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Bioengineering, Volume 4, Issue 2 (June 2017)

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Cover Story (view full-size image) Biotinylation and multivalent presentation of N-acetyllactosamine- and [...] Read more.
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Open AccessArticle The Bistable Behaviour of Pseudomonas putida KT2440 during PHA Depolymerization under Carbon Limitation
Bioengineering 2017, 4(2), 58; https://doi.org/10.3390/bioengineering4020058
Received: 14 May 2017 / Revised: 12 June 2017 / Accepted: 13 June 2017 / Published: 19 June 2017
Cited by 3 | PDF Full-text (2423 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Poly(hydroxyalkanoates) (PHAs) are bacterial polyesters offering a biodegradable alternative to petrochemical plastics. The intracellular formation and degradation of PHAs is a dynamic process that strongly depends on the availability of carbon and other nutrients. Carbon excess and nitrogen limitation are considered to favor
[...] Read more.
Poly(hydroxyalkanoates) (PHAs) are bacterial polyesters offering a biodegradable alternative to petrochemical plastics. The intracellular formation and degradation of PHAs is a dynamic process that strongly depends on the availability of carbon and other nutrients. Carbon excess and nitrogen limitation are considered to favor PHA accumulation, whereas carbon limitation triggers PHA depolymerization when all other essential nutrients are present in excess. We studied the population dynamics of Pseudomonas putida KT2440 at the single cell level during different physiological conditions, favoring first PHA polymerization during growth on octanoic acid, and then PHA depolymerization during carbon limitation. PHAs accumulate intracellularly in granules, and were proposed to separate preferentially together with nucleic acids, leading to two daughter cells containing approximately equal amounts of PHA. However, we could show that such P. putida KT2440 cells show bistable behavior when exposed to carbon limitation, and separate into two subpopulations: one with high and one with low PHA. This suggests an asymmetric PHA distribution during cell division under carbon limitation, which has a significant influence on our understanding of PHA mobilization. Full article
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Open AccessReview A Review of Microwave-Assisted Reactions for Biodiesel Production
Bioengineering 2017, 4(2), 57; https://doi.org/10.3390/bioengineering4020057
Received: 18 May 2017 / Revised: 9 June 2017 / Accepted: 12 June 2017 / Published: 15 June 2017
Cited by 5 | PDF Full-text (4750 KB) | HTML Full-text | XML Full-text
Abstract
The conversion of biomass into chemicals and biofuels is an active research area as trends move to replace fossil fuels with renewable resources due to society’s increased concern towards sustainability. In this context, microwave processing has emerged as a tool in organic synthesis
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The conversion of biomass into chemicals and biofuels is an active research area as trends move to replace fossil fuels with renewable resources due to society’s increased concern towards sustainability. In this context, microwave processing has emerged as a tool in organic synthesis and plays an important role in developing a more sustainable world. Integration of processing methods with microwave irradiation has resulted in a great reduction in the time required for many processes, while the reaction efficiencies have been increased markedly. Microwave processing produces a higher yield with a cleaner profile in comparison to other methods. The microwave processing is reported to be a better heating method than the conventional methods due to its unique thermal and non-thermal effects. This paper provides an insight into the theoretical aspects of microwave irradiation practices and highlights the importance of microwave processing. The potential of the microwave technology to accomplish superior outcomes over the conventional methods in biodiesel production is presented. A green process for biodiesel production using a non-catalytic method is still new and very costly because of the supercritical condition requirement. Hence, non-catalytic biodiesel conversion under ambient pressure using microwave technology must be developed, as the energy utilization for microwave-based biodiesel synthesis is reported to be lower and cost-effective. Full article
