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1.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445471

RESUMO

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is activated by the small G-protein, Ras homolog enriched in brain (RHEB-GTPase). On lysosome, RHEB activates mTORC1 by binding the domains of N-heat, M-heat, and the focal adhesion targeting (FAT) domain, which allosterically regulates ATP binding in the active site for further phosphorylation. The crucial role of RHEB in regulating growth and survival through mTORC1 makes it a targetable site for anti-cancer therapeutics. However, the binding kinetics of RHEB to mTORC1 is still unknown at the molecular level. Therefore, we studied the kinetics by in vitro and in-cell protein-protein interaction (PPI) assays. To this end, we used the split-luciferase system (NanoBiT®) for in-cell studies and prepared proteins for the in vitro measurements. Consequently, we demonstrated that RHEB binds to the whole mTOR both in the presence or absence of GTPγS, with five-fold weaker affinity in the presence of GTPγS. In addition, RHEB bound to the truncated mTOR fragments of N-heat domain (∆N, aa 60-167) or M-heat domain (∆M, aa 967-1023) with the same affinity in the absence of GTP. The reconstructed binding site of RHEB, ∆N-FAT-M, however, bound to RHEB with the same affinity as ∆N-M, indicating that the FAT domain (∆FAT, aa 1240-1360) is dispensable for RHEB binding. Furthermore, RHEB bound to the truncated kinase domain (∆ATP, aa 2148-2300) with higher affinity than to ∆N-FAT-M. In conclusion, RHEB engages two different binding sites of mTOR, ∆N-FAT-M and ∆ATP, with higher affinity for ∆ATP, which likely regulates the kinase activity of mTOR through multiple different biding modes.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Técnicas In Vitro , Cinética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Fosforilação , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética
2.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445472

RESUMO

Background: Platelet-Rich Plasma (PRP) induces bone regeneration; however, there is low evidence supporting its efficacy in bone healing. The lack of a standardized protocol of administration represents the main obstacle to its use in the clinical routine for bone defects' treatment. The purpose of this study was to characterize PRP and elucidate its osteogenic potential. Methods: Platelet count, fibrinogen levels, and growth factors concentration were measured in PRP obtained by four apheresis procedures. HOB-01-C1, a pre-osteocytic cell line, was used to examine the effects of different PRP dilutions (from 1% to 50%) on cell viability, growth, and differentiation. Gene expression of RUNX2, PHEX, COL1A1, and OCN was also assayed. Results: PRP showed a mean 4.6-fold increase of platelets amount compared to whole blood. Among the 36 proteins evaluated, we found the highest concentrations for PDGF isoforms, EGF, TGF-ß and VEGF-D. PDGF-AA positively correlated with platelet counts. In three of the four tested units, 25% PRP induced a growth rate comparable to the positive control (10% FBS); whereas, for all the tested units, 10% PRP treatment sustained differentiation. Conclusions: This study showed that PRP from apheresis stimulates proliferation and differentiation of pre-osteocyte cells through the release of growth factors from platelets.


Assuntos
Remoção de Componentes Sanguíneos/métodos , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osteócitos/citologia , Osteogênese , Plasma Rico em Plaquetas/metabolismo , Medicina Regenerativa , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Perfilação da Expressão Gênica , Humanos , Técnicas In Vitro , Osteócitos/metabolismo
3.
Parasite ; 28: 62, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34410223

RESUMO

Considerable research has been directed towards optimising in vitro tests that can diagnose resistance in pre-parasitic stages of parasites. The objective of this study was to compare the in vivo faecal egg count reduction test (FECRT), the in vitro egg hatch test (EHT), and the molecular determination of the frequency of a codon 200 allele of ß-tubulin isotype 1 associated with benzimidazole resistance in larval stages of Haemonchus contortus obtained from infected goats. Animals were infected with composite infective doses representing 10, 20, 30, 40, 60, and 80% resistant alleles. Faecal samples for the EHT were collected on 28, 33, and 35 days post-infection. The results of the in vivo FECRT indicated that albendazole treatment reduced infections consisting of composite doses of 10, 20, 30, 40, 60, and 80% larvae of the resistant isolate by 91.3, 78.0, 63.3, 48.4, 36.5, and 41.4%, respectively. The drug concentration at which 50% of the eggs were prevented from developing hatching larvae (ED50) in the in vitro EHT varied from 0.09 ± 0.01 to 15.63 ± 12.10 µg/mL thiabendazole. The results of the in vitro EHT indicated that the test could estimate in vivo resistance well. The EHT could thus accurately estimate the in vivo efficacy of the drug and percentage of the resistance allele in the population using hatching parameters in delineation doses. This finding was also supported by comparing the FECRT data to the hatching percentages in the EHT on 30 goat farms in Slovakia with natural mixed infections of gastrointestinal parasites.


