pinecone/core-2020-05-10-deduplication huggingface dataset:
Querying quantitative logic models (Q2LM) to study intracellular signaling networks and cell-cytokine interactions

Mathematical models have substantially improved our ability to predict the response of a complex biological system to perturbation, but their use is typically limited by difficulties in specifying model topology and parameter values. Additionally, incorporating entities across different biological scales ranging from molecular to organismal in the same model is not trivial. Here, we present a framework called “querying quantitative logic models” (Q2LM) for building and asking questions of constrained fuzzy logic (cFL) models. cFL is a recently developed modeling formalism that uses logic gates to describe influences among entities, with transfer functions to describe quantitative dependencies. Q2LM does not rely on dedicated data to train the parameters of the transfer functions, and it permits straight-forward incorporation of entities at multiple biological scales. The Q2LM framework can be employed to ask questions such as: Which therapeutic perturbations accomplish a designated goal, and under what environmental conditions will these perturbations be effective? We demonstrate the utility of this framework for generating testable hypotheses in two examples: (i) a intracellular signaling network model; and (ii) a model for pharmacokinetics and pharmacodynamics of cell-cytokine interactions; in the latter, we validate hypotheses concerning molecular design of granulocyte colony stimulating factor.National Institutes of Health (U.S.) (Grant P50-GM068762)National Institutes of Health (U.S.) (Grant R24-DK090963)United States. Army Research Office (Institute for Collaborative Biotechnologies Grant W911NF-09-0001
Wound-healing evaluation of entrapped active agents into protein microspheres over cellulosic gauzes

The use of active ingredients in wound management have evolved alongside the pharmaceutical agents and dressings used to deliver them. However, the development of gauzes, dressings with specific properties, still remains a challenge for several medical applications. A new methodology for the controlled release of active components for the healing of burn wounds is proposed herein. Cotton and non-woven bandages have been cationised to promote the attachment of protein microspheres. The active agents, piroxicam and vegetable oil, were entrapped into the microspheres using ultrasound energy. Active agents were released from the microspheres by a change in pH. Wound healing was assessed through the use of standardised burn wounds induced by a cautery in human full-thickness skin equivalents (EpidermFT). The best re-epithelialisation and fastest wound closure was observed in wounds treated with proteinaceous microspheres attached to gauzes, after six days of healing, in comparison with commercial collagen dressing and other controls. Furthermore, the ability of these materials to reduce the inflammation process, together with healing improvement, makes these biomaterials suitable for wound-dressing applications.We would like to acknowledge financial support from the European project Lidwine (contract no. NMP2-CT-2006-026741), and POPH/FSE for co-financing and Fundacao para a Ciencia e a Tecnologia (FCT) for fellowship SFRH/BPD/38939/2007 awarded to Helena Ferreira. The work at CBMA was supported by FEDER through POFC-COMPETE and by national funds from FCT through the project PEst-C/BIA/UI4050/2011
A quantitative approach for understanding small-scale human mesenchymal stem cell culture implications for large-scale bioprocess development

This is the peer reviewed version of the following article: RAFIQ, Q.A. ... et al, 2013. A quantitative approach for understanding small-scale human mesenchymal stem cell culture implications for large-scale bioprocess development. Biotechnology Journal, 8 (4), pp. 459 - 471, which has been published in final form at http://dx.doi.org/10.1002/biot.201200197. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Human mesenchymal stem cell (hMSC) therapies have the potential to revolutionise the healthcare industry and replicate the success of the therapeutic protein industry; however, for this to be achieved there is a need to apply key bioprocessing engineering principles and adopt a quantitative approach for large-scale reproducible hMSC bioprocess development. Here we provide a quantitative analysis of the changes in concentration of glucose, lactate and ammonium with time during hMSC monolayer culture over 4 passages, under 100% and 20% dissolved oxgen (dO2), where either a 100%, 50% or 0% growth medium exchange was performed after 72h in culture. Yield coefficients, specific growth rates (h-1) and doubling times (h) were calculated for all cases. The 100% dO2 flasks outperformed the 20% dO2 flasks with respect to cumulative cell number, with the latter consuming more glucose and producing more lactate and ammonium. Furthermore, the 100% and 50% medium exchange conditions resulted in similar cumulative cell numbers, whilst the 0% conditions were significantly lower. Cell immunophenotype and multipotency were not affected by the experimental culture conditions. This study demonstrates the importance of determining optimal culture conditions for hMSC expansion and highlights a potential cost savings from only making a 50% medium exchange, which may prove significant for large-scale bioprocessing
Chassis organism from Corynebacterium glutamicum - a top-down approach to identify and delete irrelevant gene clusters