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Open AccessArticle Three-Dimensional Culture Model of Skeletal Muscle Tissue with Atrophy Induced by Dexamethasone
Bioengineering 2017, 4(2), 56; https://doi.org/10.3390/bioengineering4020056
Received: 12 April 2017 / Revised: 12 June 2017 / Accepted: 12 June 2017 / Published: 15 June 2017
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Abstract
Drug screening systems for muscle atrophy based on the contractile force of cultured skeletal muscle tissues are required for the development of preventive or therapeutic drugs for atrophy. This study aims to develop a muscle atrophy model by inducing atrophy in normal muscle
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Drug screening systems for muscle atrophy based on the contractile force of cultured skeletal muscle tissues are required for the development of preventive or therapeutic drugs for atrophy. This study aims to develop a muscle atrophy model by inducing atrophy in normal muscle tissues constructed on microdevices capable of measuring the contractile force and to verify if this model is suitable for drug screening using the contractile force as an index. Tissue engineered skeletal muscles containing striated myotubes were prepared on the microdevices for the study. The addition of 100 µM dexamethasone (Dex), which is used as a muscle atrophy inducer, for 24 h reduced the contractile force significantly. An increase in the expression of Atrogin-1 and MuRF-1 in the tissues treated with Dex was established. A decrease in the number of striated myotubes was also observed in the tissues treated with Dex. Treatment with 8 ng/mL Insulin-like Growth Factor (IGF-I) for 24 h significantly increased the contractile force of the Dex-induced atrophic tissues. The same treatment, though, had no impact on the force of the normal tissues. Thus, it is envisaged that the atrophic skeletal muscle tissues induced by Dex can be used for drug screening against atrophy. Full article
(This article belongs to the Special Issue Human Organs-on-Chips for In Vitro Disease Models)
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Open AccessReview Recent Advances and Challenges towards Sustainable Polyhydroxyalkanoate (PHA) Production
Bioengineering 2017, 4(2), 55; https://doi.org/10.3390/bioengineering4020055
Received: 30 April 2017 / Revised: 29 May 2017 / Accepted: 9 June 2017 / Published: 11 June 2017
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Abstract
Sustainable biofuels, biomaterials, and fine chemicals production is a critical matter that research teams around the globe are focusing on nowadays. Polyhydroxyalkanoates represent one of the biomaterials of the future due to their physicochemical properties, biodegradability, and biocompatibility. Designing efficient and economic bioprocesses,
[...] Read more.
Sustainable biofuels, biomaterials, and fine chemicals production is a critical matter that research teams around the globe are focusing on nowadays. Polyhydroxyalkanoates represent one of the biomaterials of the future due to their physicochemical properties, biodegradability, and biocompatibility. Designing efficient and economic bioprocesses, combined with the respective social and environmental benefits, has brought together scientists from different backgrounds highlighting the multidisciplinary character of such a venture. In the current review, challenges and opportunities regarding polyhydroxyalkanoate production are presented and discussed, covering key steps of their overall production process by applying pure and mixed culture biotechnology, from raw bioprocess development to downstream processing. Full article
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Open AccessArticle Polyhydroxyalkanoate Production on Waste Water Treatment Plants: Process Scheme, Operating Conditions and Potential Analysis for German and European Municipal Waste Water Treatment Plants
Bioengineering 2017, 4(2), 54; https://doi.org/10.3390/bioengineering4020054
Received: 28 April 2017 / Revised: 2 June 2017 / Accepted: 3 June 2017 / Published: 6 June 2017
Cited by 3 | PDF Full-text (3550 KB) | HTML Full-text | XML Full-text
Abstract
This work describes the production of polyhydroxyalkanoates (PHA) as a side stream process on a municipal waste water treatment plant (WWTP) and a subsequent analysis of the production potential in Germany and the European Union (EU). Therefore, tests with different types of sludge
