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1.
Yakugaku Zasshi ; 140(10): 1199-1206, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32999198

RESUMO

Potential risks to the fetus or infant should be considered prior to medication during pregnancy and lactation. It is essential to evaluate the exposure levels of drugs and their related factors in addition to toxicological effects. Epilepsy is one of the most common neurological complications in pregnancy; some women continue to use antiepileptic drugs (AEDs) to control seizures. Benzodiazepines (BZDs) are widely prescribed for several women who experience symptoms such as anxiety and insomnia during the postpartum period. In this review, we describe the 1) transport mechanisms of AEDs across the placenta and the effects of these drugs on placental transporters, and 2) the transfer of BZDs into breast milk. Our findings indicated that carrier systems were involved in the uptake of gabapentin (GBP) and lamotrigine (LTG) in placental trophoblast cell lines. SLC7A5 was the main contributor to GBP transport in placental cells. LTG was transported by a carrier that was sensitive to chloroquine, imipramine, quinidine, and verapamil. Short-term exposure to 16 AEDs had no effect on folic acid uptake in placental cells. However, long-term exposure to valproic acid (VPA) affected the expression of folate carriers (FOLR1, SLC46A1). Furthermore, VPA administration changed the expression levels of various transporters in rat placenta, suggesting that sensitivity to VPA differed across gestational stages. Lastly, we developed a method for quantifying eight BZDs in human breast milk and plasma using LC/MS/MS, and successfully applied it to quantify alprazolam in breast milk and plasma donated by a lactating woman.


Assuntos
Anticonvulsivantes/metabolismo , Benzodiazepinas/metabolismo , Transporte Biológico/genética , Aleitamento Materno , Gabapentina/metabolismo , Lactação/metabolismo , Lamotrigina/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/fisiologia , Troca Materno-Fetal , Leite Humano/metabolismo , Placenta/metabolismo , Ácido Valproico/metabolismo , Anticonvulsivantes/efeitos adversos , Benzodiazepinas/efeitos adversos , Benzodiazepinas/uso terapêutico , Linhagem Celular , Epilepsia/tratamento farmacológico , Feminino , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Gabapentina/efeitos adversos , Expressão Gênica/efeitos dos fármacos , Humanos , Lamotrigina/efeitos adversos , Gravidez , Complicações na Gravidez/tratamento farmacológico , Transportador de Folato Acoplado a Próton/genética , Transportador de Folato Acoplado a Próton/metabolismo , Ácido Valproico/efeitos adversos
2.
Mol Cell ; 80(1): 140-155.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007254

RESUMO

The tissue-specific deployment of highly extended neural 3' UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well understood. Here, we show that three ELAV/Hu RNA binding proteins (Elav, Rbp9, and Fne) have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne toward the nucleus in elav mutants. We use genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, we demonstrate how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape.


Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas ELAV/metabolismo , Neurônios/metabolismo , Processamento Alternativo/genética , Motivos de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas ELAV/química , Larva/metabolismo , Mutação/genética , Poli A/metabolismo , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
3.
Nat Commun ; 11(1): 4964, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009394

RESUMO

Thrombosis leads to platelet activation and subsequent degradation; therefore, replenishment of platelets from hematopoietic stem/progenitor cells (HSPCs) is needed to maintain the physiological level of circulating platelets. Platelet-derived microparticles (PMPs) are protein- and RNA-containing vesicles released from activated platelets. We hypothesized that factors carried by PMPs might influence the production of platelets from HSPCs, in a positive feedback fashion. Here we show that, during mouse acute liver injury, the density of megakaryocyte in the bone marrow increases following an increase in circulating PMPs, but without thrombopoietin (TPO) upregulation. In vitro, PMPs are internalized by HSPCs and drive them toward a megakaryocytic fate. Mechanistically, miR-1915-3p, a miRNA highly enriched in PMPs, is transported to target cells and suppresses the expression levels of Rho GTPase family member B, thereby inducing megakaryopoiesis. In addition, direct injection of PMPs into irradiated mice increases the number of megakaryocytes and platelets without affecting TPO levels. In conclusion, our data reveal that PMPs have a role in promoting megakaryocytic differentiation and platelet production.


