15 results on '"Duarte Miguel F, Prazeres"'
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2. Manufacturing of non-viral protein nanocages for biotechnological and biomedical applications
- Author
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Jorge João and Duarte Miguel F. Prazeres
- Subjects
biomanufacturing ,bottom-up synthesis ,downstream processing ,drug delivery ,nanostructure engineering ,protein nanocages ,Biotechnology ,TP248.13-248.65 - Abstract
Protein nanocages are highly ordered nanometer scale architectures, which are typically formed by homo- or hetero-self-assembly of multiple monomers into symmetric structures of different size and shape. The intrinsic characteristics of protein nanocages make them very attractive and promising as a biological nanomaterial. These include, among others, a high surface/volume ratio, multi-functionality, ease to modify or manipulate genetically or chemically, high stability, mono-dispersity, and biocompatibility. Since the beginning of the investigation into protein nanocages, several applications were conceived in a variety of areas such as drug delivery, vaccine development, bioimaging, biomineralization, nanomaterial synthesis and biocatalysis. The ability to generate large amounts of pure and well-folded protein assemblies is one of the keys to transform nanocages into clinically valuable products and move biomedical applications forward. This calls for the development of more efficient biomanufacturing processes and for the setting up of analytical techniques adequate for the quality control and characterization of the biological function and structure of nanocages. This review concisely covers and overviews the progress made since the emergence of protein nanocages as a new, next-generation class of biologics. A brief outline of non-viral protein nanocages is followed by a presentation of their main applications in the areas of bioengineering, biotechnology, and biomedicine. Afterwards, we focus on a description of the current processes used in the manufacturing of protein nanocages with particular emphasis on the most relevant aspects of production and purification. The state-of-the-art on current characterization techniques is then described and future alternative or complementary approaches in development are also discussed. Finally, a critical analysis of the limitations and drawbacks of the current manufacturing strategies is presented, alongside with the identification of the major challenges and bottlenecks.
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- 2023
- Full Text
- View/download PDF
3. Minicircle Biopharmaceuticals–An Overview of Purification Strategies
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Cláudia P. A. Alves, Duarte Miguel F. Prazeres, and Gabriel A. Monteiro
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minicircle DNA ,non-viral DNA vector ,gene therapy ,biopharmaceuticals ,purification ,chromatography ,Technology ,Chemical technology ,TP1-1185 - Abstract
Minicircles are non-viral delivery vectors with promising features for biopharmaceutical applications. These vectors are plasmid-derived circular DNA molecules that are obtained in vivo in Escherichia coli by the intramolecular recombination of a parental plasmid, which generates a minicircle containing the eukaryotic therapeutic cassette of interest and a miniplasmid containing the prokaryotic backbone. The production process results thus in a complex mixture, which hinders the isolation of minicircle molecules from other DNA molecules. Several strategies have been proposed over the years to meet the challenge of purifying and obtaining high quality minicircles in compliance with the regulatory guidelines for therapeutic use. In minicircle purification, the characteristics of the strain and parental plasmid used have a high impact and strongly affect the purification strategy that can be applied. This review summarizes the different methods developed so far, focusing not only on the purification method itself but also on its dependence on the upstream production strategy used.
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- 2021
- Full Text
- View/download PDF
4. Functionalization of Cellulose-Based Hydrogels with Bi-Functional Fusion Proteins Containing Carbohydrate-Binding Modules
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Mariana Barbosa, Hélvio Simões, and Duarte Miguel F. Prazeres
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biomolecular recognition ,carbohydrate-binding module ,cellulose ,hydrogel ,ionic liquid ,Technology ,Electrical engineering. Electronics. Nuclear engineering ,TK1-9971 ,Engineering (General). Civil engineering (General) ,TA1-2040 ,Microscopy ,QH201-278.5 ,Descriptive and experimental mechanics ,QC120-168.85 - Abstract
Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.
