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1.
Virus Res ; 305: 198563, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34530046

RESUMO

This study compared the lethality of severe acute respiratory syndrome coronavirus 2 variants belonging to the S, V, L, G, GH, and GR clades using K18-human angiotensin-converting enzyme 2 heterozygous mice. To estimate the 50% lethal dose (LD50) of each variant, increasing viral loads (100-104 plaque-forming units [PFU]) were administered intranasally. Mouse weight and survival were monitored for 14 days. The LD50 of the GH and GR clades was significantly lower than that of other clades at 50 PFU. These findings suggest that the GH and GR clades, which are prevalent worldwide, are more virulent than the other clades.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/mortalidade , Receptores Virais/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Carga Viral/genética , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Sequência de Bases , Peso Corporal , COVID-19/patologia , COVID-19/virologia , Chlorocebus aethiops , Expressão Gênica , Humanos , Dose Letal Mediana , Masculino , Camundongos , Camundongos Transgênicos , Filogenia , Receptores Virais/metabolismo , SARS-CoV-2/classificação , SARS-CoV-2/metabolismo , Índice de Gravidade de Doença , Análise de Sobrevida , Transgenes , Células Vero , Ensaio de Placa Viral , Virulência
2.
Viruses ; 13(7)2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34372571

RESUMO

Anti-cancer activity can be improved by engineering immune cells to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Retroviral vector gene transfer strategies allow stable and durable transgene expression. Here, we used alpharetroviral vectors to modify NK-92 cells, a natural killer cell line, with a third-generation CAR designed to target the IL-3 receptor subunit alpha (CD123), which is strongly expressed on the surface of acute myeloid leukemia (AML) cells. Alpharetroviral vectors also contained a transgene cassette to allow constitutive expression of human IL-15 for increased NK cell persistence in vivo. The anti-AML activity of CAR-NK-92 cells was tested via in vitro cytotoxicity assays with the CD123+ AML cell line KG-1a and in vivo in a patient-derived xenotransplantation CD123+ AML model. Unmodified NK-92 cells or NK-92 cells modified with a truncated version of the CAR that lacked the signaling domain served as controls. Alpharetroviral vector-modified NK-92 cells stably expressed the transgenes and secreted IL-15. Anti-CD123-CAR-NK-92 cells exhibited enhanced anti-AML activity in vitro and in vivo as compared to control NK-92 cells. Our data (1) shows the importance of IL-15 expression for in vivo persistence of NK-92 cells, (2) supports continued investigation of anti-CD123-CAR-NK cells to target AML, and (3) points towards potential strategies to further improve CAR-NK anti-AML activity.


Assuntos
Imunoterapia Adotiva/métodos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Idoso , Alpharetrovirus/genética , Animais , Linhagem Celular Tumoral , Feminino , Terapia Genética , Vetores Genéticos/genética , Humanos , Subunidade alfa de Receptor de Interleucina-3/genética , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Cultura Primária de Células , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Transdução Genética , Transgenes , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Biomolecules ; 11(8)2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34439801

RESUMO

The potential, multifaceted therapeutic profile of cannabidiol (CBD), a major constituent derived from the Cannabis sativa plant, covers a wide range of neurological and psychiatric disorders, ranging from anxiety to pediatric epilepsy and drug addiction. However, the molecular targets responsible for these effects have been only partially identified. In this view, the involvement of the orexin system, the key regulator in arousal and the sleep/wake cycle, and in motivation and reward processes, including drug addiction, prompted us to explore, using computational and experimental approaches, the possibility that CBD could act as a ligand of orexin receptors, orexin 1 receptor of type 1 (OX1R) and type 2 (OX2R). Ligand-binding assays showed that CBD is a selective ligand of OX1R in the low micromolar range (Ki 1.58 ± 0.2 µM) while in vitro functional assays, carried out by intracellular calcium imaging and mobilization assays, showed that CBD acts as an antagonist at this receptor. Finally, the putative binding mode of CBD has been inferred by molecular docking and molecular dynamics simulations and its selectivity toward the OX1R subtype rationalized at the molecular level. This study provides the first evidence that CBD acts as an OX1R antagonist, supporting its potential use in addictive disorders and/or body weight regulation.


