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2.
Science ; 368(6494): 993-1001, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32467389

RESUMO

Sophisticated devices for remote-controlled medical interventions require an electrogenetic interface that uses digital electronic input to directly program cellular behavior. We present a cofactor-free bioelectronic interface that directly links wireless-powered electrical stimulation of human cells to either synthetic promoter-driven transgene expression or rapid secretion of constitutively expressed protein therapeutics from vesicular stores. Electrogenetic control was achieved by coupling ectopic expression of the L-type voltage-gated channel CaV1.2 and the inwardly rectifying potassium channel Kir2.1 to the desired output through endogenous calcium signaling. Focusing on type 1 diabetes, we engineered electrosensitive human ß cells (Electroß cells). Wireless electrical stimulation of Electroß cells inside a custom-built bioelectronic device provided real-time control of vesicular insulin release; insulin levels peaked within 10 minutes. When subcutaneously implanted, this electrotriggered vesicular release system restored normoglycemia in type 1 diabetic mice.


Assuntos
Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Estimulação Elétrica/instrumentação , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Tecnologia sem Fio/instrumentação , Animais , Biônica , Canais de Cálcio Tipo L/genética , Sinalização do Cálcio , Engenharia Celular , Células HEK293 , Humanos , Masculino , Camundongos , Canais de Potássio Corretores do Fluxo de Internalização/genética , Próteses e Implantes , Transcrição Genética , Transgenes
3.
PLoS One ; 15(4): e0231664, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302338

RESUMO

Natural killer (NK) cells are innate lymphocytes with functions that include target cell killing, inflammation and regulation. NK cells integrate incoming activating and inhibitory signals through an array of germline-encoded receptors to gauge the health of neighbouring cells. The reactive potential of NK cells is influenced by microRNA (miRNA), small non-coding sequences that interfere with mRNA expression. miRNAs are highly conserved between species, and a single miRNA can have hundreds to thousands of targets and influence entire cellular programs. Two miRNA species, miR-155-5p and miR-146a-5p are known to be important in controlling NK cell function, but research to best understand the impacts of miRNA species within NK cells has been bottlenecked by a lack of techniques for altering miRNA concentrations efficiently and without off-target effects. Here, we describe a non-viral and straightforward approach for increasing or decreasing expression of miRNA in primary human NK cells. We achieve >90% transfection efficiency without off-target impacts on NK cell viability, education, phenotype or function. This opens the opportunity to study and manipulate NK cell miRNA profiles and their impacts on NK cellular programs which may influence outcomes of cancer, inflammation and autoimmunity.


Assuntos
Engenharia Celular/métodos , Células Matadoras Naturais/metabolismo , MicroRNAs/genética , Transfecção/métodos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Meios de Cultura Livres de Soro/farmacologia , Voluntários Saudáveis , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , Cultura Primária de Células
4.
Nat Commun ; 11(1): 1881, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312967

RESUMO

Cells maintain reserves in their metabolic and translational capacities as a strategy to quickly respond to changing environments. Here we quantify these reserves by stepwise reducing nitrogen availability in yeast steady-state chemostat cultures, imposing severe restrictions on total cellular protein and transcript content. Combining multi-omics analysis with metabolic modeling, we find that seven metabolic superpathways maintain >50% metabolic capacity in reserve, with glucose metabolism maintaining >80% reserve capacity. Cells maintain >50% reserve in translational capacity for 2490 out of 3361 expressed genes (74%), with a disproportionately large reserve dedicated to translating metabolic proteins. Finally, ribosome reserves contain up to 30% sub-stoichiometric ribosomal proteins, with activation of reserve translational capacity associated with selective upregulation of 17 ribosomal proteins. Together, our dataset provides a quantitative link between yeast physiology and cellular economics, which could be leveraged in future cell engineering through targeted proteome streamlining.