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- 2021
- Full Text
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5. A Cellulose Paper-Based Fluorescent Lateral Flow Immunoassay for the Quantitative Detection of Cardiac Troponin I
- Author
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Satheesh Natarajan, Joseph Jayaraj, and Duarte Miguel F. Prazeres
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biomarker ,carbon nanofiber ,cellulose ,diagnostics ,immunoassay ,lateral flow assays ,Biotechnology ,TP248.13-248.65 - Abstract
This paper presents a lateral flow assay (LFA) for the quantitative, fluorescence-based detection of the cardiac biomarker troponin I (cTnI) that features an analytical strip made of cellulose filter paper. The results show that the wicking and test time are comparable to those obtained with conventional nitrocellulose (NC)-based LFAs. Further, the cellulose paper provides an excellent background with no auto-fluorescence that is very adequate in detecting fluorescent lines. While fluorescence that was generated with cellulose strips was lower when compared to that generated in NC strips, signals could be improved by layering carbon nanofibers (CNF) on the cellulose. A nonlinear behavior of the concentration–response relationship was observed for the LFA architectures with NC, cellulose, and cellulose-CNF in the 0 to 200 ng/mL cTnI concentration range. The measurements were consistent and characterized by coefficients of variation lower than 2.5%. Detection and quantitation limits that were in the range 1.28–1.40 ng/mL and 2.10–2.75 ng/mL were obtained for LFA with cellulose and cellulose CNF strips that are equivalent to the limits obtained with the standard NC LFA. Overall, we showed that commercially available filter paper can be used in the analytical strip of LFA.
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- 2021
- Full Text
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6. Plasmid Biopharmaceuticals: Basics, Applications, and Manufacturing
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Duarte Miguel F. Prazeres and Duarte Miguel F. Prazeres
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- 2011
7. Scalable purification of single stranded DNA scaffolds for biomanufacturing DNA-origami nanostructures: Exploring anion-exchange and multimodal chromatography
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A. Rita Silva-Santos, Pedro M.R. Paulo, and Duarte Miguel F. Prazeres
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Filtration and Separation ,Analytical Chemistry - Published
- 2022
8. Monitoring Intracellular Calcium in Response to GPCR Activation: Comparison Between Microtiter Plates and Microfluidic Assays
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Sofia Aires M, Martins, Duarte Miguel F, Prazeres, Virginia, Chu, and João P, Conde
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HEK293 Cells ,Microfluidics ,Receptor, Muscarinic M1 ,Animals ,Humans ,Calcium ,Carbachol ,Muscarinic Antagonists ,Pirenzepine ,Cholinergic Agonists ,Receptors, G-Protein-Coupled ,Signal Transduction - Abstract
Microfluidic strategies combined with transduction and electronic integration have the promise of enabling miniaturized, combinatorial assays at higher speeds and lower costs, while at the same time mimicking the local chemical concentrations and force fields of the cellular in vivo environment. In this chapter we introduce a microfluidic structure with hydrodynamic cell traps and a culture volume in the nanoliter range (50 nL), to quantitatively evaluate the transient calcium response of the endogenous Muscarinic type 1 receptor (M1) in HEK 293 T cells. The microfluidic fabrication protocol is described as well as a methodology to monitor the cell response in real time, after stimulation with M1 agonists (e.g., carbachol) and antagonists (e.g., Pirenzepine).
- Published
- 2021
9. Purification of Plasmid DNA by Multimodal Chromatography
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A Rita, Silva-Santos, Sara Sousa, Rosa, Duarte Miguel F, Prazeres, and Ana M, Azevedo
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Chromatography ,DNA, Superhelical ,Escherichia coli ,DNA, Circular ,Dialysis ,Plasmids - Abstract
Multimodal (MM) chromatography can be described as a chromatographic method that uses more than one mode of interaction between the target molecule and the ligand to achieve a particular separation. Owing to its advantages over traditional chromatography, such as higher selectivity and capacity, its application for the purification of biomolecules with therapeutic interest has been widely studied. The potential of MM chromatography for the purification of plasmid DNA has been demonstrated. In this chapter, a downstream process for the purification of supercoiled plasmid DNA using MM chromatography with two different ligands-Capto™ adhere and PPA HyperCell™-is described. In both the cases, the purification process yields a high purity and highly homogeneous sc plasmid product.