Assuntos
Ansiolíticos/farmacologia , Anticonvulsivantes/farmacologia , Canabidiol/farmacologia , Receptores de Orexina/química , Orexinas/química , Animais , Ansiolíticos/química , Ansiolíticos/metabolismo , Anticonvulsivantes/química , Anticonvulsivantes/metabolismo , Sítios de Ligação , Células CHO , Cálcio/metabolismo , Canabidiol/química , Canabidiol/metabolismo , Cricetulus , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Imagem Molecular , Antagonistas dos Receptores de Orexina , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Ensaio Radioligante , Transgenes
4.
Biomolecules ; 11(8)2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34439829

RESUMO

Retinitis pigmentosa (RP) is a hereditary disease of the retina that results in complete blindness. Currently, there are very few treatments for the disease and those that exist work only for the recessively inherited forms. To better understand the pathogenesis of RP, multiple mouse models have been generated bearing mutations found in human patients including the human Q344X rhodopsin knock-in mouse. In recent years, the immune system was shown to play an increasingly important role in RP degeneration. By way of electroretinography, optical coherence tomography, funduscopy, fluorescein angiography, and fluorescent immunohistochemistry, we show degenerative and vascular phenotypes, microglial activation, photoreceptor phagocytosis, and upregulation of proinflammatory pathway proteins in the retinas of the human Q344X rhodopsin knock-in mouse. We also show that an FDA-approved pharmacological agent indicated for the treatment of rheumatoid arthritis is able to halt activation of pro-inflammatory signaling in cultured retinal cells, setting the stage for pre-clinical trials using these mice to inhibit proinflammatory signaling in an attempt to preserve vision. We conclude from this work that pro- and autoinflammatory upregulation likely act to enhance the progression of the degenerative phenotype of rhodopsin Q344X-mediated RP and that inhibition of these pathways may lead to longer-lasting vision in not only the Q344X rhodopsin knock-in mice, but humans as well.


Assuntos
Antirreumáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Fator Inibidor de Leucemia/farmacologia , Mutação , Retina/efeitos dos fármacos , Retinite Pigmentosa/tratamento farmacológico , Rodopsina/genética , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Janus Quinases/antagonistas & inibidores , Janus Quinases/genética , Janus Quinases/imunologia , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/imunologia , Microglia/patologia , NF-kappa B/genética , NF-kappa B/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Retina/imunologia , Retina/patologia , Retinite Pigmentosa/genética , Retinite Pigmentosa/imunologia , Retinite Pigmentosa/patologia , Rodopsina/deficiência , Fatores de Transcrição STAT/antagonistas & inibidores , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/imunologia , Transdução de Sinais , Transgenes , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
5.
Poult Sci ; 100(10): 101365, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34375836

RESUMO

Virus injection into EGK-X embryos is a well-defined approach in avian transgenesis. This system uses a chicken ovalbumin gene promoter to induce transgene expression in the chicken oviduct. Although a reconstructed chicken ovalbumin promoter that links an ovalbumin promoter and estrogen-responsive enhancer element (ERE) is useful, a large viral vector containing the ovalbumin promoter and a target gene restricts viral packaging capacity and produces low-titer virus particles. We newly developed recombinant chicken promoters by linking regulatory regions of ovalbumin and other oviduct-specific genes. Putative enhancer fragments of the genes, such as ovotransferrin (TF), ovomucin alpha subunit (OVOA), and ovalbumin-related protein X (OVALX), were placed at the 5`-flanking region of the 2.8-kb ovalbumin promoter. Basal promoter fragments of the genes, namely, pTF, lysozyme (pLYZ), and ovomucoid (pOVM), were placed at the 3`-flanking region of the 1.6-kb ovalbumin ERE. The recombinant promoters cloned into each reporter vector were evaluated using a dual luciferase assay in human and chicken somatic cells, and LMH/2A cells treated with 0-1,000 nM estrogen, and cultured primary chicken oviduct cells. The recombinant promoters with linking ovalbumin and TF, OVOA, pOVM, and pLYZ regulatory regions had 2.1- to 19.5-fold (P < 0.05) higher luciferase activity than the reconstructed ovalbumin promoter in chicken oviduct cells. Therefore, recombinant promoters may be used to efficiently drive transgene expression in transgenic chickens.


