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1.
Nat Commun ; 11(1): 5448, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116131

RESUMO

Compartmentalization is a ubiquitous building principle in cells, which permits segregation of biological elements and reactions. The carboxysome is a specialized bacterial organelle that encapsulates enzymes into a virus-like protein shell and plays essential roles in photosynthetic carbon fixation. The naturally designed architecture, semi-permeability, and catalytic improvement of carboxysomes have inspired rational design and engineering of new nanomaterials to incorporate desired enzymes into the protein shell for enhanced catalytic performance. Here, we build large, intact carboxysome shells (over 90 nm in diameter) in the industrial microorganism Escherichia coli by expressing a set of carboxysome protein-encoding genes. We develop strategies for enzyme activation, shell self-assembly, and cargo encapsulation to construct a robust nanoreactor that incorporates catalytically active [FeFe]-hydrogenases and functional partners within the empty shell for the production of hydrogen. We show that shell encapsulation and the internal microenvironment of the new catalyst facilitate hydrogen production of the encapsulated oxygen-sensitive hydrogenases. The study provides insights into the assembly and formation of carboxysomes and paves the way for engineering carboxysome shell-based nanoreactors to recruit specific enzymes for diverse catalytic reactions.


Assuntos
Proteínas de Bactérias/metabolismo , Reatores Biológicos , Hidrogênio/metabolismo , Organelas/metabolismo , Proteínas de Bactérias/genética , Biocatálise , Bioengenharia , Reatores Biológicos/microbiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Halothiobacillus/genética , Halothiobacillus/metabolismo , Hidrogenase/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Nanocápsulas/química , Nanocápsulas/ultraestrutura , Organelas/genética , Organelas/ultraestrutura , Fotossíntese , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo
3.
Nature ; 585(7825): 383-389, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939070

RESUMO

Insect eyes have an anti-reflective coating, owing to nanostructures on the corneal surface creating a gradient of refractive index between that of air and that of the lens material1,2. These nanocoatings have also been shown to provide anti-adhesive functionality3. The morphology of corneal nanocoatings are very diverse in arthropods, with nipple-like structures that can be organized into arrays or fused into ridge-like structures4. This diversity can be attributed to a reaction-diffusion mechanism4 and patterning principles developed by Alan Turing5, which have applications in numerous biological settings6. The nanocoatings on insect corneas are one example of such Turing patterns, and the first known example of nanoscale Turing patterns4. Here we demonstrate a clear link between the morphology and function of the nanocoatings on Drosophila corneas. We find that nanocoatings that consist of individual protrusions have better anti-reflective properties, whereas partially merged structures have better anti-adhesion properties. We use biochemical analysis and genetic modification techniques to reverse engineer the protein Retinin and corneal waxes as the building blocks of the nanostructures. In the context of Turing patterns, these building blocks fulfil the roles of activator and inhibitor, respectively. We then establish low-cost production of Retinin, and mix this synthetic protein with waxes to forward engineer various artificial nanocoatings with insect-like morphology and anti-adhesive or anti-reflective function. Our combined reverse- and forward-engineering approach thus provides a way to economically produce functional nanostructured coatings from biodegradable materials.


Assuntos
Bioengenharia , Córnea/anatomia & histologia , Córnea/fisiologia , Proteínas de Drosophila/química , Drosophila/anatomia & histologia , Proteínas do Olho/química , Nanoestruturas/química , Ceras/química , Adesividade , Análise de Variância , Animais , Córnea/química , Difusão , Drosophila/química , Drosophila/classificação , Drosophila/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas do Olho/genética , Técnicas de Silenciamento de Genes , Nanomedicina , Ligação Proteica , Engenharia de Proteínas , Dobramento de Proteína
4.
Chimia (Aarau) ; 74(9): 704-709, 2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-32958108

RESUMO

Protein sequences inhabit a discrete set in macromolecular space with incredible capacity to treat human disease. Despite our ability to program and manipulate protein sequences, the vast majority of protein development efforts are still done heuristically without a unified set of guiding principles. This article highlights work in understanding biophysical stability and function of proteins, developing new biophysical measurement tools and building high-throughput screening platforms to explore functional protein sequences. We highlight two primary areas. First, molecular biomechanics is a subfield concerned with the response of proteins to mechanical forces, and how we can leverage mechanical force to control protein function. The second subfield investigates the use of polymers and hydrogels in protein engineering and directed evolution in pursuit of new molecular systems with therapeutic applications. These two subdisciplines complement each other by shedding light onto sequence and structural features that can be used to impart stability into therapeutic proteins.