For synthetic biology applications, a robust structural basis is required, which can be constructed either from scratch or in a top-down approach starting from any existing organism. In this study, we initiated the top-down construction of a chassis organism from Corynebacterium glutamicum ATCC 13032, aiming for the relevant gene set to maintain its fast growth on defined medium. We evaluated each native gene for its essentiality considering expression levels, phylogenetic conservation, and knockout data. Based on this classification, we determined 41 gene clusters ranging from 3.7 to 49.7 kbp as target sites for deletion. 36 deletions were successful and 10 genome-reduced strains showed impaired growth rates, indicating that genes were hit, which are relevant to maintain biological fitness at wild-type level. In contrast, 26 deleted clusters were found to include exclusively irrelevant genes for growth on defined medium. A combinatory deletion of all irrelevant gene clusters would, in a prophage-free strain, decrease the size of the native genome by about 722 kbp (22%) to 2561 kbp. Finally, five combinatory deletions of irrelevant gene clusters were investigated. The study introduces the novel concept of relevant genes and demonstrates general strategies to construct a chassis suitable for biotechnological application
Indole generates quiescent and metabolically active E. coli cultures

An inherent problem with bacterial cell factories used to produce recombinant proteins or
metabolites is that resources are channeled into unwanted biomass as well as product. Over
several years attempts have been made to increase efficiency by unlinking biomass and
product generation. One example was the quiescent cell (Q-Cell) expression system that
generated non-growing but metabolically active E. coli by over-expressing a regulatory RNA
(Rcd) in a defined genetic background. Although effective at increasing the efficiency with
which resources are converted to product, the technical complexity of the Rcd-based Q-Cell
system limited its use. We describe here an alternative method for generating Q-Cells by the
direct addition of indole, or related indole derivatives, to the culture medium of an E. coli
strain carrying defined mutations in the hns gene. This simple and effective approach is
shown to be functional in both shake-flask and fermenter culture. The method provides a
simple way to generate quiescent cells in both shake-flask and fermenter culture. They
remain metabolically active and analysis of their performance in the fermenter suggests that
they may be particularly suitable for the production of cellular metabolites.This is the accepted manuscript. The final version is available from Wiley at http://onlinelibrary.wiley.com/doi/10.1002/biot.201400381/abstract
Dynamic metabolic engineering: New strategies for developing responsive cell factories

Metabolic engineering strategies have enabled improvements in yield and titer for a variety of valuable small molecules produced naturally in microorganisms, as well as those produced via heterologous pathways. Typically, the approaches have been focused on up- and downregulation of genes to redistribute steady-state pathway fluxes, but more recently a number of groups have developed strategies for dynamic regulation, which allows rebalancing of fluxes according to changing conditions in the cell or the fermentation medium. This review highlights some of the recently published work related to dynamic metabolic engineering strategies and explores how advances in high-throughput screening and synthetic biology can support development of new dynamic systems. Dynamic gene expression profiles allow trade-offs between growth and production to be better managed and can help avoid build-up of undesired intermediates. The implementation is more complex relative to static control, but advances in screening techniques and DNA synthesis will continue to drive innovation in this field.National Science Foundation (U.S.) (CBET-0954986)United States. National Institutes of Health (T32GM008334
Production of curcuminoids from tyrosine by a metabolically engineered Escherichia coli using caffeic acid as an intermediate

Curcuminoids are phenylpropanoids with high pharmaceutical potential. Herein, we report an engineered artificial pathway in Escherichia coli to produce natural curcuminoids through caffeic acid. Arabidopsis thaliana 4-coumaroyl-CoA ligase and Curcuma longa diketide-CoA synthase (DCS) and curcumin synthase (CURS1) were used to produce curcuminoids and 70 mg/L of curcumin was obtained from ferulic acid. Bisdemethoxycurcumin and demethoxycurcumin were also produced, but in lower concentrations, by feeding p-coumaric acid or a mixture of p-coumaric acid and ferulic acid, respectively. Additionally, curcuminoids were produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase from Rhodotorula glutinis and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis were used. Caffeoyl-CoA 3-O-methyltransferase from Medicago sativa was used to convert caffeoyl-CoA to feruloyl-CoA. Using caffeic acid, p-coumaric acid or tyrosine as a substrate, 3.9, 0.3, and 0.2 mg/L of curcumin were produced, respectively. This is the first time DCS and CURS1 were used in vivo to produce curcuminoids and that curcumin was produced by feeding tyrosine. We have shown that curcumin can be produced using a pathway involvoing caffeic acid. This alternative pathway represents a step forward in the heterologous production of curcumin using E. coli.Fundacao para a Ciencia e a Tecnologia (Project FCOMP-01-0124- FEDER-027462)Fundacao para a Ciencia e a Tecnologia (Project PEst-OE/EQB/LA0023/2013)Fundacao para a Ciencia e a Tecnologia (Project NORTE-07- 0124-FEDER-000028)Fundacao para a Ciencia e a Tecnologia (Project NORTE-07-0124-FEDER-000027)Fundacao para a Ciencia e a Tecnologia (Grant SFRH/BD/51187/2010)Fundacao para a Ciencia e a Tecnologia (SYNBIOBACTHER Project PTDC/EBB-BIO/102863/2008
Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost-effective manufacturing processes. Microcarriers enable the culture of anchorage-dependent cells in stirred-tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow-derived MSC (hBM-MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM-MSC1 underwent more cumulative population doublings over three passages in comparison to hBM-MSC2 and hBM-MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM-MSC expansion. HBM-MSCs were successfully harvested and characterised, demonstrating hBM-MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier-based allogeneic cell therapy manufacture
Cell-to-cell heterogeneity emerges as consequence of metabolic cooperation in a synthetic yeast community