Assuntos
Anti-Helmínticos , Haemonchus , Doenças dos Ovinos , Animais , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Resistência a Medicamentos , Fezes , Haemonchus/genética , Técnicas In Vitro , Contagem de Ovos de Parasitas/veterinária , Ovinos
4.
Nat Commun ; 12(1): 4713, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354054

RESUMO

Maize (Zea mays L.) is a cold-sensitive species that often faces chilling stress, which adversely affects growth and reproduction. However, the genetic basis of low-temperature adaptation in maize remains unclear. Here, we demonstrate that natural variation in the type-A Response Regulator 1 (ZmRR1) gene leads to differences in chilling tolerance among maize inbred lines. Association analysis reveals that InDel-35 of ZmRR1, encoding a protein harboring a mitogen-activated protein kinase (MPK) phosphorylation residue, is strongly associated with chilling tolerance. ZmMPK8, a negative regulator of chilling tolerance, interacts with and phosphorylates ZmRR1 at Ser15. The deletion of a 45-bp region of ZmRR1 harboring Ser15 inhibits its degradation via the 26 S proteasome pathway by preventing its phosphorylation by ZmMPK8. Transcriptome analysis indicates that ZmRR1 positively regulates the expression of ZmDREB1 and Cellulose synthase (CesA) genes to enhance chilling tolerance. Our findings thus provide a potential genetic resource for improving chilling tolerance in maize.


Assuntos
Zea mays/genética , Zea mays/fisiologia , Alelos , Temperatura Baixa , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Técnicas In Vitro , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Fosforilação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico/genética
5.
Nat Commun ; 12(1): 4728, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354065

RESUMO

Understanding how diet and gut microbiota interact in the context of human health is a key question in personalized nutrition. Genome-scale metabolic networks and constraint-based modeling approaches are promising to systematically address this complex problem. However, when applied to nutritional questions, a major issue in existing reconstructions is the limited information about compounds in the diet that are metabolized by the gut microbiota. Here, we present AGREDA, an extended reconstruction of diet metabolism in the human gut microbiota. AGREDA adds the degradation pathways of 209 compounds present in the human diet, mainly phenolic compounds, a family of metabolites highly relevant for human health and nutrition. We show that AGREDA outperforms existing reconstructions in predicting diet-specific output metabolites from the gut microbiota. Using 16S rRNA gene sequencing data of faecal samples from Spanish children representing different clinical conditions, we illustrate the potential of AGREDA to establish relevant metabolic interactions between diet and gut microbiota.


Assuntos
Dieta , Microbioma Gastrointestinal/fisiologia , Redes e Vias Metabólicas , Modelos Biológicos , Algoritmos , Criança , Fenômenos Fisiológicos da Nutrição Infantil , Dieta Mediterrânea , Fermentação , Microbioma Gastrointestinal/genética , Humanos , Técnicas In Vitro , Lens (Planta)/química , Valor Nutritivo , RNA Ribossômico 16S/genética , Espanha
6.
Nat Commun ; 12(1): 4709, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354080

RESUMO

Allostery represents a fundamental mechanism of biological regulation that involves long-range communication between distant protein sites. It also provides a powerful framework for novel therapeutics. NMDA receptors (NMDARs), glutamate-gated ionotropic receptors that play central roles in synapse maturation and plasticity, are prototypical allosteric machines harboring large extracellular N-terminal domains (NTDs) that provide allosteric control of key receptor properties with impact on cognition and behavior. It is commonly thought that GluN2A and GluN2B receptors, the two predominant NMDAR subtypes in the adult brain, share similar allosteric transitions. Here, combining functional and structural interrogation, we reveal that GluN2A and GluN2B receptors utilize different long-distance allosteric mechanisms involving distinct subunit-subunit interfaces and molecular rearrangements. NMDARs have thus evolved multiple levels of subunit-specific allosteric control over their transmembrane ion channel pore. Our results uncover an unsuspected diversity in NMDAR molecular mechanisms with important implications for receptor physiology and precision drug development.


Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica , Animais , Feminino , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Fotoquímica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas , Ratos , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevis
7.
FASEB J ; 35(9): e21788, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34425031

RESUMO

Hypoxia increases fetal hepatic insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation mediated by mechanistic target of rapamycin (mTOR) inhibition. Whether maternal nutrient restriction (MNR) causes fetal hypoxia remains unclear. We used fetal liver from a baboon (Papio sp.) model of intrauterine growth restriction due to MNR (70% global diet of Control) and liver hepatocellular carcinoma (HepG2) cells as a model for human fetal hepatocytes and tested the hypothesis that mTOR-mediated IGFBP-1 hyperphosphorylation in response to hypoxia requires hypoxia-inducible factor-1α (HIF-1α) and regulated in development and DNA-damage responses-1 (REDD-1) signaling. Western blotting (n = 6) and immunohistochemistry (n = 3) using fetal liver indicated greater expression of HIF-1α, REDD-1 as well as erythropoietin and its receptor, and vascular endothelial growth factor at GD120 (GD185 term) in MNR versus Control. Moreover, treatment of HepG2 cells with hypoxia (1% pO2 ) (n = 3) induced REDD-1, inhibited mTOR complex-1 (mTORC1) activity and increased IGFBP-1 secretion/phosphorylation (Ser101/Ser119/Ser169). HIF-1α inhibition by echinomycin or small interfering RNA silencing prevented the hypoxia-mediated inhibition of mTORC1 and induction of IGFBP-1 secretion/phosphorylation. dimethyloxaloylglycine (DMOG) induced HIF-1α and also REDD-1 expression, inhibited mTORC1 and increased IGFBP-1 secretion/phosphorylation. Induction of HIF-1α (DMOG) and REDD-1 by Compound 3 inhibited mTORC1, increased IGFBP-1 secretion/ phosphorylation and protein kinase PKCα expression. Together, our data demonstrate that HIF-1α induction, increased REDD-1 expression and mTORC1 inhibition represent the mechanistic link between hypoxia and increased IGFBP-1 secretion/phosphorylation. We propose that maternal undernutrition limits fetal oxygen delivery, as demonstrated by increased fetal liver expression of hypoxia-responsive proteins in baboon MNR. These findings have important implications for our understanding of the pathophysiology of restricted fetal growth.


Assuntos
Técnicas de Cultura de Células , Modelos Animais de Doenças , Retardo do Crescimento Fetal/metabolismo , Feto/metabolismo , Hipóxia/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Animais , Eritropoetina/metabolismo , Peso Fetal , Feto/química , Células Hep G2 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas In Vitro , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Microscopia de Fluorescência , Tamanho do Órgão , Papio , Fosforilação , Proteína Quinase C-alfa/metabolismo , Receptores da Eritropoetina/metabolismo , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Molecules ; 26(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34443486

RESUMO

Phosphorylation events catalyzed by protein kinases represent one of the most prevalent as well as important regulatory posttranslational modifications, and dysregulation of protein kinases is associated with the pathogenesis of different diseases. Therefore, interest in developing potent small molecule kinase inhibitors has increased enormously within the last two decades. A critical step in the development of new inhibitors is cell-free in vitro testing with the intention to determine comparable parameters like the commonly used IC50 value. However, values described in the literature are often biased as experimental setups used for determination of kinase activity lack comparability due to different readout parameters, insufficient normalization or the sheer number of experimental approaches. Here, we would like to hold a brief for highly sensitive, radioactive-based in vitro kinase assays especially suitable for kinases exhibiting autophosphorylation activity. Therefore, we demonstrate a systematic workflow for complementing and validating results from high-throughput screening as well as increasing the comparability of enzyme-specific inhibitor parameters for radiometric as well as non-radiometric assays. Using members of the CK1 family of serine/threonine-specific protein kinases and established CK1-specific inhibitors as examples, we clearly demonstrate the power of our proposed workflow, which has the potential to support the generation of more comparable data for biological characterization of kinase inhibitors.


