Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield

<p>Abstract</p> <p>Background</p> <p><it>Pichia pastoris </it>is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.</p> <p>Results</p> <p>Addition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of <it>P. pastoris </it>increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (μg OD₅₉₅^-1) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (<it>k_La</it>) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium.</p> <p>Conclusions</p> <p>We show that addition of a range of antifoaming agents to shake flask cultures of <it>P. pastoris </it>increases the total yield of the recombinant protein being produced. This is not only a simple method to increase the amount of protein in the culture, but our study also provides insight into how antifoams interact with microbial cell factories. Two mechanisms are apparent: one group of antifoams (Antifoam A, Antifoam C and J673A) increases the specific yield of GFP by increasing the total amount of protein produced and secreted per cell, whilst the second (P2000 or SB2121) increases the total yield by increasing the density of the culture.</p

Relationship between Greenland Ice Sheet surface speed and modeled effective pressure

Author Posting. © American Geophysical Union, 2018. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research: Earth Surface 123 (2018): 2258-2278, doi:10.1029/2017JF004581.We use a numerical subglacial hydrology model and remotely sensed observations of Greenland Ice Sheet surface motion to test whether the inverse relationship between effective pressure and regional melt season surface speeds observed at individual sites holds on a regional scale. The model is forced with daily surface runoff estimates for 2009 and 2010 across an ~8,000‐km2 region on the western margin. The overall subglacial drainage system morphology develops similarly in both years, with subglacial channel networks growing inland from the ice sheet margin and robust subglacial pathways forming over bedrock ridges. Modeled effective pressures are compared to contemporaneous regional surface speeds derived from TerraSAR‐X imagery to investigate spatial relationships. Our results show an inverse spatial relationship between effective pressure and ice speed in the mid‐melt season, when surface speeds are elevated, indicating that effective pressure is the dominant control on surface velocities in the mid‐melt season. By contrast, in the early and late melt seasons, when surface speeds are slower, effective pressure and surface speed have a positive relationship. Our results suggest that outside of the mid‐melt season, the influence of effective pressures on sliding speeds may be secondary to the influence of driving stress and spatially variable bed roughness.National Aeronautics and Space Administration (NASA). Grant Number: NXX10AI30G National Science Foundation (NSF) American Geophysical Union Horton Research Grant; National Science Foundation Graduate Research Fellowship; National Science Foundation's Office of Polar Programs (NSF‐OPP) Grant Numbers: PLR‐1418256, ARC‐1023364, ARC‐0520077; Woods Hole Oceanographic Institution's Ocean and Climate Change Institute (OCCI)2019-03-2

Effectiveness of interventions to support the early detection of skin cancer through skin self-examination: a systematic review and meta-analysis.

BACKGROUND: As skin cancer incidence rises, there is a need to evaluate early detection interventions by the public using skin self-examination (SSE); however, the literature focuses on primary prevention. No systematic reviews have evaluated the effectiveness of such SSE interventions. OBJECTIVES: To systematically examine, map, appraise and synthesize, qualitatively and quantitatively, studies evaluating the early detection of skin cancer, using SSE interventions. METHODS: This is a systematic review (narrative synthesis and meta-analysis) examining randomized controlled trials (RCTs) and quasiexperimental, observational and qualitative studies, published in English, using PRISMA and National Institute for Health and Care Excellence guidance. The MEDLINE, Embase and PsycINFO databases were searched through to April 2015 (updated in April 2018 using MEDLINE). Risk-of-bias assessment was conducted. RESULTS: Included studies (n = 18), totalling 6836 participants, were derived from 22 papers; these included 12 RCTs and five quasiexperiments and one complex-intervention development. More studies (n = 10) focused on targeting high-risk groups (surveillance) than those at no higher risk (screening) (n = 8). Ten (45%) study interventions were theoretically underpinned. All of the study outcomes were self-reported, behaviour related and nonclinical in nature. Meta-analysis demonstrated the impact of the intervention on the degree of SSE activity from five studies, especially in the short term (up to 4 months) (odds ratio 2·31, 95% confidence interval 1·90-2·82), but with small effect sizes. Risk-of-bias assessment indicated that 61% of the studies (n = 11) were of weak quality. CONCLUSIONS: Four RCTs and a quasiexperimental study indicate that some interventions can enhance SSE activity and so are more likely to aid early detection of skin cancer. However, the actual clinical impact remains unclear, and this is based on overall weak study (evidence) quality

