The Experts below are selected from a list of 6441 Experts worldwide ranked by ideXlab platform
Paul S Freemont - One of the best experts on this subject based on the ideXlab platform.
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al pha beads bioplastic based protease biosensors for global health applications
Materials Today, 2021Co-Authors: Richard Kelwick, Alexander J Webb, Yizhou Wang, Amelie Heliot, Fiona Allan, Aidan M Emery, Michael R Templeton, Paul S FreemontAbstract:Abstract Proteases are multi-functional proteolytic enzymes that have complex roles in human health and disease. Therefore, the development of protease biosensors can be beneficial to global health applications. To this end, we developed Advanced proteoLytic detector Polyhydroxyalkanoates (AL-PHA) beads – a library of over 20 low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured polyhydroxyalkanoate beads. In the presence of a specific protease, superfolder green fluorescent reporter proteins are cleaved from the AL-PHA beads – resulting in a loss of bead fluorescence. The Tobacco Etch Virus (TEV) AL-PHA biosensor detected the proteolytic activity of at least 1.85 pM of AcTEV. AL-PHA beads were also engineered to detect cercarial elastase from Schistosoma mansoni-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be further developed for use in resource-limited settings.
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al pha beads bioplastic based protease biosensors for global health applications
bioRxiv, 2020Co-Authors: Richard Kelwick, Alexander J Webb, Yizhou Wang, Amelie Heliot, Fiona Allan, Aidan M Emery, Michael R Templeton, Paul S FreemontAbstract:ABSTRACT Proteases are multi-functional, proteolytic enzymes that have complex roles in human health and disease. Detecting the activities of proteases can lead to important insights into communicable and non-communicable diseases. Therefore, the development of protease detection strategies can be beneficial to an array of global health applications. To this end, we developed Advanced proteoLytic detector Polyhydroxyalkanoates (AL-PHA) beads – a library of low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured polyhydroxyalkanoate (PHA) bioplastic beads. These PhaC-fusions also incorporate modular specific protease cleavage sites. In the presence of a specific protease, superfolder green fluorescent (sfGFP) reporter proteins are cleaved off of the AL-PHA beads - resulting in a loss of bead fluorescence. These AL-PHA biosensors were initially optimised using a commercially available Tobacco Etch Virus (TEV) protease. Our third generation TEV biosensor (PhaC-112L-T-G) detected 0.5 U (1.85 pM) of AcTEV activity and 10 units of AcTEV protease activity resulted in a visually noticeable loss in AL-PHA bead fluorescence. AL-PHA beads also detected cercarial elastase from Schistosoma mansoni-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be adapted towards a low-cost and high-throughput protease detection assay for global health applications.
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Cell-free prototyping strategies for enhancing the sustainable production of Polyhydroxyalkanoates bioplastics
2017Co-Authors: Richard Kelwick, Alexander J Webb, Luca Ricci, Soo Mei Chee, Derek Bell, Paul S FreemontAbstract:The Polyhydroxyalkanoates are a group of microbially-produced biopolymers that have been proposed as sustainable alternatives to several oil-derived plastics. However, Polyhydroxyalkanoates are currently more expensive to produce than oil-derived plastics and therefore, more efficient production processes would be desirable. Cell-free transcription-translation-based metabolic engineering strategies have been previously used to optimise several different biosynthetic pathways but not the Polyhydroxyalkanoates biosynthetic pathways. Here we have developed several Escherichia coli cell-free transcription-translation-based systems for in vitro prototyping of Polyhydroxyalkanoates biosynthetic operons, and also for screening relevant metabolite recycling enzymes. These cell-free transcription-translation reactions were customised through the addition of whey permeate, an industrial waste that has been previously used as a low-cost feedstock for optimising in vivo Polyhydroxyalkanoates production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ~20% compared to control reactions that did not include whey permeate. An analysis of pH in our cell-free reactions suggests that the observed increase in GFPmut3b production was likely through enhanced ATP generation, as a consequence of the glycolytic processing of lactose present in whey permeate. We also found that whey permeate enhanced cell-free reactions produced ~3μM (R)-3HB-CoA, whilst, coupled cell-free biotransformation/transcription-translation reactions produced a ten-fold greater yield of (R)-3HB-CoA. These reactions were also used to characterise a Clostridium propionicum propionyl CoA transferase enzyme that can recycle Acetyl-CoA. Together our data demonstrate that cell-free approaches can be used to complement in vivo workflows for identifying additional strategies for optimising Polyhydroxyalkanoates production.