Assuntos
Caseína Quinase I/antagonistas & inibidores , Ensaios Enzimáticos/métodos , Inibidores de Proteínas Quinases/química , Técnicas In Vitro , Concentração Inibidora 50 , Cinética , Fosforilação
9.
Oxid Med Cell Longev ; 2021: 4786227, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34457112

RESUMO

The anti-cancer, anti-aging, anti-inflammatory, antioxidant, and anti-diabetic effects of zinc oxide nanoparticles (ZnO-NPs) produced from aqueous leaf extract of Aquilegia pubiflora were evaluated in this study. Several methods were used to characterize ZnO-NPs, including SEM, FTIR, XRD, DLS, PL, Raman, and HPLC. The nanoparticles that had a size of 34.23 nm as well as a strong aqueous dispersion potential were highly pure, spherical or elliptical in form, and had a mean size of 34.23 nm. According to FTIR and HPLC studies, the flavonoids and hydroxycinnamic acid derivatives were successfully capped. Synthesized ZnO-NPs in water have a zeta potential of -18.4 mV, showing that they are stable solutions. The ZnO-NPs proved to be highly toxic for the HepG2 cell line and showed a reduced cell viability of 23.68 ± 2.1% after 24 hours of ZnO-NP treatment. ZnO-NPs also showed excellent inhibitory potential against the enzymes acetylcholinesterase (IC50: 102 µg/mL) and butyrylcholinesterase (IC50: 125 µg/mL) which are involved in Alzheimer's disease. Overall, the enzymes involved in aging, diabetes, and inflammation showed a moderate inhibitory response to ZnO-NPs. Given these findings, these biosynthesized ZnO-NPs could be a good option for the cure of deadly diseases such as cancer, diabetes, Alzheimer's, and other inflammatory diseases due to their strong anticancer potential and efficient antioxidant properties.


Assuntos
Antineoplásicos/farmacologia , Aquilegia/química , Nanopartículas Metálicas/administração & dosagem , Extratos Vegetais/farmacologia , Folhas de Planta/química , Espécies Reativas de Oxigênio/farmacologia , Óxido de Zinco/química , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Proliferação de Células , Inibidores da Colinesterase/farmacologia , Células Hep G2 , Humanos , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Nanopartículas Metálicas/química
10.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360539

RESUMO

Vascular ischemia/reperfusion injury (IRI) contributes to graft failure and adverse clinical outcomes following coronary artery bypass grafting. Sodium-glucose-cotransporter (SGLT)-2-inhibitors have been shown to protect against myocardial IRI, irrespective of diabetes. We hypothesized that adding canagliflozin (CANA) (an SGLT-2-inhibitor) to saline protects vascular grafts from IRI. Aortic rings from non-diabetic rats were isolated and immediately mounted in organ bath chambers (control, n = 9-10 rats) or underwent cold ischemic preservation in saline, supplemented either with a DMSO vehicle (IR, n = 8-10 rats) or 50µM CANA (IR + CANA, n = 9-11 rats). Vascular function was measured, the expression of 88 genes using PCR-array was analyzed, and feature selection using machine learning was applied. Impaired maximal vasorelaxation to acetylcholine in the IR-group compared to controls was significantly ameliorated by CANA (IR 31.7 ± 3.2% vs. IR + CANA 51.9 ± 2.5%, p < 0.05). IR altered the expression of 17 genes. Ccl2, Ccl3, Ccl4, CxCr4, Fos, Icam1, Il10, Il1a and Il1b have been found to have the highest interaction. Compared to controls, IR significantly upregulated the mRNA expressions of Il1a and Il6, which were reduced by 1.5- and 1.75-fold with CANA, respectively. CANA significantly prevented the upregulation of Cd40, downregulated NoxO1 gene expression, decreased ICAM-1 and nitrotyrosine, and increased PECAM-1 immunoreactivity. CANA alleviates endothelial dysfunction following IRI.