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This work describes the production of polyhydroxyalkanoates (PHA) as a side stream process on a municipal waste water treatment plant (WWTP) and a subsequent analysis of the production potential in Germany and the European Union (EU). Therefore, tests with different types of sludge from a WWTP were investigated regarding their volatile fatty acids (VFA) production-potential. Afterwards, primary sludge was used as substrate to test a series of operating conditions (temperature, pH, retention time (RT) and withdrawal (WD)) in order to find suitable settings for a high and stable VFA production. In a second step, various tests regarding a high PHA production and stable PHA composition to determine the influence of substrate concentration, temperature, pH and cycle time of an installed feast/famine-regime were conducted. Experiments with a semi-continuous reactor operation showed that a short RT of 4 days and a small WD of 25% at pH = 6 and around 30 °C is preferable for a high VFA production rate (PR) of 1913 mgVFA/(L×d) and a stable VFA composition. A high PHA production up to 28.4% of cell dry weight (CDW) was reached at lower substrate concentration, 20 °C, neutral pH-value and a 24 h cycle time. A final step a potential analysis, based on the results and detailed data from German waste water treatment plants, showed that the theoretically possible production of biopolymers in Germany amounts to more than 19% of the 2016 worldwide biopolymer production. In addition, a profound estimation regarding the EU showed that in theory about 120% of the worldwide biopolymer production (in 2016) could be produced on European waste water treatment plants. Full article
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Open AccessArticle Production of Polyhydroxyalkanoates Using Hydrolyzates of Spruce Sawdust: Comparison of Hydrolyzates Detoxification by Application of Overliming, Active Carbon, and Lignite
Bioengineering 2017, 4(2), 53; https://doi.org/10.3390/bioengineering4020053
Received: 28 April 2017 / Revised: 24 May 2017 / Accepted: 25 May 2017 / Published: 28 May 2017
Cited by 4 | PDF Full-text (777 KB) | HTML Full-text | XML Full-text
Abstract
Polyhydroxyalkanoates (PHAs) are bacterial polyesters which are considered biodegradable alternatives to petrochemical plastics. PHAs have a wide range of potential applications, however, the production cost of this bioplastic is several times higher. A major percentage of the final cost is represented by the
[...] Read more.
Polyhydroxyalkanoates (PHAs) are bacterial polyesters which are considered biodegradable alternatives to petrochemical plastics. PHAs have a wide range of potential applications, however, the production cost of this bioplastic is several times higher. A major percentage of the final cost is represented by the price of the carbon source used in the fermentation. Burkholderia cepacia and Burkholderia sacchari are generally considered promising candidates for PHA production from lignocellulosic hydrolyzates. The wood waste biomass has been subjected to hydrolysis. The resulting hydrolyzate contained a sufficient amount of fermentable sugars. Growth experiments indicated a strong inhibition by the wood hydrolyzate. Over-liming and activated carbon as an adsorbent of inhibitors were employed for detoxification. All methods of detoxification had a positive influence on the growth of biomass and PHB production. Furthermore, lignite was identified as a promising alternative sorbent which can be used for detoxification of lignocellulose hydrolyzates. Detoxification using lignite instead of activated carbon had lower inhibitor removal efficiency, but greater positive impact on growth of the bacterial culture and overall PHA productivity. Moreover, lignite is a significantly less expensive adsorbent in comparison with activated charcoal and; moreover, used lignite can be simply utilized as a fuel to, at least partially, cover heat and energetic demands of fermentation, which should improve the economic feasibility of the process. Full article
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Open AccessArticle Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria
Bioengineering 2017, 4(2), 52; https://doi.org/10.3390/bioengineering4020052
Received: 24 April 2017 / Revised: 12 May 2017 / Accepted: 19 May 2017 / Published: 25 May 2017