Richard Kelwick - One of the best experts on this subject based on the ideXlab platform.
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al pha beads bioplastic based protease biosensors for global health applications
Materials Today, 2021Co-Authors: Richard Kelwick, Alexander J Webb, Yizhou Wang, Amelie Heliot, Fiona Allan, Aidan M Emery, Michael R Templeton, Paul S FreemontAbstract:Abstract Proteases are multi-functional proteolytic enzymes that have complex roles in human health and disease. Therefore, the development of protease biosensors can be beneficial to global health applications. To this end, we developed Advanced proteoLytic detector Polyhydroxyalkanoates (AL-PHA) beads – a library of over 20 low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured polyhydroxyalkanoate beads. In the presence of a specific protease, superfolder green fluorescent reporter proteins are cleaved from the AL-PHA beads – resulting in a loss of bead fluorescence. The Tobacco Etch Virus (TEV) AL-PHA biosensor detected the proteolytic activity of at least 1.85 pM of AcTEV. AL-PHA beads were also engineered to detect cercarial elastase from Schistosoma mansoni-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be further developed for use in resource-limited settings.
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al pha beads bioplastic based protease biosensors for global health applications
bioRxiv, 2020Co-Authors: Richard Kelwick, Alexander J Webb, Yizhou Wang, Amelie Heliot, Fiona Allan, Aidan M Emery, Michael R Templeton, Paul S FreemontAbstract:ABSTRACT Proteases are multi-functional, proteolytic enzymes that have complex roles in human health and disease. Detecting the activities of proteases can lead to important insights into communicable and non-communicable diseases. Therefore, the development of protease detection strategies can be beneficial to an array of global health applications. To this end, we developed Advanced proteoLytic detector Polyhydroxyalkanoates (AL-PHA) beads – a library of low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured polyhydroxyalkanoate (PHA) bioplastic beads. These PhaC-fusions also incorporate modular specific protease cleavage sites. In the presence of a specific protease, superfolder green fluorescent (sfGFP) reporter proteins are cleaved off of the AL-PHA beads - resulting in a loss of bead fluorescence. These AL-PHA biosensors were initially optimised using a commercially available Tobacco Etch Virus (TEV) protease. Our third generation TEV biosensor (PhaC-112L-T-G) detected 0.5 U (1.85 pM) of AcTEV activity and 10 units of AcTEV protease activity resulted in a visually noticeable loss in AL-PHA bead fluorescence. AL-PHA beads also detected cercarial elastase from Schistosoma mansoni-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be adapted towards a low-cost and high-throughput protease detection assay for global health applications.