Assuntos
Galinhas , Oviductos , Animais , Galinhas/genética , Tubas Uterinas , Feminino , Humanos , Ovalbumina/genética , Regiões Promotoras Genéticas , Transgenes
6.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361005

RESUMO

Although the development of gene delivery systems based on non-viral vectors is advancing, it remains a challenge to deliver plasmid DNA into human blood cells. The current "gold standard", namely linear polyethyleneimine (l-PEI 25 kDa), in particular, is unable to produce transgene expression levels >5% in primary human B lymphocytes. Here, it is demonstrated that a well-defined 24-armed poly(2-dimethylamino) ethyl methacrylate (PDMAEMA, 755 kDa) nano-star is able to reproducibly elicit high transgene expression (40%) at sufficient residual viability (69%) in primary human B cells derived from tonsillar tissue. Moreover, our results indicate that the length of the mitogenic stimulation prior to transfection is an important parameter that must be established during the development of the transfection protocol. In our hands, four days of stimulation with rhCD40L post-thawing led to the best transfection results in terms of TE and cell survival. Most importantly, our data argue for an impact of the B cell subsets on the transfection outcomes, underlining that the complexity and heterogeneity of a given B cell population pre- and post-transfection is a critical parameter to consider in the multiparametric approach required for the implementation of the transfection protocol.


Assuntos
Linfócitos B/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Linfócitos B/citologia , Células Cultivadas , Humanos , Metacrilatos/química , Nylons/química , Transgenes
7.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361789

RESUMO

Isobavachalcone (IBC) is an active substance from the medicinal plant Psoralea corylifolia. This prenylated chalcone was reported to possess antioxidative, anti-inflammatory, antibacterial, and anticancer activities. Multidrug resistance (MDR) associated with the over-expression of the transporters of vast substrate specificity such as ABCB1 (P-glycoprotein) belongs to the main causes of cancer chemotherapy failure. The cytotoxic, MDR reversing, and ABCB1-inhibiting potency of isobavachalcone was studied in two cellular models: human colorectal adenocarcinoma HT29 cell line and its resistant counterpart HT29/Dx in which doxorubicin resistance was induced by prolonged drug treatment, and the variant of MDCK cells transfected with the human gene encoding ABCB1. Because MDR modulators are frequently membrane-active substances, the interaction of isobavachalcone with model phosphatidylcholine bilayers was studied by means of differential scanning calorimetry. Molecular modeling was employed to characterize the process of membrane permeation by isobavachalcone. IBC interacted with ABCB1 transporter, being a substrate and/or competitive inhibitor of ABCB1. Moreover, IBC intercalated into model membranes, significantly affecting the parameters of their main phospholipid phase transition. It was concluded that isobavachalcone interfered both with the lipid phase of cellular membrane and with ABCB1 transporter, and for this reason, its activity in MDR cancer cells was presumptively beneficial.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Chalconas/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Psoralea/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Ligação Competitiva , Linhagem Celular Tumoral , Chalconas/química , Chalconas/isolamento & purificação , Cães , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Células HT29 , Humanos , Concentração Inibidora 50 , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Células Madin Darby de Rim Canino , Membranas Artificiais , Modelos Moleculares , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Extratos Vegetais/química , Plantas Medicinais , Ligação Proteica , Transgenes , Verapamil/farmacologia
8.
Anim Genet ; 52(5): 759-761, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34339052

RESUMO

Gene doping is prohibited in horseracing. In a previous study, we developed a method for non-targeted transgene detection using DELLY, which is based on split-read (SR) and paired-end (PE) algorithms to detect structural variants, on WGS data. In this study, we validated the detection sensitivity of DELLY using artificially generated sequence data of 12 target genes. With DELLY, at least one intron was detected as a deletion in eight targeted genes using the 150 bp PE read WGS data, whereas all targeted genes were detected by DELLY using the 100 bp PE read data. The detection sensitivity was higher in 100 bp PE reads than in 150 bp PE reads, despite a lower total sequence coverage, probably because of mismatch tolerance between the mapped reads and reference genome. In addition, it was observed that the average intron size detected by SR alone was 293 bp and that that detected by both SR and PE was 8924 bp. Thus, we showed that transgenes with various intron-exon structures could be detected using DELLY, suggesting its application in gene-doping control in horses.