Cooking for communities, children and cows: lessons learned from institutional cookstoves in Nepal

Despite a long history of Improved Cookstove (ICS) interventions by Non-Governmental Organizations, International Development partners and the Government of Nepal, the majority of rural Nepalese people cook on a traditional open fire for their large-scale cooking needs due to a significant lack of approved institutional-scale cooking solutions. Whilst 65.8% of rural Nepalese households cook with biomass as their primary fuel source to satisfy their personal energy needs, there is no information collected on institutional cooking use by the Government of Nepal. In this paper our main objective was to design, implement and evaluate a novel Institutional Improved Cookstove (IICS) to satisfy this gap and following its manufacturing and testing in a Government of Nepal approved test center, to identify the complex contextual factors that often override the technical capabilities of IICS. Our three-phase method combined qualitative and quantitative research approaches, as well as north-south collaborations involving a transdisciplinary research team to create an integrated systems approach taking into account the voices of all key energy stakeholders. Phase 1 included UK based co-design and testing at the University of Nottingham in 2017 to develop a novel IICS that could be used in rural Nepal. Phase 2 involved adapting the design to accommodate contextual factors highlighted by Nepalese partners and to meet testing requirements at a Government of Nepal approved testing center in late 2017. Phase 3 was conducted between December 2017 and April 2020 and focused on piloting the novel IICS in a range of locations, altitudes, socio-economic and cultural settings, monitoring sustained use and obtaining user feedback. We present our results through three case studies that highlight the highly contextualized nature of IICS adoption and sustained use, the importance of stacking, usability and cost savings, and a number of pathways to scale in an institutional setting

Understanding the current market enablers for Nepal’s biomass cookstove industry

This paper applies the market map tool to the Nepalese biomass cookstove market highlighting existing weaknesses in government policy, cookstove market chains and providing a framework for cookstove interventions that better address the social, economic and cultural needs of users. Findings indicate government policy actively promotes cooking technologies however, this may not result in sustained use. Tracking mechanisms focus on numbers of stoves disseminated rather than numbers used. Attention is given to links between the underdeveloped nature of the institutional cookstove market and restrictive government policy. The methodological approach involved semi-structured interviews, field observations and analysis of government policy documents. Abstract This paper applies the Market Map tool to the Nepalese biomass Improved Cookstove (ICS) sector highlighting existing weaknesses in government policy and biomass cookstove market chains to provide recommendations to better address the social, economic and cultural needs of users. This addresses the problem of low adoption rates of biomass ICS in Nepal. Our research objectives set out to explore the effectiveness of market maps designed for East Africa's ICS sector (Stevens et al., 2019) in Nepal, co-develop a revised market map for Nepal's biomass ICS sector, conduct a parallel process for institutional-scale biomass ICS and draw on the co-produced market map to inform policy and regulatory frameworks relating to biomass-fuelled ICS. The methodological approach involved reviewing cookstove-related policy documents and regulatory frameworks, undertaking 31 semi-structured interviews, analysing findings from an Institutional Top-Loading Down-Draft (TLUD) Natural Draft Gasifier Pilot study and co-developing the final market map in collaboration with key ICS stakeholders. The results indicate that although government policy actively promotes biomass ICS, this often results in cookstove 'stacking' rather than the sustained and exclusive use of clean cooking solutions necessary to promote health benefits. Attention is also focused on the underdeveloped nature of the institutional cookstove market. Our conclusions highlight the usefulness of market maps with a monitoring and evaluation element for identifying barriers to clean cooking uptake and facilitating product improvement by integrating end-user feedback

Phase Changes in Internally Mixed Maleic Acid/Ammonium Sulfate Aerosols

A temperature controlled flow tube system equipped with Fourier transform infrared (FTIR) detection of particle phase and relative humidity was used to measure the deliquescence and efflorescence of ammonium sulfate, maleic acid, and internally mixed maleic acid/ammonium sulfate particles. Our results indicate that maleic acid aerosols begin to take up water starting at a low relative humidity, ∼20%, and continue the constant uptake of water until the final deliquescence relative humidity (DRH), 89%, is reached. Internally mixed particles containing maleic acid and ammonium sulfate were found to deliquesce at a lower relative humidity (RH) than either of the pure species. Efflorescence studies indicated that while pure maleic acid particles crystallize at ∼18% RH, pure ammonium sulfate and all mixed aerosols effloresce at or just below 30% RH. Taken together, our results suggest that the presence of water-soluble organics internally mixed with ammonium sulfate aerosol could increase the range of conditions under which the aerosol is a solution