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Cell-free prototyping strategies for enhancing the sustainable production of Polyhydroxyalkanoates bioplastics
2017Co-Authors: Richard Kelwick, Alexander J Webb, Luca Ricci, Soo Mei Chee, Derek Bell, Paul S FreemontAbstract:The Polyhydroxyalkanoates are a group of microbially-produced biopolymers that have been proposed as sustainable alternatives to several oil-derived plastics. However, Polyhydroxyalkanoates are currently more expensive to produce than oil-derived plastics and therefore, more efficient production processes would be desirable. Cell-free transcription-translation-based metabolic engineering strategies have been previously used to optimise several different biosynthetic pathways but not the Polyhydroxyalkanoates biosynthetic pathways. Here we have developed several Escherichia coli cell-free transcription-translation-based systems for in vitro prototyping of Polyhydroxyalkanoates biosynthetic operons, and also for screening relevant metabolite recycling enzymes. These cell-free transcription-translation reactions were customised through the addition of whey permeate, an industrial waste that has been previously used as a low-cost feedstock for optimising in vivo Polyhydroxyalkanoates production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ~20% compared to control reactions that did not include whey permeate. An analysis of pH in our cell-free reactions suggests that the observed increase in GFPmut3b production was likely through enhanced ATP generation, as a consequence of the glycolytic processing of lactose present in whey permeate. We also found that whey permeate enhanced cell-free reactions produced ~3μM (R)-3HB-CoA, whilst, coupled cell-free biotransformation/transcription-translation reactions produced a ten-fold greater yield of (R)-3HB-CoA. These reactions were also used to characterise a Clostridium propionicum propionyl CoA transferase enzyme that can recycle Acetyl-CoA. Together our data demonstrate that cell-free approaches can be used to complement in vivo workflows for identifying additional strategies for optimising Polyhydroxyalkanoates production.
Ralf Sodian - One of the best experts on this subject based on the ideXlab platform.
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early in vivo experience with tissue engineered trileaflet heart valves
Circulation, 2000Co-Authors: Ralf Sodian, Simon P Hoerstrup, Jason S Sperling, Sabine Daebritz, David P Martin, Frederick J Schoen, Adrian M Moran, Byung S Kim, Joseph P VacantiAbstract:BACKGROUND: Tissue engineering is a new approach in which techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional autologous tissue. Workers at our laboratory have focused on tissue engineering of heart valves. The present study was designed to evaluate the implantation of a whole trileaflet tissue-engineered heart valve in the pulmonary position in a lamb model. METHODS AND RESULTS: We constructed a biodegradable and biocompatible trileaflet heart valve scaffold that was fabricated from a porous polyhydroxyalkanoate (pore size 180 to 240 microm; Tepha Inc). Vascular cells were harvested from ovine carotid arteries, expanded in vitro, and seeded onto our heart valve scaffold. With the use of cardiopulmonary bypass, the native pulmonary leaflets were resected, and 2-cm segments of pulmonary artery were replaced by autologous cell-seeded heart valve constructs (n=4). One animal received an acellular valved conduit. No animal received any anticoagulation therapy. Animals were killed at 1, 5, 13, and 17 weeks. Explanted valves were examined histologically with scanning electron microscopy, biochemically, and biomechanically. All animals survived the procedure. The valves showed minimal regurgitation, and valve gradients were <20 mm Hg on echocardiography. The maximum gradient was 10 mm Hg with direct pressures. Macroscopically, the tissue-engineered constructs were covered with tissue, and there was no thrombus formation on any of the specimens. Scanning electron microscopy showed smooth flow surfaces during the follow-up period. Histological examination demonstrated laminated fibrous tissue with predominant glycosaminoglycans as extracellular matrix. 4-Hydroxyproline assays demonstrated an increase in collagen content as a percentage of native pulmonary artery (1 week 45.8%, 17 weeks 116%). DNA assays showed a comparable number of cells in all explanted samples. There was no tissue formation in the acellular control. CONCLUSIONS: Tissue-engineered heart valve scaffolds fabricated from Polyhydroxyalkanoates can be used for implantation in the pulmonary position with an appropriate function for 120 days in lambs.
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tissue engineering of heart valves in vitro experiences
The Annals of Thoracic Surgery, 2000Co-Authors: Ralf Sodian, Simon P Hoerstrup, Jason S Sperling, Sabine Daebritz, David P Martin, Frederick J Schoen, Joseph P Vacanti, John E MayerAbstract:Abstract Background . Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. Methods . We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 × 15 × 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 μm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. Results . It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. Conclusions . Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.