Assuntos
Animais Geneticamente Modificados , Doping nos Esportes , Cavalos/genética , Íntrons , Esportes , Transgenes , Algoritmos , Animais , Éxons
9.
Cell Host Microbe ; 29(9): 1437-1453.e8, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34428428

RESUMO

The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and can be deployed easily. Here, AAVCOVID-1, an adeno-associated viral (AAV), spike-gene-based vaccine candidate demonstrates potent immunogenicity in mouse and non-human primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques. Peak neutralizing antibody titers are sustained at 1 year and complemented by functional memory T cell responses. The AAVCOVID vector has no relevant pre-existing immunity in humans and does not elicit cross-reactivity to common AAVs used in gene therapy. Vector genome persistence and expression wanes following injection. The single low-dose requirement, high-yield manufacturability, and 1-month stability for storage at room temperature may make this technology well suited to support effective immunization campaigns for emerging pathogens on a global scale.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/genética , Dependovirus/genética , Dependovirus/metabolismo , Feminino , Humanos , Imunogenicidade da Vacina/imunologia , Memória Imunológica/imunologia , Macaca fascicularis , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Transgenes/genética , Vacinação/métodos , Carga Viral/imunologia
10.
Biomolecules ; 11(8)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34439791

RESUMO

CHO is the cell line of choice for the manufacturing of many complex biotherapeutics. The constant upgrading of cell productivity is needed to meet the growing demand for these life-saving drugs. Manipulation of small non-coding RNAs-miRNAs-is a good alternative to a single gene knockdown approach due to their post-transcriptional regulation of entire cellular pathways without posing translational burden to the production cell. In this study, we performed a high-throughput screening of 2042-human miRNAs and identified several candidates able to increase cell-specific and overall production of Erythropoietin and Etanercept in CHO cells. Some of these human miRNAs have not been found in Chinese hamster cells and yet were still effective in them. We identified miR-574-3p as being able, when overexpressed in CHO cells, to improve overall productivity of Erythropoietin and Etanercept titers from 1.3 to up to 2-fold. In addition, we validated several targets of miR-574-3p and identified p300 as a main target of miR-574-3p in CHO cells. Furthermore, we demonstrated that stable CHO cell overexpressing miRNAs from endogenous CHO pri-miRNA sequences outperform the cells with human pri-miRNA sequences. Our findings highlight the importance of flanking genomic sequences, and their secondary structure features, on pri-miRNA processing offering a novel, cost-effective and fast strategy as a valuable tool for efficient miRNAs engineering in CHO cells.


Assuntos
Eritropoetina/genética , Etanercepte/metabolismo , Engenharia Genética/métodos , MicroRNAs/genética , Transgenes , Animais , Células CHO , Cricetulus , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Eritropoetina/biossíntese , Etanercepte/química , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
11.
Biomolecules ; 11(8)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34439793

RESUMO

The histamine H4 receptor (H4R) is a G protein-coupled receptor that is predominantly expressed on immune cells and considered to be an important drug target for various inflammatory disorders. Like most GPCRs, the H4R activates G proteins and recruits ß-arrestins upon phosphorylation by GPCR kinases to induce cellular signaling in response to agonist stimulation. However, in the last decade, novel GPCR-interacting proteins have been identified that may regulate GPCR functioning. In this study, a split-ubiquitin membrane yeast two-hybrid assay was used to identify H4R interactors in a Jurkat T cell line cDNA library. Forty-three novel H4R interactors were identified, of which 17 have also been previously observed in MYTH screens to interact with other GPCR subtypes. The interaction of H4R with the tetraspanin TSPAN4 was confirmed in transfected cells using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and co-immunoprecipitation. Histamine stimulation reduced the interaction between H4R and TSPAN4, but TSPAN4 did not affect H4R-mediated G protein signaling. Nonetheless, the identification of novel GPCR interactors by MYTH is a starting point to further investigate the regulation of GPCR signaling.


Assuntos
Receptores Histamínicos H4/metabolismo , Tetraspaninas/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Expressão Gênica , Biblioteca Gênica , Células HEK293 , Histamina/metabolismo , Histamina/farmacologia , Humanos , Células Jurkat , Fosforilação/efeitos dos fármacos , Ligação Proteica , Mapeamento de Interação de Proteínas , Receptores Histamínicos H4/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Tetraspaninas/genética , Transgenes , Técnicas do Sistema de Duplo-Híbrido
12.
Bioresour Technol ; 340: 125676, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34365302

RESUMO

Transgene expression in microalgae can be hampered by transgene silencing and unstable expression due to position effects. To overcome this, "safe harboring" transgene expression system was established for Nannochloropsis. Initially, transformants were obtained expressing a sfGFP reporter, followed by screening for high expression of sfGFP with fluorescence-activated cell sorter (FACS). 'T1' transcriptional hotspot was identified from a mutant showing best expression of sfGFP, but did not affect growth or lipid contents. By using a Cas9 editor strain, FAD12 gene, encoding Δ12-fatty acid desaturase (FAD12), was successfully knocked-in at the T1 locus, resulting in significantly higher expression of FAD12 than those of random integration. Importantly, the "safe harbored" FAD12 transformants showed four-fold higher production of linoleic acid (LA), the product of FAD12, leading to 1.5-fold increase in eicosapentaenoic acid (EPA). This safe harboring principle provide excellent proof of the concept for successful genetic/metabolic engineering of microalgae and other organisms.