Assuntos
Canagliflozina/farmacologia , Endotélio Vascular/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Traumatismo por Reperfusão/complicações , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Doenças Vasculares/prevenção & controle , Vasodilatação/efeitos dos fármacos , Animais , Endotélio Vascular/patologia , Técnicas In Vitro , Masculino , Neovascularização Patológica/etiologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Ratos , Ratos Wistar , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia
11.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360580

RESUMO

Melanin causes melasma, freckles, age spots, and chloasma. Anti-melanogenic agents can prevent disease-related hyperpigmentation. In the present study, the dose-dependent tyrosinase inhibitory activity of Avenanthramide (Avn)-A-B-C was demonstrated, and 100 µM Avn-A-B-C produced the strongest competitive inhibition against inter-cellular tyrosinase and melanin synthesis. Avn-A-B-C inhibits the expression of melanogenesis-related proteins, such as TRP1 and 2. Molecular docking simulation revealed that AvnC (-7.6 kcal/mol) had a higher binding affinity for tyrosinase than AvnA (-7.3 kcal/mol) and AvnB (-6.8 kcal/mol). AvnC was predicted to interact with tyrosinase through two hydrogen bonds at Ser360 (distance: 2.7 Å) and Asn364 (distance: 2.6 Å). In addition, AvnB and AvnC were predicted to be skin non-sensitizers in mammals by the Derek Nexus Quantitative Structure-Activity Relationship system.


Assuntos
Simulação por Computador , Melaninas/biossíntese , Melanoma/tratamento farmacológico , Monofenol Mono-Oxigenase/antagonistas & inibidores , Pele/efeitos dos fármacos , alfa-MSH/farmacologia , ortoaminobenzoatos/farmacologia , Hormônios/farmacologia , Humanos , Técnicas In Vitro , Melanoma/metabolismo , Melanoma/patologia , Simulação de Acoplamento Molecular , Células Tumorais Cultivadas
12.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360671

RESUMO

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.


Assuntos
Proteases Virais 3C/metabolismo , Núcleo Celular/patologia , Ferroptose , Mitocôndrias/patologia , Proteases Virais 3C/genética , Células A549 , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Técnicas In Vitro , Peroxidação de Lipídeos , Mitocôndrias/metabolismo
13.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361066

RESUMO

Ceramides, a class of sphingolipids containing a backbone of sphingoid base, are the most important and effective structural component for the formation of the epidermal permeability barrier. While ceramides comprise approximately 50% of the epidermal lipid content by mass, the content is substantially decreased in certain inflammatory skin diseases, such as atopic dermatitis (AD), causing improper barrier function. It is widely accepted that the endocannabinoid system (ECS) can modulate a number of biological responses in the central nerve system, prior studies revealed that activation of endocannabinoid receptor CB1, a key component of ECS, triggers the generation of ceramides that mediate neuronal cell fate. However, as the impact of ECS on the production of epidermal ceramide has not been studied, we here investigated whether the ECS stimulates the generation of epidermal ceramides in an IL-4-treated in vitro model of skin inflammation using N-palmitoyl serinol (PS), an analog of the endocannabinoid N-palmitoyl ethanolamine. Accordingly, an IL-4-mediated decrease in cellular ceramide levels was significantly stimulated in human epidermal keratinocytes (KC) following PS treatment through both de novo ceramide synthesis- and sphingomyelin hydrolysis-pathways. Importantly, PS selectively increases ceramides with long-chain fatty acids (FAs) (C22-C24), which mainly account for the formation of the epidermal barrier, through activation of ceramide synthase (CerS) 2 and Cer3 in IL-4-mediated inflamed KC. Furthermore, blockade of cannabinoid receptor CB1 activation by AM-251 failed to stimulate the production of total ceramide as well as long-chain ceramides in response to PS. These studies demonstrate that an analog of endocannabinoid, PS, stimulates the generation of specific ceramide species as well as the total amount of ceramides via the endocannabinoid receptor CB1-dependent mechanism, thereby resulting in the enhancement of epidermal permeability barrier function.