Guoqiang Chen - One of the best experts on this subject based on the ideXlab platform.
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Polyhydroxyalkanoates pha for therapeutic applications
Materials Science and Engineering: C, 2018Co-Authors: Junyu Zhang, T. G. Volova, Ekaterina I Shishatskaya, Luiziana Ferreira Da Silva, Guoqiang ChenAbstract:As intracellular carbon and energy storage materials, Polyhydroxyalkanoates (PHA) are a diverse biopolyesters synthesized by many bacteria. PHA have been produced in large quantity for various application research including medical implants for approximately 30years. Many studies demonstrated that PHA are promising implant materials due to their diverse and ascendant mechanical, biodegradable and tissue compatible properties. Importantly, common PHA biodegradation products including oligomers and monomers are also not toxic to the cells and tissues. Pharmaceutical applications of some PHA degradation products also have been reported. So far, no study has been reported to have any carcinogenesis result induced by any PHA or their biodegradation products. All results suggest that PHA could be developed into various bio-implant products.
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microbial Polyhydroxyalkanoates as medical implant biomaterials
Artificial Cells Nanomedicine and Biotechnology, 2018Co-Authors: Guoqiang Chen, Junyu ZhangAbstract:Polyhydroxyalkanoates (PHAs), a diverse biopolyester synthesized by many bacteria as intracellular carbon and energy storage materials, have been produced in large quantity for various application researches including medical implants for approximately 30 years. It has been demonstrated by many studies that PHAs possess the required mechanical, biodegradable and tissue-compatible properties for implant applications. Very importantly, common PHA biodegradation products including oligomers and monomers are also not toxic to the cells and tissues. Some PHA degradation products have been studied for pharmaceutical applications. Mechanisms of PHA that stimulate cell growth were revealed. So far, no study has been reported to have any carcinogenesis result induced by any PHA or their biodegradation products. All results point to the feasibility of PHA to be developed into various bio-implant products.
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engineering biosynthesis mechanisms for diversifying Polyhydroxyalkanoates
Trends in Biotechnology, 2015Co-Authors: Guoqiang Chen, Ivan HajnalAbstract:Polyhydroxyalkanoates (PHA) are a family of diverse biopolyesters synthesized by bacteria. PHA diversity, as reflected by its monomers, homopolymers, random and block copolymers, as well as functional polymers, can now be generated by engineering the three basic synthesis pathways including the acetoacetyl-CoA pathway, in situ fatty acid synthesis, and/or β-oxidation cycles, as well as PHA synthase specificity. It is now possible to tailor the PHA structures via genome editing or process engineering. The increasing PHA diversity and maturing PHA production technology should lead to more focused research into their low-cost and/or high-value applications.
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Polyhydroxyalkanoates challenges and opportunities
Current Opinion in Biotechnology, 2014Co-Authors: Ying Wang, Jin Yin, Guoqiang ChenAbstract:Microbial Polyhydroxyalkanoates (PHA) have been developed as biodegradable plastics for the past many years. However, PHA still have only a very limited market. Because of the availability of large amount of shale gas, petroleum will not raise dramatically in price, this situation makes PHA less competitive compared with low cost petroleum based plastics. Therefore, two strategies have been adopted to meet this challenge: first, the development of a super PHA production strain combined with advanced fermentation processes to produce PHA at a low cost; second, the construction of functional PHA production strains with technology to control the precise structures of PHA molecules, this will allow the resulting PHA with high value added applications. The recent systems and synthetic biology approaches allow the above two strategies to be implemented. In the not so distant future, the new technology will allow PHA to be produced with a competitive price compared with petroleum-based plastics.