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Abstract
The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC)
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The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC) gene, which is involved in the production of polyhydroxyalkanoate (PHA)—a biodegradable, renewable biopolymer. Primers were designed based on multiple alignments of phaC gene sequences from 218 species that have their genomes already analyzed and deposited at Biocyc databank. The combination of oligos phaCF3/phaCR1 allowed the amplification of the expected product (PHA synthases families types I and IV) from reference organisms used as positive control (PHA producer). The method was also tested in a multiplex system with two combinations of initiators, using 16 colonies of marine bacteria (pre-characterized for PHA production) as a DNA template. All amplicon positive organisms (n = 9) were also PHA producers, thus no false positives were observed. Amplified DNA was sequenced (n = 4), allowing for the confirmation of the phaC gene identity as well its diversity among marine bacteria. Primers were also tested for screening purposes using 37 colonies from six different environments. Almost 30% of the organisms presented the target amplicon. Thus, the proposed primers are an efficient tool for screening bacteria with potential for the production of PHA as well to study PHA genetic diversity. Full article
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Open AccessArticle Development and Characterization of a Parallelizable Perfusion Bioreactor for 3D Cell Culture
Bioengineering 2017, 4(2), 51; https://doi.org/10.3390/bioengineering4020051
Received: 29 March 2017 / Revised: 11 May 2017 / Accepted: 23 May 2017 / Published: 25 May 2017
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Abstract
The three dimensional (3D) cultivation of stem cells in dynamic bioreactor systems is essential in the context of regenerative medicine. Still, there is a lack of bioreactor systems that allow the cultivation of multiple independent samples under different conditions while ensuring comprehensive control
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The three dimensional (3D) cultivation of stem cells in dynamic bioreactor systems is essential in the context of regenerative medicine. Still, there is a lack of bioreactor systems that allow the cultivation of multiple independent samples under different conditions while ensuring comprehensive control over the mechanical environment. Therefore, we developed a miniaturized, parallelizable perfusion bioreactor system with two different bioreactor chambers. Pressure sensors were also implemented to determine the permeability of biomaterials which allows us to approximate the shear stress conditions. To characterize the flow velocity and shear stress profile of a porous scaffold in both bioreactor chambers, a computational fluid dynamics analysis was performed. Furthermore, the mixing behavior was characterized by acquisition of the residence time distributions. Finally, the effects of the different flow and shear stress profiles of the bioreactor chambers on osteogenic differentiation of human mesenchymal stem cells were evaluated in a proof of concept study. In conclusion, the data from computational fluid dynamics and shear stress calculations were found to be predictable for relative comparison of the bioreactor geometries, but not for final determination of the optimal flow rate. However, we suggest that the system is beneficial for parallel dynamic cultivation of multiple samples for 3D cell culture processes. Full article
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Open AccessArticle Utilization of Sugarcane Bagasse by Halogeometricum borinquense Strain E3 for Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
Bioengineering 2017, 4(2), 50; https://doi.org/10.3390/bioengineering4020050
Received: 29 March 2017 / Revised: 17 May 2017 / Accepted: 22 May 2017 / Published: 25 May 2017
Cited by 4 | PDF Full-text (2879 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Sugarcane bagasse (SCB), one of the major lignocellulosic agro-industrial waste products, was used as a substrate for biosynthesis of polyhydroxyalkanoates (PHA) by halophilic archaea. Among the various wild-type halophilic archaeal strains screened, Halogeometricum borinquense strain E3 showed better growth and PHA accumulation as
[...] Read more.