Assuntos
Sistemas CRISPR-Cas , Estramenópilas , Sistemas CRISPR-Cas/genética , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Engenharia Genética , Estramenópilas/genética , Estramenópilas/metabolismo , Transgenes
13.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445105

RESUMO

In grafted plants, the movement of long-distance signals from rootstocks can modulate the development and function of the scion. To understand the mechanisms by which tolerant rootstocks improve scion responses to osmotic stress (OS) conditions, mRNA transport of osmotic responsive genes (ORGs) was evaluated in a tomato/potato heterograft system. In this system, Solanum tuberosum was used as a rootstock and Solanum lycopersicum as a scion. We detected changes in the gene expression levels of 13 out of the 21 ORGs tested in the osmotically stressed plants; of these, only NPR1 transcripts were transported across the graft union under both normal and OS conditions. Importantly, OS increased the abundance of StNPR1 transcripts in the tomato scion. To examine mRNA mobility in transgrafted plants, StNPR1 and StDREB1 genes representing the mobile and non-mobile transcripts, respectively, were overexpressed in tobacco (Nicotiana tabacum). The evaluation of transgenic tobacco plants indicated that overexpression of these genes enhanced the growth and improved the physiological status of transgenic plants growing under OS conditions induced by NaCl, mannitol and polyethylene glycol (PEG). We also found that transgenic tobacco rootstocks increased the OS tolerance of the WT-scion. Indeed, WT scions on transgenic rootstocks had higher ORGs transcript levels than their counterparts on non-transgenic rootstocks. However, neither StNPR1 nor StDREB1 transcripts were transported from the transgenic rootstock to the wild-type (WT) tobacco scion, suggesting that other long-distance signals downstream these transgenes could have moved across the graft union leading to OS tolerance. Overall, our results signify the importance of StNPR1 and StDREB1 as two anticipated candidates for the development of stress-resilient crops through transgrafting technology.


Assuntos
Lycopersicon esculentum/genética , Osmose/fisiologia , Pressão Osmótica/fisiologia , Solanum tuberosum/genética , Tabaco/genética , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/genética , Transgenes/genética
14.
Int J Mol Sci ; 22(16)2021 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-34445759

RESUMO

The dogma of engineering oncolytic viral vectors has shifted from emphasizing the viral lysis of individual cancer cells to the recruitment and coordination of the adaptive immune system to clear the tumor. To accomplish this, researchers have been adding several classes of transgenes to their preferred viral platforms. The most prevalent of these include antibodies and targeting moieties, interleukins and cytokines, and genes which rely on small molecule co-administration for tumor killing. Most current vectors rely exclusively on one of these types of transgenes to elicit the desired immune response to clear tumors, but are not mutually exclusive, with several larger OVs armed with several of these factors. The common theme of emerging armed vectors is to simply initiate or enhance infiltration of effector CD8+ T cells to clear the tumor locally at OV infection sites, and systemically throughout the body where the OV has not infected tumor cells. The precision of oncolytic vectors to target a cell type or tissue remains its key advantage over small-molecule drugs. Unlike chemo- and other drug therapies, viral vectors can be made to specifically infect and grow within tumor cells. This ensures localized expression of the therapeutic transgene to the diseased tissue, thereby limiting systemic toxicity. This review will examine the immunomodulating transgenes of current OVs, describe their general effect on the immune system, and provide the rationale for each vector's use in clearing its targeted tumor.


Assuntos
Vetores Genéticos , Imunomodulação , Neoplasias/terapia , Terapia Viral Oncolítica , Animais , Anticorpos/administração & dosagem , Citocinas/metabolismo , Humanos , Transgenes
15.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445358

RESUMO

The human dopamine receptors D2S and D3 belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D2S and D3 receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate KD-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.