Assuntos
Bioengenharia , Nanotecnologia , Humanos , Hidrogéis , Engenharia de Proteínas , Proteínas
5.
Nat Commun ; 11(1): 4440, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895374

RESUMO

Traditionally engineered genetic circuits have almost exclusively used naturally occurring transcriptional repressors. Recently, non-natural transcription factors (repressors) have been engineered and employed in synthetic biology with great success. However, transcriptional anti-repressors have largely been absent with regard to the regulation of genes in engineered genetic circuits. Here, we present a workflow for engineering systems of non-natural anti-repressors. In this study, we create 41 inducible anti-repressors. This collection of transcription factors respond to two distinct ligands, fructose (anti-FruR) or D-ribose (anti-RbsR); and were complemented by 14 additional engineered anti-repressors that respond to the ligand isopropyl ß-d-1-thiogalactopyranoside (anti-LacI). In turn, we use this collection of anti-repressors and complementary genetic architectures to confer logical control over gene expression. Here, we achieved all NOT oriented logical controls (i.e., NOT, NOR, NAND, and XNOR). The engineered transcription factors and corresponding series, parallel, and series-parallel genetic architectures represent a nascent anti-repressor based transcriptional programming structure.


Assuntos
Bioengenharia/métodos , Repressores Lac/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Expressão Gênica/fisiologia , Redes Reguladoras de Genes , Repressores Lac/síntese química , Ligantes , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/síntese química , Biologia Sintética/métodos , Fatores de Transcrição/síntese química , Fatores de Transcrição/metabolismo
6.
J Environ Manage ; 273: 111096, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32734892

RESUMO

Environmental sustainability criteria and rising energy demands, exhaustion of conventional resources of energy followed by environmental degradation due to abrupt climate changes have shifted the attention of scientists to seek renewable sources of green and clean energy for sustainable development. Bioenergy is an excellent alternative since it can be applied for several energy-requirements after utilizing suitable conversion methodology. This review elucidates all aspects of biofuels (bioethanol, biodiesel, and butanol) and their sustainability criteria. The principal focus is on the latest developments in biofuel production chiefly stressing on the role of nanotechnology. A plethora of investigations regarding the emerging techniques for process improvement like integration methods, less energy-intensive distillation techniques, and bioengineering of microorganisms are discussed. This can assist in making biofuel-production in a real-world market more economically and environmentally viable.


Assuntos
Biocombustíveis , Microalgas , Bioengenharia , Biotecnologia , Conservação dos Recursos Naturais
7.
Theranostics ; 10(16): 7034-7052, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32641977

RESUMO

This review provides an update for the international research community on the cell modeling tools that could accelerate the understanding of SARS-CoV-2 infection mechanisms and could thus speed up the development of vaccines and therapeutic agents against COVID-19. Many bioengineering groups are actively developing frontier tools that are capable of providing realistic three-dimensional (3D) models for biological research, including cell culture scaffolds, microfluidic chambers for the culture of tissue equivalents and organoids, and implantable windows for intravital imaging. Here, we review the most innovative study models based on these bioengineering tools in the context of virology and vaccinology. To make it easier for scientists working on SARS-CoV-2 to identify and apply specific tools, we discuss how they could accelerate the discovery and preclinical development of antiviral drugs and vaccines, compared to conventional models.


Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Vacinas Virais/isolamento & purificação , Vacinas Virais/farmacologia , Betacoronavirus/química , Betacoronavirus/genética , Betacoronavirus/imunologia , Bioengenharia/métodos , Bioengenharia/tendências , Reatores Biológicos , Técnicas de Cultura de Células , Simulação por Computador , Infecções por Coronavirus/imunologia , Descoberta de Drogas/métodos , Descoberta de Drogas/tendências , Avaliação de Medicamentos/métodos , Avaliação de Medicamentos/tendências , Farmacorresistência Viral , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Modelos Biológicos , Organoides/citologia , Organoides/virologia , Pneumonia Viral/imunologia , Nanomedicina Teranóstica
8.
Surg Clin North Am ; 100(4): 741-756, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32681874

RESUMO

Cellular and/or tissue-based products (CTPs) have advanced greatly in the past several decades and improve the ability to heal wounds more efficiently. Products can be characterized as nonviable cells, tissue based, animal; nonviable cells, tissue based, human; viable human cells, cultured in vitro, animal substrate; viable human cells, cultured in vitro, synthetic substrate; viable human cells, noncultured, intact tissue. There are approximately 77 different CTPs at the time of this writing, with many more being investigated. Cellular and/or tissue-based product selection, application, postapplication course, and patient selection depend on patient attributes, CTP specifications, and surgeon preference.


