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Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort

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Synthetic and Systems Biotechnology 2020年第3期5卷 137-144页

作者： Alex J.Spice Rochelle Aw Daniel G.Bracewell karen m.polizzi Department of Chemical Engineering Imperial College LondonLondonUK Imperial College Centre for Synthetic Biology Imperial College LondonUK Department of Biochemical Engineering University College LondonLondonUK

A renaissance in cell-free protein synthesis(CFPS)is underway,enabled by the acceleration and adoption of synthetic biology *** has emerged as a powerful platform technology for synthetic gene network design,biosensing and on-demand *** primarily of bacterial origin,cell-free extracts derived from a variety of host organisms have been explored,aiming to capitalise on cellular diversity and the advantageous properties associated with those ***,cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields,despite the potential for improved protein folding and posttranslational *** we describe further development of a Pichia pastoris cell-free platform,a widely used expression host in both academia and the biopharmaceutical *** a minimised Design of Experiments(DOE)approach,we were able to increase the productivity of the system by improving the composition of the complex reaction *** was achieved in a minimal number of experimental runs,within the constraints of the design and without the need for liquid-handling *** doing so,we were able to estimate the main effects impacting productivity in the system and increased the protein synthesis of firefly luciferase and the biopharmaceutical HSA by 4.8-fold and 3.5-fold,*** study highlights the ***-based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs.

关键词： Cell-free protein synthesis Pichia pastoris Synthetic biology Design of experiments(DOE)

Investigating the consequences of asymmetric endoplasmic reticulum inheritance in Saccharomyces cerevisiae under stress using a combination of single cell measurements and mathematical modelling

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Synthetic and Systems Biotechnology 2018年第1期3卷 64-75页

作者： Felix R.H.Jonas Kate E.Royle Rochelle Aw Guy-Bart V.Stan karen m.polizzi Department of Bioengineering Imperial College LondonLondonSW72AZUnited Kingdom Imperial College Centre for Synthetic Biology LondonSW72AZUnited Kingdom Department of Life Sciences Imperial College LondonLondonSW72AZUnited Kingdom

Adaptation allows organisms to maintain a constant internal environment,which is optimised for *** unfolded protein response(UPR)is an example of a feedback loop that maintains endoplasmic reticulum(ER)homeostasis,and is characteristic of how adaptation is often mediated by transcriptional *** more recent discovery of asymmetric division in maintaining ER homeostasis,however,is an example of how alternative non-transcriptional pathways can exist,but are overlooked by gold standard transcriptomic or proteomic population-based *** this study,we have used a combination of fluorescent reporters,flow cytometry and mathematical modelling to explore the relative roles of asymmetric cell division and the UPR in maintaining ER *** low ER stress,asymmetric division leaves daughter cells with an ER deficiency,necessitating activation of the UPR and prolonged cell cycle during which they can recover ER functionality before *** analysis of and simulation results from our mathematical model reinforce the experimental observations that low ER stress primarily impacts the growth rate of the daughter *** results demonstrate the interplay between homeostatic pathways and the importance of exploring sub-population dynamics to understand population adaptation to quantitatively different stresses.

关键词： Adaptation Asymmetric cell division Endoplasmic reticulum stress Unfolded protein response Saccharomyces cerevisiae

Evaluation of transfection methods for transient gene expression in Chinese hamster ovary cells

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Advances in Bioscience and Biotechnology 2013年第12期4卷 1013-1019页

作者： Si Nga Sou karen m. polizzi Cleo Kontoravdi Centre for Process Systems Engineering Department of Chemical Engineering Imperial College London London UK Department of Life Sciences Imperial College London London UK Centre for Synthetic Biology and Innovation Imperial College London London UK Department of Life Sciences Imperial College London London UK Centre for Synthetic Biology and Innovation Imperial College London London UK Centre for Process Systems Engineering Department of Chemical Engineering Imperial College London London UK

Three transfection reagents, Lipofectamine® 2000, TransIT-PRO® and linear 25 kDa polyethylenimine were evaluated for transient expression of enhanced green fluorescent protein in Chinese hamster ovary cells. TransIT-PRO® was found to be more efficient under the examined conditions, but comes at an increased cost compared to the widely used PEI.