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- 2020
10. Primary Purification of Plasmid DNA Using Differential Isopropanol Precipitation
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Alexandra, Wagner, A Rita, Silva-Santos, Sara Sousa, Rosa, Sophie, Gierak, Ana M, Azevedo, and Duarte Miguel F, Prazeres
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2-Propanol ,Escherichia coli ,Chemical Precipitation ,Urea ,Electrophoresis, Polyacrylamide Gel ,DNA ,Plasmids - Abstract
A method for the intermediate recovery of plasmid DNA (pDNA) from alkaline lysates is described that comprises differential isopropanol precipitation steps. In a first low-cut precipitation, a smaller amount of isopropanol (20% v/v) is used so that only high molecular weight RNA precipitates. After solid liquid separation, a high-cut precipitation is performed by bringing isopropanol concentration to 70% v/v to precipitate pDNA. Tests made with lysates show that the differential precipitation increases purity threefold compared to the conventional one-step precipitation at 70% v/v without affecting pDNA recovery (80%).
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- 2020
11. Plasmid Biopharmaceuticals
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Duarte Miguel F. Prazeres and Gabriel A. Monteiro
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- 2015
12. Use of ImageJ to recover information from individual cells in a G protein-coupled receptor assay
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João R C, Trabuco, Sofia Aires M, Martins, and Duarte Miguel F, Prazeres
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Aniline Compounds ,Receptor, Muscarinic M1 ,Video Recording ,Gene Expression ,Fluorescence ,HEK293 Cells ,Xanthenes ,Image Processing, Computer-Assisted ,Humans ,Calcium ,Carbachol ,Calcium Signaling ,Single-Cell Analysis ,Software ,Fluorescent Dyes - Abstract
Live-cell assays used in GPCR research often rely on fluorescence techniques that generate large amounts of raw image data. Consequently, the capacity to accurately and timely extract useful information from image and video data has become more and more important. Image J is an open-source program that provides powerful tools with a simple interface designed to fit the needs of image analysis of most researchers. In this chapter, Image J routines to extract information from individual cells in a calcium GPCR assay are described. In these routines, individual cells in the same image/video data can be separated using either a progressive threshold or a local threshold method. Both methods can be optimized to either a maximum number of selection or maximum area selected resulting in conceptually distinct selections.
- Published
- 2015
13. G protein-coupled receptors: an overview of signaling mechanisms and screening assays
- Author
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Duarte Miguel F, Prazeres and Sofia Aires M, Martins
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Radioligand Assay ,Animals ,Gene Expression ,Humans ,Biological Assay ,History, 19th Century ,Cloning, Molecular ,History, 20th Century ,Crystallography, X-Ray ,History, 21st Century ,High-Throughput Screening Assays ,Receptors, G-Protein-Coupled ,Signal Transduction - Abstract
The existence of cellular receptors, a group of specialized biomolecules to which endogenous and exogenous compounds bind and exert an effect, is one of the most exciting aspects of cell biology. Among the different receptor types recognized today, G-protein-coupled receptors (GPCRs) constitute, undoubtedly, one of the most important classes, in part due to their versatility, but particularly, due to their central role in a multitude of physiological states. The unveiling of GPCR function and mode of action is a challenging task that prevails until our days, as the full potential of these receptors is far from being established. Such an undertaking calls for a joint effort of multidisciplinary teams that must combine state-of-the-art technologies with in-depth knowledge of cell biology to probe such specialized molecules. This review provides a concise coverage of the scientific progress that has been made in GPCR research to provide researchers with an updated overview of the field. A brief outline of the historical breakthroughs is followed by a discussion of GPCR signaling mechanisms and by a description of the role played by assay technologies.
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- 2015
14. Plasmid Biopharmaceuticals
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Duarte Miguel F, Prazeres and Gabriel A, Monteiro
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Microbiology (medical) ,Drug Carriers ,General Immunology and Microbiology ,Ecology ,Physiology ,Genetic Therapy ,Cell Biology ,Infectious Diseases ,Vaccines, DNA ,Genetics ,Humans ,Technology, Pharmaceutical ,Biotechnology ,Plasmids - Abstract
Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.
- Published
- 2014
15. DNA Vaccines
- Author
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Duarte Miguel F. Prazeres and Gabriel Amaro Monteiro
- Published
- 2007
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