Assuntos
Bandagens , Cicatrização/fisiologia , Ferimentos e Lesões/terapia , Bioengenharia , Curativos Biológicos , Queimaduras/fisiopatologia , Queimaduras/terapia , Humanos , Pele Artificial , Infecção dos Ferimentos/fisiopatologia , Infecção dos Ferimentos/prevenção & controle , Ferimentos e Lesões/fisiopatologia
10.
Nat Commun ; 11(1): 3658, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694613

RESUMO

Biological systems organize multiple hierarchical structures in parallel, and create dynamic assemblies and functions by energy dissipation. In contrast, emerging artificial non-equilibrium self-assembling systems have remained relatively simplistic concerning hierarchical design, and non-equilibrium multi-component systems are uncharted territory. Here we report a modular DNA toolbox allowing to program transient non-equilibrium multicomponent systems across hierarchical length scales by introducing chemically fueled molecular recognition orchestrated by reaction networks of concurrent ATP-powered ligation and cleavage of freely programmable DNA building blocks. Going across hierarchical levels, we demonstrate transient side-chain functionalized nucleic acid polymers, and further introduce the concept of transient cooperative multivalency as a key to bridge length scales to pioneer fuel-driven encapsulation, self-assembly of colloids, and non-equilibrium transient narcissistic colloidal self-sorting on a systems level. The fully programmable and functionalizable DNA components pave the way to design chemically fueled 4D (3 space, 1 time) molecular multicomponent systems and autonomous materials.


Assuntos
Trifosfato de Adenosina/química , Bioengenharia/métodos , DNA/química , Nanotecnologia/métodos , Coloides , DNA Ligases/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Conformação de Ácido Nucleico , Polimerização , Polímeros/química
11.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1249-1256, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597075

RESUMO

Microbial genetics and breeding is a compulsory course for "Bioengineering Excellence Talents Experimental Class" and "Bioengineering International Student Class". However, the traditional teaching model has many deficiencies in terms of content selection, teaching methods and examination forms. At Tianjin University of Science and Technology, to improve the quality and effectiveness of teaching, especially in the field of microbiology, innovative leaders who meet the needs of national and international communities are highly needed. This article describes the reformed teaching content, teaching methods, and curriculum assessment methods of microbial genetics and breeding. With the help of the latest scientific research progress, pre-class preview system, video display, and diversified assessment methods, teaching mode has been innovatively reformed. As such, students not only mastered the relevant professional knowledge of microbial genetics and breeding, but also exercised their subjective initiative, teamwork consciousness, professional foreign language expression level, and cultivated their interest in scientific knowledge related to microbial genetics.


Assuntos
Cruzamento , Currículo , Genética Microbiana , Bioengenharia/educação , Currículo/normas , Genética Microbiana/educação , Genética Microbiana/tendências , Humanos , Estudantes
12.
Nat Rev Cardiol ; 17(11): 685-697, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32483304

RESUMO

Extracellular vesicles (EVs) are a heterogeneous group of natural particles that are relevant to the treatment of cardiovascular diseases. These endogenous vesicles have certain properties that allow them to survive in the extracellular space, bypass biological barriers and deliver their biologically active molecular cargo to recipient cells. Moreover, EVs can be bioengineered to increase their stability, bioactivity, presentation to acceptor cells and capacity for on-target binding at both cell-type-specific and tissue-specific levels. Bioengineering of EVs involves the modification of the donor cell before EV isolation or direct modification of the EV properties after isolation. The therapeutic potential of native EVs and bioengineered EVs has been only minimally explored in the context of cardiovascular diseases. Efforts to harness the therapeutic potential of EVs will require innovative approaches and a comprehensive integration of knowledge gathered from decades of research into molecular-compound delivery. In this Review, we outline the endogenous properties of EVs that make them natural delivery agents as well as the features that can be improved by bioengineering. We also discuss the therapeutic applications of native and bioengineered EVs to cardiovascular diseases and examine the opportunities and challenges that need to be addressed to advance this research area, with an emphasis on clinical translation.