Assuntos
Biossíntese de Proteínas , Proteômica , Saccharomyces cerevisiae/metabolismo , Reatores Biológicos , Engenharia Celular , Fermentação , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Redes e Vias Metabólicas , Nitrogênio/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Proteínas Ribossômicas/metabolismo
5.
Nucleic Acids Res ; 48(8): 4081-4099, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32187373

RESUMO

Cytosine methylation is a ubiquitous modification in mammalian DNA generated and maintained by several DNA methyltransferases (DNMTs) with partially overlapping functions and genomic targets. To systematically dissect the factors specifying each DNMT's activity, we engineered combinatorial knock-in of human DNMT genes in Komagataella phaffii, a yeast species lacking endogenous DNA methylation. Time-course expression measurements captured dynamic network-level adaptation of cells to DNMT3B1-induced DNA methylation stress and showed that coordinately modulating the availability of S-adenosyl methionine (SAM), the essential metabolite for DNMT-catalyzed methylation, is an evolutionarily conserved epigenetic stress response, also implicated in several human diseases. Convolutional neural networks trained on genome-wide CpG-methylation data learned distinct sequence preferences of DNMT3 family members. A simulated annealing interpretation method resolved these preferences into individual flanking nucleotides and periodic poly(A) tracts that rotationally position highly methylated cytosines relative to phased nucleosomes. Furthermore, the nucleosome repeat length defined the spatial unit of methylation spreading. Gene methylation patterns were similar to those in mammals, and hypo- and hypermethylation were predictive of increased and decreased transcription relative to control, respectively, in the absence of mammalian readers of DNA methylation. Introducing controlled epigenetic perturbations in yeast thus enabled characterization of fundamental genomic features directing specific DNMT3 proteins.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Epigênese Genética , Saccharomycetales/genética , Engenharia Celular , Centrômero , Cromatina/química , DNA (Citosina-5-)-Metiltransferases/genética , Técnicas de Introdução de Genes , Genoma Fúngico , Humanos , Redes Neurais de Computação , S-Adenosilmetionina/metabolismo , Saccharomycetales/metabolismo , Estresse Fisiológico/genética , Telômero , Transcrição Genética
6.
Nat Commun ; 11(1): 1193, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132536

RESUMO

The last decade has seen bacteria at the forefront of biotechnological innovation, with applications including biomolecular computing, living therapeutics, microbiome engineering and microbial factories. These emerging applications are all united by the need to precisely control complex microbial dynamics in spatially extended environments, requiring tools that can bridge the gap between intracellular and population-level coordination. To address this need, we engineer an inducible quorum sensing system which enables precise tunability of bacterial dynamics both at the population and community level. As a proof-of-principle, we demonstrate the advantages of this system when genetically equipped for cargo delivery. In addition, we exploit the absence of cross-talk with respect to the majority of well-characterized quorum sensing systems to demonstrate inducibility of multi-strain communities. More broadly, this work highlights the unexplored potential of remotely inducible quorum sensing systems which, coupled to any gene of interest, may facilitate the translation of circuit designs into applications.


Assuntos
Engenharia Celular/métodos , Escherichia coli/fisiologia , Microbiota/fisiologia , Percepção de Quorum/genética , Estudo de Prova de Conceito , Biologia Sintética/métodos
8.
Mol Cell ; 78(1): 184-191.e3, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32027839

RESUMO

The ability to integrate biological signals and execute a functional response when appropriate is critical for sophisticated cell engineering using synthetic biology. Although the CRISPR-Cas system has been harnessed for synthetic manipulation of the genome, it has not been fully utilized for complex environmental signal sensing, integration, and actuation. Here, we develop a split dCas12a platform and show that it allows for the construction of multi-input, multi-output logic circuits in mammalian cells. The system is highly programmable and can generate expandable AND gates with two, three, and four inputs. It can also incorporate NOT logic by using anti-CRISPR proteins as an OFF switch. By coupling the split dCas12a design to multiple tumor-relevant promoters, we provide a proof of concept that the system can implement logic gating to specifically detect breast cancer cells and execute therapeutic immunomodulatory responses.


Assuntos
Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Engenharia Celular , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Dimerização , Feminino , Células HEK293 , Humanos , Ativação Transcricional
9.
Nat Biotechnol ; 38(4): 426-432, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32015549

RESUMO

Approaches to increase the activity of chimeric antigen receptor (CAR)-T cells against solid tumors may also increase the risk of toxicity and other side effects. To improve the safety of CAR-T-cell therapy, we computationally designed a chemically disruptable heterodimer (CDH) based on the binding of two human proteins. The CDH self-assembles, can be disrupted by a small-molecule drug and has a high-affinity protein interface with minimal amino acid deviation from wild-type human proteins. We incorporated the CDH into a synthetic heterodimeric CAR, called STOP-CAR, that has an antigen-recognition chain and a CD3ζ- and CD28-containing endodomain signaling chain. We tested STOP-CAR-T cells specific for two antigens in vitro and in vivo and found similar antitumor activity compared to second-generation (2G) CAR-T cells. Timed administration of the small-molecule drug dynamically inactivated the activity of STOP-CAR-T cells. Our work highlights the potential for structure-based design to add controllable elements to synthetic cellular therapies.