Assuntos
Ceramidas/metabolismo , Inflamação/metabolismo , Queratinócitos/metabolismo , Propanolaminas/farmacologia , Propilenoglicóis/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Pele/metabolismo , Células Cultivadas , Humanos , Técnicas In Vitro , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Propanolaminas/química , Propilenoglicóis/química , Pele/citologia , Pele/efeitos dos fármacos
14.
Artigo em Inglês | MEDLINE | ID: mdl-34263708

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic of the coronavirus disease in 2019. RNA-dependent RNA polymerase (RdRp) plays an essential role in RNA replication and transcription in SARS-CoV-2. In this study, we focused on the RNA template component of viral RdRp structure and analyzed human microRNAs (miRNAs) targeting specific sequences in this RNA. By examining miRNA databases and using an in vitro RNA-RNA interaction assay, we observed hsa-miR-15b-5p interacting with the RNA component of viral RdRp. Our findings provide evidence that hsa-miR15b-5p may suppresses viral infection and proliferation by targeting the RNA template component of SARS-CoV-2 RdRp.


Assuntos
COVID-19/metabolismo , Regulação Viral da Expressão Gênica , MicroRNAs/fisiologia , RNA Polimerase Dependente de RNA/metabolismo , Biomarcadores/metabolismo , COVID-19/enzimologia , COVID-19/genética , COVID-19/virologia , Bases de Dados Genéticas , Humanos , Técnicas In Vitro , SARS-CoV-2 , Transcrição Genética
15.
Methods Mol Biol ; 2287: 3-22, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270023

RESUMO

Doubled haploids (DH) have become a powerful tool to assist in different basic research studies, and also in applied research. The principal (but not the only) and routine use of DH by breeding companies is to produce pure lines for hybrid seed production in different crop species. Several decades after the discovery of haploid inducer lines in maize and of anther culture as a method to produce haploid plants from pollen precursors, the biotechnological revolution of the last decades allowed to the development of a variety of approaches to pursue the goal of doubled haploid production. Now, it is possible to produce haploids and DHs in many different species, because when a method does not work properly, there are several others to test. In this chapter, we overview the currently available approaches used to produce haploids and DHs by using methods based on in vitro culture, or involving the in vivo induction of haploid embryo development, or a combination of both.


Assuntos
Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/genética , Melhoramento Vegetal/métodos , Haploidia , Técnicas In Vitro , Fenótipo , Pólen/genética , Pólen/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimento
16.
Methods Mol Biol ; 2287: 151-169, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270028

RESUMO

Onion (A. cepa L.) is an outcrossing biennial species with a very large genome. Development of genetically uniform (inbred) lines highly desired by onion breeders is a difficult process due to high level of heterozygosity. Inbred onion development may take up to five generations (~10 years) by classical selfing technique. Onion shows severe inbreeding depression, which further complicates production of lines with stabilized important agronomic traits. When applied successfully, haploidization technology can be useful in the development of fully homozygous onion lines in 2 years. Although production of haploid and doubled haploid (DH) onions via gynogenesis was reported more than three decades ago, successful production and utilization of DHs in onion breeding is still far behind of expectations of breeders. The main obstacles in front of the success include high variation in the response of donor materials to gynogenesis induction and difficulties faced in the process of obtaining DHs from haploid plants. We use a DH production procedure enabling us to develop DH plants from a wide range of onion donor materials. This procedure is based on production of haploid plants via single step culture of unopened flower buds, detection of haploid plants among gynogenic regenerants, and converting these plants to fecund DHs using a combination of ploidy manipulation techniques. The bulbs of DHs are produced in about 1 year after the initiation of induction cultures and selfed seeds are produced from fecund DH plants when they flower in the second year.


Assuntos
Cromossomos de Plantas , Gametogênese Vegetal , Cebolas/crescimento & desenvolvimento , Cebolas/genética , Melhoramento Vegetal/métodos , Haploidia , Técnicas In Vitro , Fenótipo , Sementes/genética , Sementes/crescimento & desenvolvimento
17.
Methods Mol Biol ; 2287: 171-184, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270029