Sugarcane bagasse (SCB), one of the major lignocellulosic agro-industrial waste products, was used as a substrate for biosynthesis of polyhydroxyalkanoates (PHA) by halophilic archaea. Among the various wild-type halophilic archaeal strains screened, Halogeometricum borinquense strain E3 showed better growth and PHA accumulation as compared to Haloferaxvolcanii strain BBK2, Haloarcula japonica strain BS2, and Halococcus salifodinae strain BK6. Growth kinetics and bioprocess parameters revealed the maximum PHA accumulated by strain E3 to be 50.4 ± 0.1 and 45.7 ± 0.19 (%) with specific productivity (qp) of 3.0 and 2.7 (mg/g/h) using NaCl synthetic medium supplemented with 25% and 50% SCB hydrolysate, respectively. PHAs synthesized by strain E3 were recovered in chloroform using a Soxhlet apparatus. Characterization of the polymer using crotonic acid assay, X-ray diffraction (XRD), differential scanning calorimeter (DSC), Fourier transform infrared (FT-IR), and proton nuclear magnetic resonance (1H-NMR) spectroscopy analysis revealed the polymer obtained from SCB hydrolysate to be a co-polymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] comprising of 13.29 mol % 3HV units. Full article
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Open AccessArticle Additive Manufacturing of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/poly(ε-caprolactone) Blend Scaffolds for Tissue Engineering
Bioengineering 2017, 4(2), 49; https://doi.org/10.3390/bioengineering4020049
Received: 27 April 2017 / Revised: 19 May 2017 / Accepted: 21 May 2017 / Published: 24 May 2017
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Abstract
Additive manufacturing of scaffolds made of a polyhydroxyalkanoate blended with another biocompatible polymer represents a cost-effective strategy for combining the advantages of the two blend components in order to develop tailored tissue engineering approaches. The aim of this study was the development of
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Additive manufacturing of scaffolds made of a polyhydroxyalkanoate blended with another biocompatible polymer represents a cost-effective strategy for combining the advantages of the two blend components in order to develop tailored tissue engineering approaches. The aim of this study was the development of novel poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/ poly(ε-caprolactone) (PHBHHx/PCL) blend scaffolds for tissue engineering by means of computer-aided wet-spinning, a hybrid additive manufacturing technique suitable for processing polyhydroxyalkanoates dissolved in organic solvents. The experimental conditions for processing tetrahydrofuran solutions containing the two polymers at different concentrations (PHBHHx/PCL weight ratio of 3:1, 2:1 or 1:1) were optimized in order to manufacture scaffolds with predefined geometry and internal porous architecture. PHBHHx/PCL scaffolds with a 3D interconnected network of macropores and a local microporosity of the polymeric matrix, as a consequence of the phase inversion process governing material solidification, were successfully fabricated. As shown by scanning electron microscopy, thermogravimetric, differential scanning calorimetric and uniaxial compressive analyses, blend composition significantly influenced the scaffold morphological, thermal and mechanical properties. In vitro biological characterization showed that the developed scaffolds were able to sustain the adhesion and proliferation of MC3T3-E1 murine preosteoblast cells. The additive manufacturing approach developed in this study, based on a polymeric solution processing method avoiding possible material degradation related to thermal treatments, could represent a powerful tool for the development of customized PHBHHx-based blend scaffolds for tissue engineering. Full article
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Open AccessArticle Assessing and Resolving Model Misspecifications in Metabolic Flux Analysis
Bioengineering 2017, 4(2), 48; https://doi.org/10.3390/bioengineering4020048
Received: 14 March 2017 / Revised: 30 April 2017 / Accepted: 22 May 2017 / Published: 24 May 2017
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Abstract
Metabolic flux analysis (MFA) is an indispensable tool in metabolic engineering. The simplest variant of MFA relies on an overdetermined stoichiometric model of the cell’s metabolism under the pseudo-steady state assumption to evaluate the intracellular flux distribution. Despite its long history, the issue
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Metabolic flux analysis (MFA) is an indispensable tool in metabolic engineering. The simplest variant of MFA relies on an overdetermined stoichiometric model of the cell’s metabolism under the pseudo-steady state assumption to evaluate the intracellular flux distribution. Despite its long history, the issue of model error in overdetermined MFA, particularly misspecifications of the stoichiometric matrix, has not received much attention. We evaluated the performance of statistical tests from linear least square regressions, namely Ramsey’s Regression Equation Specification Error Test (RESET), the F-test, and the Lagrange multiplier test, in detecting model misspecifications in the overdetermined MFA, particularly missing reactions. We further proposed an iterative procedure using the F-test to correct such an issue. Using Chinese hamster ovary and random metabolic networks, we demonstrated that: (1) a statistically significant regression does not guarantee high accuracy of the flux estimates; (2) the removal of a reaction with a low flux magnitude can cause disproportionately large biases in the flux estimates; (3) the F-test could efficiently detect missing reactions; and (4) the proposed iterative procedure could robustly resolve the omission of reactions. Our work demonstrated that statistical analysis and tests could be used to systematically assess, detect, and resolve model misspecifications in the overdetermined MFA. Full article