Cells that grow together respond heterogeneously to stress even when they are genetically similar. Metabolism, a key determinant of cellular stress tolerance, may be one source of this phenotypic heterogeneity, however, this relationship is largely unclear. We used self-establishing metaboli-cally cooperating (SeMeCo) yeast communities, in which metabolic cooperation can be followed on the basis of genotype, as a model to dissect the role of metabolic cooperation in cell-to-cell heterogeneity.  Cells  within  SeMeCo  communities  showed  to  be  highly  heterogeneous  in  their stress tolerance, while the survival of each cell under heat or oxidative stress was strongly deter-mined by its metabolic specialization. This heterogeneity emerged for all metabolite exchange interactions studied (histidine, leucine, uracil, and methionine) as well as oxidants (H$_2$O$_2$ and diamide) and heat stress treatments. In contrast, the SeMeCo community collectively showed to be similarly tolerant to stress as wild-type populations. Moreover, stress heterogeneity did not establish as direct consequence of the metabolic genotype (auxotrophic background) of the single cell, but was observed only for cells that cooperated according to their metabolic capacity. We therefore conclude that phenotypic heterogeneity and cell-to-cell differences in stress tolerance are emergent properties when cells cooperate in metabolism.Wellcome Trust (Grant ID: RG 093735/Z/10/Z) and the European Research Council (Starting grant 260809)This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/biot.20150030
Exploring mild enzymatic sustainable routes for the synthesis\ud
of bio-degradable aromatic-aliphatic oligoesters

The application of Candida antarctica lipase B in enzyme-catalyzed synthesis of aromatic-aliphatic oligoesters is here reported. The aim of the present study is to systematically investigate the most favorable conditions for the enzyme catalyzed synthesis of aromatic-aliphatic oligomers using commercially available monomers. Reaction conditions and enzyme selectivity for polymerization of various commercially available monomers were considered using different inactivated/activated aromatic monomers combined with linear polyols ranging from C2 to C12. The effect of various reaction solvents in enzymatic polymerization was assessed and toluene allowed to achieve the highest conversions for the reaction of dimethyl isophthalate with 1,4-butanediol and with 1,10-decanediol (88 and 87% monomer conversion respectively). Mw as high as 1512 Da was obtained from the reaction of dimethyl isophthalate with ,10-decanediol. The obtained oligomers have potential applications as raw materials in personal and home care formulations, for the production of aliphatic-aromatic block co-polymers or can be further functionalized with various moieties for a subsequent photo- or radical polymerization
Bacterial microcompartment-directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli

Processes for the biological removal of phosphate from wastewater rely on temporary manipulation of bacterial polyphosphate levels by phased environmental stimuli. In E. coli polyphosphate levels are controlled via the polyphosphate-synthesizing enzyme polyphosphate kinase (PPK1) and exopolyphosphatases (PPX and GPPA), and are temporarily enhanced by PPK1 overexpression and reduced by PPX overexpression. We hypothesised that partitioning PPK1 from cytoplasmic exopolyphosphatases would increase and stabilise E. coli polyphosphate levels. Partitioning was achieved by co-expression of E. coli PPK1 fused with a microcompartment-targeting sequence and an artificial operon of Citrobacter freundii bacterial microcompartment genes. Encapsulation of targeted PPK1 resulted in persistent phosphate uptake and stably increased cellular polyphosphate levels throughout cell growth and into the stationary phase, while PPK1 overexpression alone produced temporary polyphosphate increase and phosphate uptake. Targeted PPK1 increased polyphosphate in microcompartments 8-fold compared with non-targeted PPK1. Co-expression of PPX polyphosphatase with targeted PPK1 had little effect on elevated cellular polyphosphate levels because microcompartments retained polyphosphate. Co-expression of PPX with non-targeted PPK1 reduced cellular polyphosphate levels. Thus, subcellular compartmentalisation of a polymerising enzyme sequesters metabolic products from competing catabolism by preventing catabolic enzyme access. Specific application of this process to polyphosphate is of potential application for biological phosphate removal
Immobilized hematopoietic growth factors onto magnetic particles offer a scalable strategy for cell therapy manufacturing in suspension cultures