RESUMO

Leek (A. ampeloprasum L.) is an economically important vegetable crop from Alliaceae family. It is a non-bulb forming biennial species grown for its pseudostem and leaves. Leek is a tetraploid with one of the largest genomes known among cultivated plant species. It has enormous economic importance all around the world for many purposes such as vegetable, medicinal herb, and food seasoning. Production and consumption of leek is in rise all around the world and breeders are trying to develop new F1 hybrid varieties with desired agronomical traits. Although self-compatible, leek shows high tendency toward outcrossing and display severe inbreeding depression when selfed with its own pollen. Therefore, inbred development through classical breeding techniques is very difficult in this crop. Traditional leek genotypes are highly heterozygous, open pollinated varieties. There is a high demand for F1 hybrid varieties with resistance to biotic and abiotic stresses and high-quality plants. Our group is trying to incorporate gynogenesis-based doubled haploid technology to leek improvement programs. Over the years, many experiments were carried out to determine the gynogenic potential of donor leek genotypes of different genetic backgrounds in different induction media. Here, we report a protocol allowing production of green gynogenic leek plants via single step culture of unopened flower buds. Ploidy levels of gynogenic regenerants are determined by flow cytometry analysis. A majority of the gynogenic leek regenerants produced survived well in vivo.


Assuntos
Allium/crescimento & desenvolvimento , Allium/genética , Cromossomos de Plantas , Gametogênese Vegetal , Melhoramento Vegetal/métodos , Flores/genética , Flores/crescimento & desenvolvimento , Haploidia , Técnicas In Vitro , Fenótipo , Pólen/genética , Pólen/crescimento & desenvolvimento
18.
Methods Mol Biol ; 2287: 257-266, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270035

RESUMO

Doubled haploid (DH) plant production belongs to modern biotechnology methods of plant breeding. The main advantage of DH plant production methods is the development of genetically homozygous lines in one generation, whilst in conventional breeding programmes, the development of homozygous lines requires more generations. The present chapter describes an efficient protocol for DH plant production in spelt wheat genotypes using in vitro anther culture.


Assuntos
Flores/crescimento & desenvolvimento , Melhoramento Vegetal/métodos , Técnicas de Cultura de Tecidos/métodos , Triticum/crescimento & desenvolvimento , Flores/genética , Haploidia , Técnicas In Vitro/métodos , Pólen/genética , Pólen/crescimento & desenvolvimento , Triticum/genética
19.
Sci Signal ; 14(690)2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230209

RESUMO

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Polifosfatos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Administração por Inalação , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Antivirais/administração & dosagem , Antivirais/química , COVID-19/metabolismo , COVID-19/virologia , Células CACO-2 , Chlorocebus aethiops , RNA-Polimerase RNA-Dependente de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/genética , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Citocinas/metabolismo , Células HEK293 , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Técnicas In Vitro , Modelos Biológicos , Simulação de Acoplamento Molecular , Nebulizadores e Vaporizadores , Polifosfatos/administração & dosagem , Polifosfatos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteólise/efeitos dos fármacos , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacos
20.
Biochem Biophys Res Commun ; 571: 152-158, 2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34325131

RESUMO

Potent neutralizing antibodies (Abs) have been proven with therapeutic efficacy for the intervention against SARS-CoV-2. Majority of these Abs function by directly interfering with the virus entry to host cells. Here, we identified a receptor binding domain (RBD) specific monoclonal Ab (mAb) 82A6 with efficient neutralizing potency against authentic SARS-CoV-2 virus. As most Abs targeting the non-receptor binding motif (RBM) region, 82A6 was incapable to block the RBD-ACE2 interaction. In particular, it actively promoted the S1 subunit shedding from the S protein, which may lead to effective reduction of intact SARS-CoV-2 viruses. Importantly, it could block potential syncytia formation associated with post-infectious cell surface expression of S proteins. Our study evidenced a RBD specific Ab with unique beneficial efficacy against SARS-CoV-2 infection, which might bring informative significance to understand the collective effects of neutralizing Abs elicited in COVID-19 patients.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , COVID-19/terapia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos , Sítios de Ligação/imunologia , COVID-19/imunologia , COVID-19/virologia , Células Gigantes/imunologia , Células Gigantes/virologia , Células HEK293 , Humanos , Imunização Passiva , Técnicas In Vitro , Domínios Proteicos , Subunidades Proteicas , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/química , Eliminação de Partículas Virais
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