(This article belongs to the Special Issue Applying Systems Biotechnology Tools to Study Cell Metabolism)
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Open AccessArticle Hypoxic Three-Dimensional Scaffold-Free Aggregate Cultivation of Mesenchymal Stem Cells in a Stirred Tank Reactor
Bioengineering 2017, 4(2), 47; https://doi.org/10.3390/bioengineering4020047
Received: 26 April 2017 / Revised: 19 May 2017 / Accepted: 21 May 2017 / Published: 23 May 2017
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Abstract
Extensive expansion of mesenchymal stem cells (MSCs) for cell-based therapies remains challenging since long-term cultivation and excessive passaging in two-dimensional conditions result in a loss of essential stem cell properties. Indeed, low survival rate of cells, alteration of surface marker profiles, and reduced
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Extensive expansion of mesenchymal stem cells (MSCs) for cell-based therapies remains challenging since long-term cultivation and excessive passaging in two-dimensional conditions result in a loss of essential stem cell properties. Indeed, low survival rate of cells, alteration of surface marker profiles, and reduced differentiation capacity are observed after in vitro expansion and reduce therapeutic success in clinical studies. Remarkably, cultivation of MSCs in three-dimensional aggregates preserve stem cell properties. Hence, the large scale formation and cultivation of MSC aggregates is highly desirable. Besides other effects, MSCs cultivated under hypoxic conditions are known to display increased proliferation and genetic stability. Therefore, in this study we demonstrate cultivation of adipose derived human MSC aggregates in a stirred tank reactor under hypoxic conditions. Although aggregates were exposed to comparatively high average shear stress of 0.2 Pa as estimated by computational fluid dynamics, MSCs displayed a viability of 78–86% and maintained their surface marker profile and differentiation potential after cultivation. We postulate that cultivation of 3D MSC aggregates in stirred tank reactors is valuable for large-scale production of MSCs or their secreted compounds after further optimization of cultivation parameters. Full article
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Open AccessReview Microtechnology-Based Multi-Organ Models
Bioengineering 2017, 4(2), 46; https://doi.org/10.3390/bioengineering4020046
Received: 19 March 2017 / Revised: 17 May 2017 / Accepted: 18 May 2017 / Published: 21 May 2017
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Abstract
Drugs affect the human body through absorption, distribution, metabolism, and elimination (ADME) processes. Due to their importance, the ADME processes need to be studied to determine the efficacy and side effects of drugs. Various in vitro model systems have been developed and used
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Drugs affect the human body through absorption, distribution, metabolism, and elimination (ADME) processes. Due to their importance, the ADME processes need to be studied to determine the efficacy and side effects of drugs. Various in vitro model systems have been developed and used to realize the ADME processes. However, conventional model systems have failed to simulate the ADME processes because they are different from in vivo, which has resulted in a high attrition rate of drugs and a decrease in the productivity of new drug development. Recently, a microtechnology-based in vitro system called “organ-on-a-chip” has been gaining attention, with more realistic cell behavior and physiological reactions, capable of better simulating the in vivo environment. Furthermore, multi-organ-on-a-chip models that can provide information on the interaction between the organs have been developed. The ultimate goal is the development of a “body-on-a-chip”, which can act as a whole body model. In this review, we introduce and summarize the current progress in the development of multi-organ models as a foundation for the development of body-on-a-chip. Full article
(This article belongs to the Special Issue Human Organs-on-Chips for In Vitro Disease Models)
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Open AccessArticle OpenDrop: An Integrated Do-It-Yourself Platform for Personal Use of Biochips
Bioengineering 2017, 4(2), 45; https://doi.org/10.3390/bioengineering4020045
Received: 1 April 2017 / Revised: 6 May 2017 / Accepted: 11 May 2017 / Published: 19 May 2017
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Abstract
Biochips, or digital labs-on-chip, are developed with the purpose of being used by laboratory technicians or biologists in laboratories or clinics. In this article, we expand this vision with the goal of enabling everyone, regardless of their expertise, to use biochips for their
[...] Read more.