Assuntos
Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos Quiméricos/química , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T/efeitos dos fármacos , Engenharia Celular , Células Cultivadas , Humanos , Imunoterapia Adotiva , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Células PC-3 , Ligação Proteica , Engenharia de Proteínas , Multimerização Proteica , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/antagonistas & inibidores , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/química , Linfócitos T/imunologia , Linfócitos T/metabolismo
10.
Nat Biotechnol ; 38(4): 460-470, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32094658

RESUMO

Generation of pancreatic ß cells from human pluripotent stem cells (hPSCs) holds promise as a cell replacement therapy for diabetes. In this study, we establish a link between the state of the actin cytoskeleton and the expression of pancreatic transcription factors that drive pancreatic lineage specification. Bulk and single-cell RNA sequencing demonstrated that different degrees of actin polymerization biased cells toward various endodermal lineages and that conditions favoring a polymerized cytoskeleton strongly inhibited neurogenin 3-induced endocrine differentiation. Using latrunculin A to depolymerize the cytoskeleton during endocrine induction, we developed a two-dimensional differentiation protocol for generating human pluripotent stem-cell-derived ß (SC-ß) cells with improved in vitro and in vivo function. SC-ß cells differentiated from four hPSC lines exhibited first- and second-phase dynamic glucose-stimulated insulin secretion. Transplantation of islet-sized aggregates of these cells rapidly reversed severe preexisting diabetes in mice at a rate close to that of human islets and maintained normoglycemia for at least 9 months.


Assuntos
Engenharia Celular/métodos , Citoesqueleto/metabolismo , Células Secretoras de Insulina/citologia , Células-Tronco Pluripotentes/citologia , Actinas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Diabetes Mellitus/terapia , Endoderma/citologia , Endoderma/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/transplante , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Pluripotentes/metabolismo , Tiazolidinas/farmacologia , Transativadores/metabolismo , Moduladores de Tubulina/farmacologia
11.
Science ; 367(6481)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32029687

RESUMO

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRß (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.


Assuntos
Transferência Adotiva , Sistemas CRISPR-Cas , Edição de Genes , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Linfócitos T/transplante , Idoso , Proteína 9 Associada à CRISPR , Engenharia Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/genética , Transgenes
12.
Cancer Immunol Immunother ; 69(5): 731-744, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32036448

RESUMO

Tumor-associated macrophages (TAMs) have been shown to both aid and hinder tumor growth, with patient outcomes potentially hinging on the proportion of M1, pro-inflammatory/growth-inhibiting, to M2, growth-supporting, phenotypes. Strategies to stimulate tumor regression by promoting polarization to M1 are a novel approach that harnesses the immune system to enhance therapeutic outcomes, including chemotherapy. We recently found that nanotherapy with mesoporous particles loaded with albumin-bound paclitaxel (MSV-nab-PTX) promotes macrophage polarization towards M1 in breast cancer liver metastases (BCLM). However, it remains unclear to what extent tumor regression can be maximized based on modulation of the macrophage phenotype, especially for poorly perfused tumors such as BCLM. Here, for the first time, a CRISPR system is employed to permanently modulate macrophage polarization in a controlled in vitro setting. This enables the design of 3D co-culture experiments mimicking the BCLM hypovascularized environment with various ratios of polarized macrophages. We implement a mathematical framework to evaluate nanoparticle-mediated chemotherapy in conjunction with TAM polarization. The response is predicted to be not linearly dependent on the M1:M2 ratio. To investigate this phenomenon, the response is simulated via the model for a variety of M1:M2 ratios. The modeling indicates that polarization to an all-M1 population may be less effective than a combination of both M1 and M2. Experimental results with the CRISPR system confirm this model-driven hypothesis. Altogether, this study indicates that response to nanoparticle-mediated chemotherapy targeting poorly perfused tumors may benefit from a fine-tuned M1:M2 ratio that maintains both phenotypes in the tumor microenvironment during treatment.


Assuntos
Paclitaxel Ligado a Albumina/administração & dosagem , Neoplasias da Mama/terapia , Neoplasias Hepáticas/terapia , Ativação de Macrófagos/genética , Macrófagos/imunologia , Modelos Biológicos , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Engenharia Celular , Linhagem Celular Tumoral/transplante , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Humanos , Lipossomos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Camundongos , Nanopartículas , Esferoides Celulares , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
13.
PLoS One ; 15(2): e0228112, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32040512

RESUMO

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αß T-cell receptors (TCRαß) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαßs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαß genes as Sleeping Beauty transposons and the determination of the introduced TCRαßs' antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.