Assuntos
Escherichia coli/genética , Engenharia de Proteínas/métodos , Receptores Dopaminérgicos/genética , Calibragem , Membrana Celular/metabolismo , Clonagem Molecular/métodos , Escherichia coli/metabolismo , Biblioteca Gênica , Humanos , Mutação , Organismos Geneticamente Modificados , Engenharia de Proteínas/normas , Receptores Dopaminérgicos/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/genética , Receptores de Dopamina D3/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformação Bacteriana , Transgenes
16.
Methods Mol Biol ; 2352: 45-55, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324179

RESUMO

Astrocytes play important roles in neurodevelopment and diseases. Previous studies described ways to derive astrocytes from somatic cells by going through iPSC or iNSC/iNPC intermediates. Here we describe a method to directly convert mouse fibroblasts into functional astrocytes using small molecules without transgenes or viral transduction. The direct chemical reprogramming method described in this study provides a more rapid way to derive astrocytes from fibroblasts.


Assuntos
Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Técnicas de Reprogramação Celular , Reprogramação Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Biomarcadores , Diferenciação Celular/genética , Separação Celular , Células Cultivadas , Reprogramação Celular/genética , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Fatores de Transcrição/genética , Transgenes
17.
Am J Hum Genet ; 108(9): 1735-1751, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34314704

RESUMO

CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect individual drug response and lead to an increased risk of hemorrhage. We developed click-seq, a pooled yeast-based activity assay, to test thousands of variants. Using click-seq, we measured the activity of 6,142 missense variants in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants in a human cell line by using variant abundance by massively parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.


Assuntos
Citocromo P-450 CYP2C9/metabolismo , Mutação de Sentido Incorreto , Medicamentos sob Prescrição/metabolismo , Saccharomyces cerevisiae/enzimologia , Xenobióticos/metabolismo , Sítios de Ligação , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP2C9/genética , Ensaios Enzimáticos , Biblioteca Gênica , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenitoína/química , Polimorfismo Genético , Medicamentos sob Prescrição/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Transgenes , Varfarina/química , Varfarina/metabolismo , Xenobióticos/química
18.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34210738

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small-molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme- and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and were proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 h postinfection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2-treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.


Assuntos
Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Inibidores de Proteases/uso terapêutico , Pirrolidinas/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Animais , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , COVID-19/patologia , Proteases 3C de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/química , Cristalografia por Raios X , Deutério , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Conformação Proteica , Pirrolidinas/química , SARS-CoV-2/enzimologia , Transgenes
19.
Methods Mol Biol ; 2312: 125-139, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228288

RESUMO

With the increasing indispensable role of smartphones in our daily lives, the mobile health care system coupled with embedded physical sensors and modern communication technologies make it an attractive technology for enabling the remote monitoring of an individual's health. Using a multidisciplinary design principle coupled with smart electronics, software, and optogenetics, the investigators constructed smartphone-controlled optogenetic switches to enable the ultraremote-control transgene expression. A custom-designed SmartController system was programmed to process wireless signals from smartphones, enabling the regulation of therapeutic outputs production by optically engineered cells via a far-red light (FRL)-responsive optogenetic interface. In the present study, the investigators describe the details of the protocols for constructing smartphone-controlled optogenetic switches, including the rational design of an FRL-triggered transgene expression circuit, the procedure for cell culture and transfection, the implementation of the smartphone-controlled far-red light-emitting diode (LED) module, and the reporter detection assay.


Assuntos
Proteínas de Bactérias/genética , Engenharia Celular/instrumentação , Regulação da Expressão Gênica/efeitos da radiação , Luz , Optogenética/instrumentação , Smartphone , Biologia Sintética/instrumentação , Tecnologia sem Fio/instrumentação , Animais , Proteínas de Bactérias/metabolismo , Células HEK293 , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Transgenes
20.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208268

RESUMO

Euglena gracilis (E. gracilis) is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of E. gracilis mediated by Agrobacterium (A. tumefaciens). E. gracilis was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. A. tumefaciens-mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the gus gene (encoding ß-glucuronidase (GUS)) were found to be GUS-negative, indicating that the gus gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene (hptII) (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of Euglena with the hptII-2A-gus gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when A. tumefaciens LBA4404 (A600 = 1.0) and E. gracilis (A750 = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer E. gracilis for producing high-value products and fundamental studies.


Assuntos
Agrobacterium tumefaciens/metabolismo , Biotecnologia , Euglena gracilis/genética , Microalgas/genética , Técnicas de Transferência Nuclear , Transformação Genética , Agrobacterium tumefaciens/efeitos dos fármacos , Antibacterianos/farmacologia , Cinamatos/farmacologia , Células Clonais , DNA Bacteriano/genética , Euglena gracilis/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Microalgas/efeitos dos fármacos , Mutagênese Insercional/genética , Transformação Genética/efeitos dos fármacos , Transgenes
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