Assuntos
Plaquetas/metabolismo , Diferenciação Celular , Micropartículas Derivadas de Células/metabolismo , Megacariócitos/citologia , MicroRNAs/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Endocitose , Perfilação da Expressão Gênica , Humanos , Fígado/lesões , Fígado/patologia , Masculino , Megacariócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Poliploidia , Proteína rhoB de Ligação ao GTP/metabolismo
4.
Phys Rev Lett ; 125(12): 128101, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-33016741

RESUMO

The efficiency of a virus to establish its infection in host cells varies broadly among viruses. It remains unclear if there is a key step in this process that controls viral infectivity. To address this question, we use single-particle tracking and Brownian dynamics simulation to examine human immunodeficiency virus type 1 (HIV-1) infection in cell culture. We find that the frequency of viral-cell encounters is consistent with diffusion-limited interactions. However, even under the most favorable conditions, only 1% of the viruses can become immobilized on cell surface and subsequently enter the cell. This is a result of weak interaction between viral surface gp120 and CD4 receptor, which is insufficient to form a stable complex the majority of the time. We provide the first direct quantitation for efficiencies of these events relevant to measured HIV-1 infectivity and demonstrate that immobilization on host cell surface post-virion-diffusion is the key step in viral infection. Variation of its probability controls the efficiency of a virus to infect its host cells. These results explain the low infectivity of cell-free HIV-1 in vitro and offer a potential rationale for the pervasive high efficiency of cell-to-cell transmission of animal viruses.


Assuntos
HIV-1/patogenicidade , Antígenos CD4/metabolismo , Linhagem Celular , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Humanos , Imagem Óptica , Imagem com Lapso de Tempo , Vírion/metabolismo , Vírion/patogenicidade
5.
Nat Commun ; 11(1): 4941, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009412

RESUMO

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.


Assuntos
Terminação Traducional da Cadeia Peptídica , Preparações Farmacêuticas/metabolismo , Linhagem Celular , Humanos , Modelos Moleculares , Mutação/genética , Conformação Proteica , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
6.
Nat Commun ; 11(1): 4905, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999288

RESUMO

Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.


Assuntos
Regulação Fúngica da Expressão Gênica , RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/genética , Fatores de Transcrição TFIII/ultraestrutura , Animais , Linhagem Celular , Microscopia Crioeletrônica , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos/genética , Insetos , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIIB/genética , Fator de Transcrição TFIIIB/isolamento & purificação , Fator de Transcrição TFIIIB/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/isolamento & purificação , Fatores de Transcrição TFIII/metabolismo , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética
7.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 45(8): 901-908, 2020 Aug 28.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-33053530

RESUMO

OBJECTIVES: To compare the effect on proliferation of osteoblasts MC3T3-E1 between the concentrated growth factor extract (CGFe) and the platelet-rich fibrin extract (PRFe). METHODS: CGFe and PRFe were prepared. MC3T3-E1 was cultured in DMEM medium containing CGFe (10%, 20%, or 30%) and PRFe (10%, 20%, or 30%). The proliferation of MC3T3-E1 was detected by MTT assay at Day 1, 3, 5, and 7. ALP activity was detected by alkaline phosphatase (ALP) staining at Day 1, 3, 5, and 7, and mRNA expressions of Runt-related transcription factor 2 (Runx2) and Osterix (Osx) were detected by quantitative RT-PCR (RT-qPCR) at Day 3 and 7. RESULTS: Compared with the control group, CGFe and PRFe promoted the proliferation of MC3T3-E1 at Day 1, 3, 5, and 7 (all P<0.05). Except for the first day, the proliferation activity in the CGFe group was higher than that in the PRFe group (all P<0.05). At Day 1, 3, 5, and 7, compared with the control group, the ALP activities in the CGFe group and the PRFe group were significantly increased (all P<0.05). Except for the first day, the ALP activity in the CGFe group was higher than that in the PRFe group (all P<0.05). At Day 3 and 7, compared with the control group, the mRNA expression levels of Osx and Runx2 in the CGFe group and the PRFe group were significantly increased (all P<0.05); compared with PRFe group, the mRNA expression level of Osx in the CGFe group was significantly higher than that in the PRFe group, and the mRNA expression level of Runx2 was significantly lower than that in the PRFe group (all P<0.05). CONCLUSIONS: CGFe could promote the proliferation of MC3T3-E1 stronger than PRFe, which might be related to the increase of ALP activity and up-regulation of Osx expression.