Assuntos
Engenharia Celular/métodos , Clonagem Molecular/métodos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Expressão Gênica , Biblioteca Gênica , Genes MHC Classe I/genética , Genes MHC da Classe II/genética , Células HEK293 , Humanos , Cinética , Receptores de Antígenos de Linfócitos T alfa-beta/genética
14.
Nat Commun ; 11(1): 304, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949141

RESUMO

Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development. These independent tools can be combined for spatial and temporal depletion of different sets of proteins, can help to distinguish immediate cellular responses from long-term adaptation effects and can facilitate the exploration of complex networks.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Técnicas Citológicas , Luz , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/efeitos da radiação , Animais , Apoptose , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/efeitos da radiação , Caspases/efeitos dos fármacos , Caspases/metabolismo , Caspases/efeitos da radiação , Engenharia Celular/métodos , Dano ao DNA , DNA Ligase Dependente de ATP , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Lamina Tipo A/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética
15.
Nat Biotechnol ; 38(2): 233-244, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907405

RESUMO

Despite the global rapid increase in the number of clinical trials employing chimeric antigen receptors (CARs), no comprehensive survey of their scope, targets and design exists. In this study, we present an interactive CAR clinical trial database, spanning 64 targets deployed in T cells (CAR-T), natural killer cells (CAR-NK) or mixtures (CAR-NK/T) from over 500 clinical trials in 20 countries, encompassing >20,000 patients. By combining these data with transcriptional and proteomic data, we create a 'targetable landscape' for CAR cell therapies based on 13,206 proteins and RNAs across 78 tissues, 124 cell types and 20 cancer types. These data suggest a landscape of over 100 single targets and over 100,000 target pairs using logical switches for CAR cell engineering. Our analysis of the CAR cellular therapeutic landscape may aid the design of future therapies, improve target-to-patient matching, and ultimately help inform our understanding of CAR therapy's safety and efficacy.


Assuntos
Engenharia Celular/métodos , Receptores de Antígenos Quiméricos/metabolismo , Terapia Baseada em Transplante de Células e Tecidos , Ensaios Clínicos como Assunto , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo
16.
Chemphyschem ; 21(2): 131, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31957181

RESUMO

The front cover artwork is provided by the group of Dr. Baojun Wang (The University of Edinburgh). The image shows an engineered bacterial cell containing a genetic amplifier circuit which transforms a weak input signal into a larger easily detectable output signal. The electronics symbols used to illustrate the genetic circuit highlight the programmability of the circuit components enabled by state-of-the-art synthetic biology tools. Read the full text of the Review at 10.1002/cphc.201900739.


Assuntos
Bactérias/citologia , Engenharia Celular , Eletrônica , Ilustração Médica , Biologia Sintética , Humanos
17.
Chemphyschem ; 21(2): 132-144, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31585026

RESUMO

Cell-based biosensors offer cheap, portable and simple methods of detecting molecules of interest but have yet to be truly adopted commercially. Issues with their performance and specificity initially slowed the development of cell-based biosensors. With the development of rational approaches to tune response curves, the performance of biosensors has rapidly improved and there are now many biosensors capable of sensing with the required performance. This has stimulated an increased interest in biosensors and their commercial potential. However the reliability, long term stability and biosecurity of these sensors are still barriers to commercial application and public acceptance. Research into overcoming these issues remains active. Here we present the state-of-the-art tools offered by synthetic biology to allow construction of cell-based biosensors with customisable performance to meet the real world requirements in terms of sensitivity and dynamic range and discuss the research progress to overcome the challenges in terms of the sensor stability and biosecurity fears.