关键词： Transient Gene Expression Transfection Efficiency Chinese Hamster Ovary Cells mRNA Stability Temperature Shift

TEACHING VISUAL SCRIPTING IN BIm:A CASE STUDY USING A PANEL CONTROLLED BY SOLAR ANGLES

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Journal of Green Building 2018年第1期13卷 115-137页

作者： karen m.Kensek University of Southern California School of Architecture Watt Hall#204Los AngelesCA 90089-029

Programming and scripting can be used to activate a 3D parametric model to create a more intelligent and flexible building information model. There has been a trend in the building industry towards the use of visual scripting that allow users to create customized, flexible, and powerful programs without having to first learn how to write traditional code. Using visual scripting, users graphically interact with program elements instead of typing lines of text-based code. Nodes are created and virtually wired together;they can be numbers, sliders for adjusting values, operators and functions, list manipulation tools, graphic creators, and other types. Text based coding programs such as Python can also be used for the creation of custom nodes when greater flexibility is desired. Examples from professional firms include scripts that help automate work in the office to increase efficiency and accuracy (e.g. create escape routes, renumber rooms by levels, create documentation), assist in form generation (e.g. parametric design of metal panels, rebar generation, coordination between Revit and Rhino), analyze BIm files (e.g. terminal airflow, visual loads and capacity), and provide analysis results (e.g. daylighting, thermal comfort, window optimization). One can learn the basic steps of learning a visual programming language through the use of Dynamo within Autodesk Revit. The example used is for a façade component that changes based on the sun’s altitude.

关键词： BIm building information modeling visual programming scripting Revit Dynamo teaching

Regulation ofβ2-adrenoceptors in brain glia:implications for neuroinflammatory and degenerative disorders

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Neural Regeneration Research 2020年第11期15卷 2035-2036页

作者： karen m.Ryan Andrew Harkin Trinity College Institute of Neuroscience Trinity College Dublin Neuropsychopharmacology Research Group School of Pharmacy and Pharmaceutical Sciences&Trinity College Institute of NeuroscienceTrinity College DublinDublinIreland

Noradrenaline:Within the central nervous system(CNS),the primary source of the catecholamine neurotransmitter noradrenaline is the locus coeruleus(LC)in the pontine tegmentum,with LC neurons projecting to almost all regions of the brain and spinal *** its release from LC neurons,noradrenaline has wide ranging *** example,noradrenaline is the endogenous agonist for G-coupledα-andβ-adrenoceptors that are expressed on many cell types,including neurons and glia,in both the peripheral nervous system and *** is via these receptors that noradrenaline exerts its anti-inflammatory and neurotrophic effects in the *** additionally has adrenoceptor-independent neuroprotective actions,and as such plays a role in free radical scavenging and reducing oxidative stress(Feinstein et al.,2016).

关键词： adrenoceptor protective endogenous

Vitamin D improves inflammatory bowel disease outcomes:Basic science and clinical review

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World Journal of Gastroenterology 2014年第17期20卷 4934-4947页

作者： Krista m Reich Richard N Fedorak karen madsen karen I Kroeker Division of Gastroenterology University of AlbertaEdmontonAlberta T6G 2X8Canada

Vitamin D deficiency is commonly diagnosed among patients with inflammatory bowel disease (IBD). Patients with IBD are at risk of low bone density and increased fractures due to low vitamin D levels, long standing disease, and frequent steroid exposures; as a result, it is well established that vitamin D supplementation in this population is important. There is increasing support for the role of vitamin D in strengthening the innate immune system by acting as an immunomodulator and reducing inflammation in experimental and human IBD. The active form of vitamin D, 1,25(OH)D3, acts on T cells to promote T helper (Th)2/regulatory T responses over Th1/Th17 responses; suppresses dendritic cell inflammatory activity; induces antibacterial activity; and regulates cytokine production in favor of an anti-inflammatory response. murine and human IBD studies support a therapeutic role of vitamin D in IBD. Risk factors for vitamin D deficiency in this population include decreased sunlight exposure, disease duration, smoking, and genetics. Vitamin D normalization is associated with reduced risk of relapse, reduced risk of IBD-related surgeries, and improvement in quality of life. Vitamin D is an inexpensive supplement which has been shown to improve IBD outcomes. However, further research is required to determine optimal serum vitamin D levels which will achieve beneficial immune effects, and stronger evidence is needed to support the role of vitamin D in inducing disease response and remission, as well as maintaining this improvement in patients’ disease states.