This is an Open Access Article. It is published by Wiley under the Creative Commons Attribution 4.0 Unported Licence (CC BY). Full details of this licence are available at: http://creativecommons.org/licenses/by/4.0/Hematopoietic therapies require high cell dosages and precise phenotype control for clinical success; scalable manufacturing processes therefore need to be economic and controllable, in particular with respect to culture medium and growth factor (GF) strategy. The aim of this work was to demonstrate the biological function, and integration within scalable systems, of a highly controllable immobilized growth factor (iGF) approach. GFs were biotinylated and attached to streptavidin coated magnetic particles. GF concentration during biotinylation, GF-biotin ratio, and GF\ud
lysine content were shown to control iGF surface concentration and enable predictable co-presentation of multiple GF on a single bead. Function was demonstrated for immobilized GMCSF, SCF, TPO and IL-3 in GF dependent cell lines TF-1 and M-07e. Immobilized GMCSF (iGMCSF) was analyzed to show sustained activity over eight days of culture, a two to three order of magnitude\ud
potency increase relative to soluble factor, and retained functionality under agitation in a microscale stirred tank bioreactor. Further, short exposure to iGMCSF demonstrated prolonged growth response relative to soluble factor. This immobilization approach has the potential to reduce the manufacturing costs of scaled cell therapy products by reducing GF quantities and offers important process control opportunities through separation of GF treatments from the bulk media
Electrospun nanofibrous meshes cultured with Wharton’s Jelly Stem Cell: an alternative for cartilage regeneration, without the need of growth factors

Many efforts are being directed worldwide to the treatment of OA-focal lesions.
The majority of those efforts comprise either the refinement of surgical
techniques or combinations of biomaterials with various autologous cells.
Herein, we tested electrospun polycaprolactone (PCL) nanofibrous meshes for
cartilage tissue engineering. For that, articular chondrocytes (hACs) isolated
from human osteoarthritic joints and Whartonâ  s Jelly Stem Cells (hWJSCs) are
cultured on electrospun nanofiber meshes, without adding external growth
factors. We observed higher glycosaminoglycans production and higher overexpression
of cartilage-related genes from hWJSCs cultured with basal medium,
when compared to hACs isolated from osteoarthritic joints. Moreover, the
presence of sulfated proteoglycans and collagen type II is observed on both
types of cell cultures. We believe that this effect is due to either the electrospun
nanofibers topography or the intrinsic chondrogenic differentiation potential of
hWJSCs. Therefore, we propose the electrospun nanofibrous scaffolds in
combination with hWJSCs as a viable alternative to the commercial membranes
used in autologous chondrogenic regeneration approaches.The authors thank the Portuguese Foundation for Science and
Technology for the Post-Doc grant of Marta Alves da Silva (SFRH/
BPD/73322/2010, financed by POPH  QREN  Tipologia 4.1 –
Advanced Formation, co-financed by Fundo Social Europeu and MEC
national funds). This work was supported by the project SPARTAN
(PTDC/CTM-BIO/4388/2014) FCT/MEC with PIDDAC funds. It was also
partly supported by the POLARIS (FP7-REGPOT-2012-2013-1) and the
Project “New methodologies for the isolation and control of stem cells
differentiation using advanced culturing conditions and/or nanomaterials”
(RL2  SCN  NORTE-01-0124-FEDER-000018), co-financed by
North Portugal Regional Operational Programme (ON.2 – O Novo
Norte), under the National Strategic Reference Framework (NSRF),
through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio
Synthetic Biology and the Gut Microbiome

The gut microbiome plays a crucial role in maintaining human health. Functions performed by gastrointestinal microbes range from regulating metabolism to modulating immune and nervous system development. Scientists have attempted to exploit this importance through the development of engineered probiotics that are capable of producing and delivering small molecule therapeutics within the gut. However, existing synthetic probiotics are simplistic and fail to replicate the complexity and adaptability of native homeostatic mechanisms. In this review, the ways in which the tools and approaches of synthetic biology have been applied to improve the efficacy of therapeutic probiotics, and the ways in which they might be applied in the future is discussed. Simple devices, such as a bistable switches and integrase memory arrays, have been successfully implemented in the mammalian gut, and models for targeted delivery in this environment have also been developed. In the future, it will be necessary to introduce concepts such as logic-gating and biocontainment mechanisms into synthetic probiotics, as well as to expand the collection of relevant biosensors. Ideally, this will bring us closer to a reality in which engineered therapeutic microbes will be able to accurately diagnose and effectively respond to a variety of disease states