Assuntos
Bactérias/citologia , Técnicas Biossensoriais , Engenharia Celular , Biologia Sintética , Técnicas Biossensoriais/instrumentação , Humanos
18.
Biochem Mol Biol Educ ; 48(1): 67-73, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31532903

RESUMO

The ability to separate, identify, and quantify proteins from complex mixtures are key foundational methods across biochemistry teaching and research. In particular, enzyme-linked immunosorbent assay (ELISA) is an important technique that is used to measure antigen concentrations in both industry and academia. There are four categories of ELISA, direct, indirect, competitive, and sandwich, each with their own applications. Sandwich ELISAs are used to determine antigen concentrations from complex mixtures of protein, such as a cell lysates, and are regularly used as medical diagnostics to diagnose illness and diseases ranging from hepatitis to celiac disease. One major problem with teaching the sandwich ELISA technique to students is the prohibitive cost due to the need to coat a 96-well plate with a capture antibody. One solution to this problem would be to significantly reduce the role of each student in the lab, but this does not adequately prepare students to perform the procedure in a research or industry lab. Instead, this laboratory exercise teaches students the procedural knowledge needed to perform a direct sandwich ELISA, but uses a simulated experience performed within a wet-lab environment. The presented scenario is the analysis of phosphorylated proteins within a synthetic signaling pathway, but because the lab uses simulated samples, it can be tailored to different topics and educational aims. The procedure is 10- to 26-fold less expensive per student to deploy than an authentic sandwich ELISA. Students in the course report that the ELISA lab significantly strengthened the connection between theory and practice. © 2019 International Union of Biochemistry and Molecular Biology, 48(1):67-73, 2020.


Assuntos
Doença Celíaca/diagnóstico , Engenharia Celular , Ensaio de Imunoadsorção Enzimática/economia , Hepatite/diagnóstico , Transdução de Sinais , Humanos , Estudantes
19.
Biotechnol Adv ; 40: 107497, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31830520

RESUMO

Long-chain polyunsaturated fatty acids (LC-PUFAs) especially ω-3 fatty acids provide significant health benefits for human beings. However, ω-3 LC-PUFAs cannot be synthesized de novo in mammals. Traditionally, ω-3 LC-PUFAs are extracted from marine fish, and their production depends on sea fishing, which has not met ever-increasing global demand. To address the challenges, innovative cellular engineering strategies need to be developed. In nature, many fungi and microalgae are rich in ω-3 LC-PUFAs, representing promising sources of ω-3 LC-PUFAs. The latest progress in developing new cellular engineering strategies toward sustainable ω-3 LC-PUFAs production using fungi and microalga has demonstrated that they can to some extent address the supply shortage. In this review, we critically summarize the recent progress in enhancing the productivity in various ω-3 LC-PUFAs-producing organisms, as well as the latest efforts of biosynthesizing PUFAs in heterogenous biosystems. In addition, we also provide future perspectives in developing genetic toolkits for LC-PUFAs producing microbes so that cut-edging biotechnology such as gene stacking and genome editing can be further applied to increase the productivity of ω-3 LC-PUFAs.


Assuntos
Engenharia Celular , Microalgas , Animais , Ácidos Graxos Ômega-3 , Ácidos Graxos Insaturados , Humanos , Engenharia Metabólica
20.
Arch Physiol Biochem ; 126(2): 139-156, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-30445857

RESUMO

Anchorage of a subset of cell surface proteins in eukaryotic cells is mediated by a glycosylphosphatidylinositol (GPI) moiety covalently attached to the carboxy-terminus of the protein moiety. Experimental evidence for the potential of GPI-anchored proteins (GPI-AP) of being released from cells into the extracellular environment has been accumulating, which involves either the loss or retention of the GPI anchor. Release of GPI-AP from donor cells may occur spontaneously or in response to endogenous or environmental signals. The experimental evidence for direct insertion of exogenous GPI-AP equipped with the complete anchor structure into the outer plasma membrane bilayer leaflets of acceptor cells is reviewed as well as the potential underlying molecular mechanisms. Furthermore, promiscuous transfer of certain GPI-AP between plasma membranes of different cells in vivo under certain (patho)physiological conditions has been reported. Engineering of target cell surfaces using chimeric GPI-AP with complete GPI anchor may be useful for therapeutic applications.


Assuntos
Engenharia Celular/métodos , Glicosilfosfatidilinositóis/metabolismo , Proteínas Ligadas a Lipídeos/uso terapêutico , Doenças Metabólicas/terapia , Neoplasias/terapia , Doenças Priônicas/terapia , Transtornos da Coagulação Sanguínea/terapia , Membrana Celular/química , Membrana Celular/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Glicosilfosfatidilinositóis/química , Hemoglobinúria Paroxística/terapia , Humanos , Imunoterapia/métodos , Proteínas Ligadas a Lipídeos/química , Proteínas Ligadas a Lipídeos/metabolismo , Transporte Proteico , Técnicas de Reprodução Assistida , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia
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