Biochips, or digital labs-on-chip, are developed with the purpose of being used by laboratory technicians or biologists in laboratories or clinics. In this article, we expand this vision with the goal of enabling everyone, regardless of their expertise, to use biochips for their own personal purposes. We developed OpenDrop, an integrated electromicrofluidic platform that allows users to develop and program their own bio-applications. We address the main challenges that users may encounter: accessibility, bio-protocol design and interaction with microfluidics. OpenDrop consists of a do-it-yourself biochip, an automated software tool with visual interface and a detailed technique for at-home operations of microfluidics. We report on two years of use of OpenDrop, released as an open-source platform. Our platform attracted a highly diverse user base with participants originating from maker communities, academia and industry. Our findings show that 47% of attempts to replicate OpenDrop were successful, the main challenge remaining the assembly of the device. In terms of usability, the users managed to operate their platforms at home and are working on designing their own bio-applications. Our work provides a step towards a future in which everyone will be able to create microfluidic devices for their personal applications, thereby democratizing parts of health care. Full article
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Open AccessArticle Comparative N-Glycosylation Analysis of the Fc Portions of a Chimeric Human Coagulation Factor VIII and Immunoglobulin G1
Bioengineering 2017, 4(2), 44; https://doi.org/10.3390/bioengineering4020044
Received: 13 April 2017 / Revised: 10 May 2017 / Accepted: 11 May 2017 / Published: 17 May 2017
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Abstract
Prevention and treatment of bleeding in patients suffering from hemophilia A are inconvenient due to repeated intravenous infusions owing to the short half-life of coagulation factor VIII (FVIII) in circulation. Besides (glyco-)pegylation of the FVIII molecule, a bioengineering approach comprises the protein fusion
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Prevention and treatment of bleeding in patients suffering from hemophilia A are inconvenient due to repeated intravenous infusions owing to the short half-life of coagulation factor VIII (FVIII) in circulation. Besides (glyco-)pegylation of the FVIII molecule, a bioengineering approach comprises the protein fusion to Fc-immunoglobulin (Ig)G that mediate protection from clearance or degradation via binding to the neonatal Fc receptor. While human-like N-glycosylation of recombinant FVIII is known to be crucial for the clotting factor’s quality and function, the particular glycosylation of the fused Fc portion has not been investigated in detail so far, despite its known impact on Fcγ receptor binding. Here, we analyzed the N-glycosylation of the Fc part of a chimeric FVIII-Fc protein compared to a commercial IgG1 purified from human plasma. Fc parts from both samples were released by enzymatic cleavage and were subsequently separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Corresponding protein bands were referred to PNGase F in-gel digestion in order to release the respective N-glycans. Analysis via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed structural differences of both N-glycan patterns. Labeling with 2-aminobenzamide (2AB) and analysis via hydrophilic interaction liquid chromatography (HILIC) allowed a quantitative comparison of the respective N-glycosylation. Observed variations in Fc glycosylation of the chimeric FVIII fusion protein and human plasma-derived IgG1, e.g., regarding terminal sialylation, are discussed, focusing on the impact of the clotting factor’s properties, most notably its binding to Fcγ receptors. Full article
(This article belongs to the Special Issue Recombinant Glycoproteins)
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