Assuntos
Fibrina Rica em Plaquetas , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Osteoblastos , Extratos Vegetais
8.
Sci Rep ; 10(1): 16099, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999356

RESUMO

SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Here, we investigated the interaction of this new coronavirus with Vero cells using high resolution scanning electron microscopy. Surface morphology, the interior of infected cells and the distribution of viral particles in both environments were observed 2 and 48 h after infection. We showed areas of viral processing, details of vacuole contents, and viral interactions with the cell surface. Intercellular connections were also approached, and viral particles were adhered to these extensions suggesting direct cell-to-cell transmission of SARS-CoV-2.


Assuntos
Betacoronavirus/ultraestrutura , Infecções por Coronavirus/transmissão , Interações Hospedeiro-Patógeno/fisiologia , Pneumonia Viral/transmissão , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Humanos , Microscopia Eletrônica de Varredura , Pandemias , Pneumonia Viral/patologia , Células Vero
9.
Shanghai Kou Qiang Yi Xue ; 29(3): 231-236, 2020 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-33043337

RESUMO

PURPOSE: The aim of this study was to investigate the molecular mechanism of autophagy and apoptosis induced by cyclic mechanical stretch and the potential role of autophagy in stretch-induced apoptosis of myoblasts. METHODS: Loading model of L6 myoblasts was established in vitro. The cells were then subjected to cyclic mechanical stretch involving 3 s of 15% stretch alternating with 3 s of relaxation. The cells were collected after mechanical stretch for 6 h, 12 h and 24 h, respectively. Control cells were cultured on the same plates without mechanical strain. Apoptosis of myoblasts was assessed by Hoechst 33258 staining and Annexin V binding and propidium iodide staining. Autophagy was determined by MDC staining and transmission electron microscopy(TEM). The level of proteins associated with apoptosis and autophagy was detected by Western blot. The data were analyzed with SPSS 17.0 software package. RESULTS: The results of Hoechst 33258 staining and Annexin V binding and propidium iodide staining indicated that mechanical stretch notably induced apoptosis of myoblasts. Caspase inhibitor z-VAD-fmk effectively abrogated apoptosis of myoblasts, indicating mechanical stretch induced caspase-dependent apoptosis. In addition, the results of TEM, MDC staining and Western blot proved that mechanical stretch elicited autophagy of myoblasts. Inhibition of autophagy using 3-MA enhanced caspase-dependent apoptosis induced by mechanical stretch. CONCLUSIONS: Cyclic mechanical stretch induced apoptosis and autophagy of myoblasts time-dependently. Protective autophagy, acting as the compensatory mechanism, inhibited caspase-dependent apoptosis induced by mechanical stretch.


Assuntos
Autofagia , Apoptose , Linhagem Celular , Mioblastos
10.
Cells ; 9(9)2020 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-32899484

RESUMO

Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). All hybrid systems, combining lipid and polymer, displayed significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. For the hybrid nanoparticles, characteristic differences regarding the internal organization, release characteristics, and activity were determined depending on the assembly route. The systems with the highest transfection efficacy were characterized by a heterogenous internal organization, accompanied by facilitated release. Such a system could be best obtained by the single step protocol, starting with a lipid and polymer mixture for nanoparticle formation.


Assuntos
Biopolímeros/química , Lipídeos/química , Nanopartículas/química , RNA Mensageiro/metabolismo , Transfecção/métodos , Animais , Linhagem Celular , Ácidos Graxos Monoinsaturados/química , Feminino , Heparina/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Imagem Óptica , Tamanho da Partícula , Compostos de Amônio Quaternário/química , RNA Mensageiro/química
11.
PLoS Negl Trop Dis ; 14(8): e0008660, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866199

RESUMO

Aedes mosquitoes can transmit dengue and several other severe vector-borne viral diseases, thereby influencing millions of people worldwide. Insects primarily control and clear the viral infections via their innate immune systems. Mitogen-Activated Protein Kinases (MAPKs) and antimicrobial peptides (AMPs) are both evolutionarily conserved components of the innate immune systems. In this study, we investigated the role of MAPKs in Aedes mosquitoes following DENV infection by using genetic and pharmacological approaches. We demonstrated that knockdown of ERK, but not of JNK or p38, significantly enhances the viral replication in Aedes mosquito cells. The Ras/ERK signaling is activated in both the cells and midguts of Aedes mosquitoes following DENV infection, and thus plays a role in restricting the viral infection, as both genetic and pharmacological activation of the Ras/ERK pathway significantly decreases the viral titers. In contrast, inhibition of the Ras/ERK pathway enhances DENV infection. In addition, we identified a signaling crosstalk between the Ras/ERK pathway and DENV-induced AMPs in which defensin C participates in restricting DENV infection in Aedes mosquitoes. Our results reveal that the Ras/ERK signaling pathway couples AMPs to mediate the resistance of Aedes mosquitoes to DENV infection, which provides a new insight into understanding the crosstalk between MAPKs and AMPs in the innate immunity of mosquito vectors during the viral infection.