Assuntos
Bioengenharia , Doenças Cardiovasculares/terapia , Vesículas Extracelulares/transplante , Isquemia/terapia , Infarto do Miocárdio/terapia , Regeneração , Células-Tronco/metabolismo , Acidente Vascular Cerebral/terapia , Encéfalo/fisiologia , Sobrevivência Celular , Vesículas Extracelulares/metabolismo , Extremidades/irrigação sanguínea , Coração/fisiologia , Humanos , MicroRNAs/metabolismo , MicroRNAs/uso terapêutico , Miócitos Cardíacos , Comunicação Parácrina
13.
MAbs ; 12(1): 1770028, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32486889

RESUMO

Many therapeutic monoclonal antibodies (mAbs) were initially developed for intravenous (IV) administration. As a means to improve mAb drug-ability and the patient experience, subcutaneous (SC) administration is an increasingly important delivery route for mAbs. Unlike IV administration, bioavailability limitations for antibodies have been reported following SC injection and can dictate whether a mAb is administered via this parenteral route. The SC bioavailability of antibodies has been difficult to predict, and it can be variable and partial, with values ranging from ~50% to 100%. The mechanisms leading to the incomplete bioavailability of some mAbs relative to others are not well understood. There are some limited data that suggest the physiochemical properties inherent to a mAb can contribute to its SC absorption, bioavailability, and in vivo fate. In this study, we evaluated the integrated influence of multiple mAb physiochemical factors on the SC absorption and bioavailability of six humanized mAbs in both rats and cynomolgus monkeys. We demonstrate the physiochemical properties of mAbs are critical to their rate and extent of SC absorption. The combination of high positive charge and hydrophobic interaction significantly reduced the rate of the evaluated mAb's SC absorption and bioavailability. Reduction or balancing of both these attributes via re-engineering the mAbs restored desirable properties of the molecules assessed. This included reduced association with SC tissue, improvements in mAb absorption from the SC space and overall SC bioavailability. Our findings point to the importance of evaluating the relative balance between various physiochemical factors, including charge, hydrophobicity, and stability, to improve the SC drug-ability of mAbs for selecting or engineering mAbs with enhanced in vivo absorption and bioavailability following SC administration.


Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Físico-Química/métodos , Animais , Anticorpos Monoclonais Humanizados/química , Bioengenharia , Disponibilidade Biológica , Desenvolvimento de Medicamentos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Injeções Subcutâneas , Macaca fascicularis , Ligação Proteica , Estabilidade Proteica , Ratos , Absorção Subcutânea
14.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 1012-1016, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567284

RESUMO

Strengthening practical teaching, together with improving innovation ability is one of the key tasks of Emerging Engineering Education. This paper is based on the revision of the training program of bioengineering in School of Chemical Engineering and Technology, Tianjin University, improved the practical teaching system and curriculum content, built a five-level teaching system for basic experiment, comprehensive experiment, course design, scientific research and practical training. In order to cultivate outstanding innovative talents with practical ability and innovative spirit, innovative teaching reform mode is proposed. Furthermore the new thought and new schemes for Emerging Engineering Education are put forward.


Assuntos
Bioengenharia , Currículo , Bioengenharia/educação
15.
PLoS One ; 15(5): e0233021, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32396571

RESUMO

The time-dependent properties of rubber-like synthesized and biological materials are crucial for their applications. Currently, this behavior is mainly measured using axial tensile test, compression test, or indentation. Limited studies performed on using multi-axial loading measurements of time-dependent material behavior exist in the literature. Therefore, the aim of this study is to investigate the viscoelastic response of rubber-like materials under multi-axial loading using cavity expansion and relaxation tests. The tests were performed on PVA hydrogel specimens. Three hyperelasitc models and one term Prony series were used to characterize the viscoelastic response of the hydrogels. Finite element (FE) simulations were performed to verify the validity of the calibrated material coefficients by reproducing the experimental results. The excellent agreement between the experimental, analytical and numerical data proves the capability of the cavity expansion technique to measure the time-dependent behavior of viscoelastic materials.


Assuntos
Álcool de Polivinil/química , Bioengenharia , Fenômenos Biomecânicos , Elasticidade , Elastômeros/química , Análise de Elementos Finitos , Hidrogéis/química , Teste de Materiais , Estresse Mecânico , Viscosidade
16.
Nat Commun ; 11(1): 2105, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32355158

RESUMO

3D-printing networks of droplets connected by interface bilayers are a powerful platform to build synthetic tissues in which functionality relies on precisely ordered structures. However, the structural precision and consistency in assembling these structures is currently limited, which restricts intricate designs and the complexity of functions performed by synthetic tissues. Here, we report that the equilibrium contact angle (θDIB) between a pair of droplets is a key parameter that dictates the tessellation and precise positioning of hundreds of picolitre-sized droplets within 3D-printed, multi-layer networks. When θDIB approximates the geometrically-derived critical angle (θc) of 35.3°, the resulting networks of droplets arrange in regular hexagonal close-packed (hcp) lattices with the least fraction of defects. With this improved control over droplet packing, we can 3D-print functional synthetic tissues with single-droplet-wide conductive pathways. Our new insights into 3D droplet packing permit the fabrication of complex synthetic tissues, where precisely positioned compartments perform coordinated tasks.