Modelling chemistry in the nocturnal boundary layer above tropical rainforest and a generalised effective nocturnal ozone deposition velocity for sub-ppbv NOx conditions

Measurements of atmospheric composition have been made over a remote rainforest landscape. A box model has previously been demonstrated to model the observed daytime chemistry well. However the box model is unable to explain the nocturnal measurements of relatively high [NO] and [O3], but relatively low observed [NO2]. It is shown that a one-dimensional (1-D) column model with simple O3 -NOx chemistry and a simple representation of vertical transport is able to explain the observed nocturnal concentrations and predict the likely vertical profiles of these species in the nocturnal boundary layer (NBL). Concentrations of tracers carried over from the end of the night can affect the atmospheric chemistry of the following day. To ascertain the anomaly introduced by using the box model to represent the NBL, vertically-averaged NBL concentrations at the end of the night are compared between the 1-D model and the box model. It is found that, under low to medium [NOx] conditions (NOx <1 ppbv), a simple parametrisation can be used to modify the box model deposition velocity of ozone, in order to achieve good agreement between the box and 1-D models for these end-of-night concentrations of NOx and O3. This parametrisation would could also be used in global climate-chemistry models with limited vertical resolution near the surface. Box-model results for the following day differ significantly if this effective nocturnal deposition velocity for ozone is implemented; for instance, there is a 9% increase in the following day’s peak ozone concentration. However under medium to high [NOx] conditions (NOx > 1 ppbv), the effect on the chemistry due to the vertical distribution of the species means no box model can adequately represent chemistry in the NBL without modifying reaction rate constants

Laser Welding of a Stent

We consider the problem of modelling the manufacture of a cylindrical Stent, in which layers of a plastic material are welded together by a Laser beam. We firstly set up the equations for this system and solve them by using a Finite Element method. We then look at various scalings which allow the equations to be simplified. The resulting equations are then solved analytically to obtain approximate solutions to the radial temperature profile and the averaged axial temperature profile

Risk of Developmental Disorders in Children Born at 32 to 38 Weeks' Gestation : A Meta-Analysis

CONTEXT: Very preterm birth (<32 weeks) is associated with increased risk of developmental disorders. Emerging evidence suggests children born 32 to 38 weeks might also be at risk. OBJECTIVES: To determine the relative risk and prevalence of being diagnosed with, or screening positive for, developmental disorders in children born moderately preterm, late preterm, and early term compared with term (≥37 weeks) or full term (39-40/41 weeks). DATA SOURCES: Medline, Embase, Psychinfo, Cumulative Index of Nursing, and Allied Health Literature. STUDY SELECTION: Reported ≥1 developmental disorder, provided estimates for children born 32 to 38 weeks. DATA EXTRACTION: A single reviewer extracted data; a 20% sample was second checked. Data were pooled using random-effects meta-analyses. RESULTS: Seventy six studies were included. Compared with term born children, there was increased risk of most developmental disorders, particularly in the moderately preterm group, but also in late preterm and early term groups: the relative risk of cerebral palsy was, for 32 to 33 weeks: 14.1 (95% confidence intervals [CI]: 12.3-16.0), 34 to 36 weeks: 3.52 (95% CI: 3.16-3.92) and 37 to 38 weeks: 1.44 (95% CI: 1.32-1.58). LIMITATIONS: Studies assessed children at different ages using varied criteria. The majority were from economically developed countries. All were published in English. Data were variably sparse; subgroup comparisons were sometimes based on single studies. CONCLUSIONS: Children born moderately preterm are at increased risk of being diagnosed with or screening positive for developmental disorders compared with term born children. This association is also demonstrated in late preterm and early term groups but effect sizes are smaller

Structure-guided design and optimization of small molecules targeting the protein-protein interaction between the von hippel-lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities

E3 ubiquitin ligases are attractive targets in the ubiquitin-proteasome system, however, the development of small-molecule ligands has been rewarded with limited success. The von Hippel-Lindau protein (pVHL) is the substrate recognition subunit of the VHL E3 ligase that targets HIF-1α for degradation. We recently reported inhibitors of the pVHL:HIF-1α interaction, however they exhibited moderate potency. Herein, we report the design and optimization, guided by X-ray crystal structures, of a ligand series with nanomolar binding affinities