关键词： Vitamin D Inflammatory bowel disease Immune response Inflammation Cytokines Supplementation

Predicting phosphorus sorption isotherm parameters in soil using data of routine laboratory tests

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Pedosphere 2021年第5期31卷 694-704页

作者： Kathleen S.DUNNE Nicholas m.HOLDEN karen DALY Teagasc Johnstown CastleEnvironmental Research CentreCo.Wexford Y35TC97(Ireland) UCD School of Biosystems and Food Engineering University College DublinBelfieldDublin 4 D04VIW8(Ireland)

Knowledge of phosphorus(P)sorption dynamics across different soil types could direct agronomic and environmental management of *** objective of this study was to predict P isotherm parameters for a national soil population using data of routine laboratory *** and Freundlich sorption parameters were calculated from two different ranges(0-25 and 0-50 mg P L^(-1))using an archive of representative agricultural soil types from *** linear regression(mLR)identified labile forms of aluminium(Al)and iron(Fe),organic matter(Om),cation exchange capacity(CEC),and clay as significant *** and Freundlich sorption capacities,Freundlich affinity constant,and Langmuir buffer capacity were predicted reliably,with R^(2) of independent validation>*** isotherm parameters were predicted from P sorbed at a single concentration of 50 mg P L^(-1)(S_(50)).An mLR prediction of P sorption maximum in the 0-50 mg P L-1 range was achieved,to an accurate standard,using S_(50),Om,and mehlich-3 Fe(R^(2) of independent calibration and validation being 0.91 and 0.95,respectively).Using Giles’four shapes of isotherms(C,L,H,and S),L non-strict-and C-shaped isotherm curves accounted for 64% and 27% of the soils,*** clustering identified a separation of isotherm curves influenced by two ranges of mehlich-3 *** with a low range of mehlich-3 Al(2.5-698 mg kg^(-1))had no incidence of rapid sorption(C shape).Single point indices,Al,or available soil data make the regression approach a feasible way of predicting Langmuir parameters that could be included with standard agronomic soil P testing.

关键词： available phosphorus binding fertilizer soil test sorption maximum

Non-steroidal anti-inflammatory drugs and statins in relation to colorectal cancer risk

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World Journal of Gastroenterology 2009年第19期15卷 2336-2339页

作者： mazyar Shadman Polly A Newcomb John m Hampton karen J Wernli Amy Trentham-Dietz Cancer Prevention Program Fred Hutchinson Cancer Research CenterSeattle 98109United States Department of Epidemiology University of WashingtonSeattle 98195United States Polly A Newcomb University of Wisconsin Paul PCarbone Comprehensive Cancer CenterMadison 53726United States

AIm： To investigate the association between individual or combined use of non-steroidal anti-inflammatory drugs （NSAIDs） or statins and colorectal cancer ***： In a population-based case-control study in women, we examined the association between NSAIDs and statin use and the risk of colorectal cancers. We further investigated whether the use of statins modifies the protective effect of NSAIDs. Female cases （n = 669）of colorectal cancer aged 50-74 years were identified from a storewide registry in Wisconsin during 1999-2001. Community control women （n = 1375） were randomly selected from lists of licensed drivers and medicare beneficiaries. medication use and risk factor information were gathered during a structured telephone interview. A multivariable logistic regression model was used to calculate odds ratio （OR） and 95% confidence interval （CI）.RESULTS： Overall, NSAIDs users had a 30% reduction in risk of colorectal cancer （95% CI： 0.56-0.88）. Statin use was not associated with colorectal cancer risk （OR = 1.17, 95% CI： 0.74-1.85）, regardless of structural type （lipophilic or hydrophilic）, duration of use, or recency. There was no evidence of an interaction between NSAIDs and statins and colorectal cancer risk （P-interaction = 0.28）.CONCLUSION： Although our results confirm the inverse association between NSAIDs use and colorectal cancer risk, they do not support a risk reduction in statin users, or an interaction effect of combined NSAIDs and statin use.

关键词： Non-steroidal anti-inflammatory drugs Statin Colorectal cancer Cancer prevention Chemoprevention

Cx26 heterozygous mutations:role in hyperacusis and vulnerability to noise

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Signal Transduction and Targeted Therapy 2023年第8期8卷 3551-3552页

作者： karen Tawk mehdi Abouzari Department of Otolaryngology-Head and Neck Surgery University of CaliforniaIrvineCAUSA

In a recent study published in Science Advances,Liu et *** that heterozygous deletion of connexin 26(Cx26)in mice is not harmless as they lead to hyperacusis-like hearing *** authors also found that children who carry a single-point heterozygous mutation in the gap junction(GJ)gene Cx26(GJB2)are susceptible to noise,which eventually leads to hearing loss(HL).1 HL affects millions of people worldwide and is considered the most prevalent sensory disorder,encompassing a wide range of heterogeneous characteristics.

关键词： Cx26 noise