Assuntos
Bioengenharia/instrumentação , Bicamadas Lipídicas/química , Impressão Tridimensional , Bioengenharia/métodos , Materiais Biomiméticos/química , Cinética , Lipídeos/química , Microscopia Confocal , Temperatura , Água/química
17.
BMJ ; 369: m718, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32349978

RESUMO

Head and neck structures govern the vital functions of breathing and swallowing. Additionally, these structures facilitate our sense of self through vocal communication, hearing, facial animation, and physical appearance. Loss of these functions can lead to loss of life or greatly affect quality of life. Regenerative medicine is a rapidly developing field that aims to repair or replace damaged cells, tissues, and organs. Although the field is largely in its nascence, regenerative medicine holds promise for improving on conventional treatments for head and neck disorders or providing therapies where no current standard exists. This review presents milestones in the research of regenerative medicine in head and neck surgery.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Otolaringologia/tendências , Procedimentos Cirúrgicos Reconstrutivos/tendências , Medicina Regenerativa/tendências , Tecidos Suporte , Bioengenharia , Transplante de Células/métodos , Transplante de Células/tendências , Cóclea , Cartilagem da Orelha , Ossos Faciais , Humanos , Laringe , Cartilagens Nasais , Procedimentos Cirúrgicos Reconstrutivos/métodos , Glândulas Salivares , Crânio , Engenharia Tecidual/métodos , Engenharia Tecidual/tendências , Traqueia , Membrana Timpânica
18.
Nat Commun ; 11(1): 2272, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385237

RESUMO

Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8 kb biosynthetic gene cluster. Here, we establish a platform for in vitro biosynthesis of lactazole A, referred to as the FIT-Laz system, via a combination of the flexible in vitro translation (FIT) system with recombinantly produced lactazole biosynthetic enzymes. Systematic dissection of lactazole biosynthesis reveals remarkable substrate tolerance of the biosynthetic enzymes and leads to the development of the minimal lactazole scaffold, a construct requiring only 6 post-translational modifications for macrocyclization. Efficient assembly of such minimal thiopeptides with FIT-Laz opens access to diverse lactazole analogs with 10 consecutive mutations, 14- to 62-membered macrocycles, and 18 amino acid-long tail regions, as well as to hybrid thiopeptides containing non-proteinogenic amino acids. This work suggests that the minimal lactazole scaffold is amenable to extensive bioengineering and opens possibilities to explore untapped chemical space of thiopeptides.


Assuntos
Bioengenharia , Peptídeos/metabolismo , Tiazóis/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas , Código Genético , Peptídeos/química , Especificidade por Substrato , Tiazóis/química
19.
Nat Commun ; 11(1): 2183, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366821

RESUMO

Coupling myoelectric and mechanical signals during voluntary muscle contraction is paramount in human-machine interactions. Spatiotemporal differences in the two signals intrinsically arise from the muscular excitation-contraction process; however, current methods fail to deliver local electromechanical coupling of the process. Here we present the locally coupled electromechanical interface based on a quadra-layered ionotronic hybrid (named as CoupOn) that mimics the transmembrane cytoadhesion architecture. CoupOn simultaneously monitors mechanical strains with a gauge factor of ~34 and surface electromyogram with a signal-to-noise ratio of 32.2 dB. The resolved excitation-contraction signatures of forearm flexor muscles can recognize flexions of different fingers, hand grips of varying strength, and nervous and metabolic muscle fatigue. The orthogonal correlation of hand grip strength with speed is further exploited to manipulate robotic hands for recapitulating corresponding gesture dynamics. It can be envisioned that such locally coupled electromechanical interfaces would endow cyber-human interactions with unprecedented robustness and dexterity.


Assuntos
Eletromiografia/métodos , Força da Mão/fisiologia , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Amplitude de Movimento Articular/fisiologia , Membros Artificiais , Bioengenharia/instrumentação , Bioengenharia/métodos , Fenômenos Biomecânicos , Eletrônica Médica/instrumentação , Eletrônica Médica/métodos , Dedos/fisiologia , Antebraço/fisiologia , Mãos/fisiologia , Humanos , Desenho de Prótese/instrumentação , Desenho de Prótese/métodos
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