrhoea, bloating and flatulence were not significant at the p < .05 level. There were no significant differences between the conditions for any of the psychological measures. CONCLUSION: Dietary nucleotide supplementation improves some of the symptoms of irritable bowel above baseline and placebo level. As expected, placebo effects were high. Apart from abdominal pain and urgency to have a bowel movement, the improvements, while consistent, are modest, and were not accompanied by improvements in any of the psychological measures. We suggest that the percentage improvement over and above the placebo effect is a physiological effect of the nucleotide supplement on the gut. The mechanisms by which these effects might improve symptoms are discussed

Dominance relationships and coalitionary aggression against conspecifics in female carrion crows

Funding: European Research Council (ERCStG-336536 FuncSpecGen to J.W.), the Swedish Research Council Vetenskapsrådet (621-2013-4510 to J.W.), Knut and Alice Wallenberg Foundation (to J.W.) and Tovetorp fieldstation through Stockholm University.Cooperation is a prevailing feature of many animal systems. Coalitionary aggression, where a group of individuals engages in coordinated behaviour to the detriment of conspecific targets, is a form of cooperation involving complex social interactions. To date, evidence has been dominated by studies in humans and other primates with a clear bias towards studies of male-male coalitions. We here characterize coalitionary aggression behaviour in a group of female carrion crows consisting of recruitment, coordinated chase, and attack. The individual of highest social rank liaised with the second most dominant individual to engage in coordinated chase and attack of a lower ranked crow on several occasions. Despite active intervention by the third most highly ranked individual opposing the offenders, the attack finally resulted in the death of the victim. All individuals were unrelated, of the same sex, and naive to the behaviour excluding kinship, reproduction, and social learning as possible drivers. Instead, the coalition may reflect a strategy of the dominant individual to secure long-term social benefits. Overall, the study provides evidence that members of the crow family engage in coordinated alliances directed against conspecifics as a possible means to manipulate their social environment.Publisher PDFPeer reviewe

Ravens Reconcile after Aggressive Conflicts with Valuable Partners

Reconciliation, a post-conflict affiliative interaction between former opponents, is an important mechanism for reducing the costs of aggressive conflict in primates and some other mammals as it may repair the opponents' relationship and reduce post-conflict distress. Opponents who share a valuable relationship are expected to be more likely to reconcile as for such partners the benefits of relationship repair should outweigh the risk of renewed aggression. In birds, however, post-conflict behavior has thus far been marked by an apparent absence of reconciliation, suggested to result either from differing avian and mammalian strategies or because birds may not share valuable relationships with partners with whom they engage in aggressive conflict. Here, we demonstrate the occurrence of reconciliation in a group of captive subadult ravens (Corvus corax) and show that it is more likely to occur after conflicts between partners who share a valuable relationship. Furthermore, former opponents were less likely to engage in renewed aggression following reconciliation, suggesting that reconciliation repairs damage caused to their relationship by the preceding conflict. Our findings suggest not only that primate-like valuable relationships exist outside the pair bond in birds, but that such partners may employ the same mechanisms in birds as in primates to ensure that the benefits afforded by their relationships are maintained even when conflicts of interest escalate into aggression. These results provide further support for a convergent evolution of social strategies in avian and mammalian species

Allocentric versus egocentric spatial memory in autism spectrum disorder

Individuals with Autism Spectrum Disorder (ASD) present difficulties in forming relations among items and context. This capacity for relational binding is also involved in spatial navigation and research on this topic in ASD is scarce and inconclusive. Using a computerised version of the Morris Water Maze task, ASD participants showed particular difficulties in performing viewpoint independent (allocentric) navigation, leaving viewpoint dependent navigation (egocentric) intact. Further analyses showed that navigation deficits were not related to poor visual short-term memory or mental rotation in the ASD group. The results further confirm the need of autistic individuals for support at retrieval and have important implications for the design of signposts and maps

GRIPS - Gamma-Ray Imaging, Polarimetry and Spectroscopy

We propose to perform a continuously scanning all-sky survey from 200 keV to 80 MeV achieving a sensitivity which is better by a factor of 40 or more compared to the previous missions in this energy range. The Gamma-Ray Imaging, Polarimetry and Spectroscopy (GRIPS) mission addresses fundamental questions in ESA's Cosmic Vision plan. Among the major themes of the strategic plan, GRIPS has its focus on the evolving, violent Universe, exploring a unique energy window. We propose to investigate $\gamma$-ray bursts and blazars, the mechanisms behind supernova explosions, nucleosynthesis and spallation, the enigmatic origin of positrons in our Galaxy, and the nature of radiation processes and particle acceleration in extreme cosmic sources including pulsars and magnetars. The natural energy scale for these non-thermal processes is of the order of MeV. Although they can be partially and indirectly studied using other methods, only the proposed GRIPS measurements will provide direct access to their primary photons. GRIPS will be a driver for the study of transient sources in the era of neutrino and gravitational wave observatories such as IceCUBE and LISA, establishing a new type of diagnostics in relativistic and nuclear astrophysics. This will support extrapolations to investigate star formation, galaxy evolution, and black hole formation at high redshifts.Comment: to appear in Exp. Astron., special vol. on M3-Call of ESA's Cosmic Vision 2010; 25 p., 25 figs; see also www.grips-mission.e

The Formation of the First Massive Black Holes

Supermassive black holes (SMBHs) are common in local galactic nuclei, and SMBHs as massive as several billion solar masses already exist at redshift z=6. These earliest SMBHs may grow by the combination of radiation-pressure-limited accretion and mergers of stellar-mass seed BHs, left behind by the first generation of metal-free stars, or may be formed by more rapid direct collapse of gas in rare special environments where dense gas can accumulate without first fragmenting into stars. This chapter offers a review of these two competing scenarios, as well as some more exotic alternative ideas. It also briefly discusses how the different models may be distinguished in the future by observations with JWST, (e)LISA and other instruments.Comment: 47 pages with 306 references; this review is a chapter in "The First Galaxies - Theoretical Predictions and Observational Clues", Springer Astrophysics and Space Science Library, Eds. T. Wiklind, V. Bromm & B. Mobasher, in pres