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plastics completely synthesized by bacteria Polyhydroxyalkanoates
2010Co-Authors: Guoqiang ChenAbstract:Polyhydroxyalkanoates (PHA) produced by many bacteria have been investigated by microbiologists, molecular biologists, biochemists, chemical engineers, chemists, polymer experts, and medical researchers over the past many years. Applications of PHA as bioplastics, fine chemicals, implant biomaterials, medicines, and biofuels have been developed. Companies have been established or involved in PHA-related R&D as well as large-scale production. PHA synthesis has been found to improve the robustness of non-PHA-producing microorganisms and to regulate bacterial metabolism, leading to yield improvement for some bacterial fermentation products. In addition, amphiphilic proteins related to PHA synthesis including PhaP, PhaZ, and PhaC have been found to be useful for achieving protein purification and even specific drug targeting. It has become clear that PHA and its related technologies are forming an industrial value chain ranging from fermentation, materials, and energy to medical fields.
Marek Kowalczuk - One of the best experts on this subject based on the ideXlab platform.
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3d printed polyester based prototypes for cosmetic applications future directions at the forensic engineering of advanced polymeric materials
Materials, 2019Co-Authors: Joanna Rydz, Wanda Sikorska, Marta Musiol, Henryk Janeczek, Jakub Wlodarczyk, Marlena Misiurskamarczak, Justyna łeczycka, Marek KowalczukAbstract:Knowledge of degradation and impairment phenomena of (bio)degradable polymeric materials under operating conditions, and thus the selection of test procedures and prediction of their behavior designates the scope and capabilities as well as possible limitations of both: the preparation of the final product and its durability. The main novelty and objective of this research was to determine the degradation pathways during testing of polylactide and polylactide/polyhydroxyalkanoate materials made with three-dimensional printing and the development of a new strategy for the comprehensive characterization of such complex systems including behavior during waste disposal. Prototype objects were subjected to tests for damage evolution performed under simulating operating conditions. The reference samples and the tested items were characterized by gel permeation chromatography and differential scanning calorimetry to determine changes in material properties. The studies showed that: polyhydroxyalkanoate component during accelerated aging and degradation in environments rich in microorganisms accelerated the degradation of the material; paraffin accelerates polylactide degradation and slows degradation of polyhydroxyalkanoate-based material; under the influence of an environment rich in enzymes, paraffin contamination accelerates biodegradation; under the influence of natural conditions, paraffin contamination slowed degradation; the processing conditions, in particular the printing orientation of individual parts of the container, influenced the material properties in its various regions, affecting the rate of degradation of individual parts.
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3D-Printed Polyester-Based Prototypes for Cosmetic Applications—Future Directions at the Forensic Engineering of Advanced Polymeric Materials
MDPI AG, 2019Co-Authors: Joanna Rydz, Wanda Sikorska, Henryk Janeczek, Marta Musioł, Jakub Włodarczyk, Marlena Misiurska-marczak, Justyna Łęczycka, Marek KowalczukAbstract:Knowledge of degradation and impairment phenomena of (bio)degradable polymeric materials under operating conditions, and thus the selection of test procedures and prediction of their behavior designates the scope and capabilities as well as possible limitations of both: the preparation of the final product and its durability. The main novelty and objective of this research was to determine the degradation pathways during testing of polylactide and polylactide/polyhydroxyalkanoate materials made with three-dimensional printing and the development of a new strategy for the comprehensive characterization of such complex systems including behavior during waste disposal. Prototype objects were subjected to tests for damage evolution performed under simulating operating conditions. The reference samples and the tested items were characterized by gel permeation chromatography and differential scanning calorimetry to determine changes in material properties. The studies showed that: polyhydroxyalkanoate component during accelerated aging and degradation in environments rich in microorganisms accelerated the degradation of the material; paraffin accelerates polylactide degradation and slows degradation of polyhydroxyalkanoate-based material; under the influence of an environment rich in enzymes, paraffin contamination accelerates biodegradation; under the influence of natural conditions, paraffin contamination slowed degradation; the processing conditions, in particular the printing orientation of individual parts of the container, influenced the material properties in its various regions, affecting the rate of degradation of individual parts