Assuntos
Aedes/virologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Vírus da Dengue/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/farmacologia , Mosquitos Vetores/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Anti-Infecciosos/farmacologia , Linhagem Celular , Sistema Digestório/virologia , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Imunidade Inata , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mosquitos Vetores/virologia , Carga Viral , Replicação Viral/efeitos dos fármacos
12.
Cell Rep ; 32(12): 108175, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32946807

RESUMO

To predict the tropism of human coronaviruses, we profile 28 SARS-CoV-2 and coronavirus-associated receptors and factors (SCARFs) using single-cell transcriptomics across various healthy human tissues. SCARFs include cellular factors both facilitating and restricting viral entry. Intestinal goblet cells, enterocytes, and kidney proximal tubule cells appear highly permissive to SARS-CoV-2, consistent with clinical data. Our analysis also predicts non-canonical entry paths for lung and brain infections. Spermatogonial cells and prostate endocrine cells also appear to be permissive to SARS-CoV-2 infection, suggesting male-specific vulnerabilities. Both pro- and anti-viral factors are highly expressed within the nasal epithelium, with potential age-dependent variation, predicting an important battleground for coronavirus infection. Our analysis also suggests that early embryonic and placental development are at moderate risk of infection. Lastly, SCARF expression appears broadly conserved across a subset of primate organs examined. Our study establishes a resource for investigations of coronavirus biology and pathology.


Assuntos
Infecções por Coronavirus/patologia , Mucosa Nasal/metabolismo , Pneumonia Viral/patologia , Receptores Virais/genética , Tropismo Viral/genética , Internalização do Vírus , Células A549 , Animais , Betacoronavirus/crescimento & desenvolvimento , Linhagem Celular , Chlorocebus aethiops , Enterócitos/metabolismo , Perfilação da Expressão Gênica , Células Caliciformes/metabolismo , Células HEK293 , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Mucosa Nasal/virologia , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Análise de Célula Única , Células Vero
13.
Nat Commun ; 11(1): 4602, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929071

RESUMO

Human behaviors are extremely sophisticated, relying on the adaptive, plastic and event-driven network of sensory neurons. Such neuronal system analyzes multiple sensory cues efficiently to establish accurate depiction of the environment. Here, we develop a bimodal artificial sensory neuron to implement the sensory fusion processes. Such a bimodal artificial sensory neuron collects optic and pressure information from the photodetector and pressure sensors respectively, transmits the bimodal information through an ionic cable, and integrates them into post-synaptic currents by a synaptic transistor. The sensory neuron can be excited in multiple levels by synchronizing the two sensory cues, which enables the manipulating of skeletal myotubes and a robotic hand. Furthermore, enhanced recognition capability achieved on fused visual/haptic cues is confirmed by simulation of a multi-transparency pattern recognition task. Our biomimetic design has the potential to advance technologies in cyborg and neuromorphic systems by endowing them with supramodal perceptual capabilities.


Assuntos
Células Receptoras Sensoriais/fisiologia , Tato/fisiologia , Visão Ocular/fisiologia , Animais , Linhagem Celular , Eletrodos , Humanos , Camundongos , Movimento (Física) , Nanotubos de Carbono/química , Reconhecimento Automatizado de Padrão
14.
Nat Commun ; 11(1): 4283, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32883967

RESUMO

Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation protocol, but also represents a promising research tool with a broad range of applications, including drug testing.


Assuntos
Matriz Extracelular/metabolismo , Fator 4 de Crescimento de Fibroblastos/metabolismo , Coração , Células-Tronco Embrionárias Murinas/metabolismo , Organoides , Potenciais de Ação , Diamino Aminoácidos/metabolismo , Animais , Biomimética/métodos , Diferenciação Celular , Linhagem Celular , Células Endoteliais , Coração/crescimento & desenvolvimento , Coração/fisiologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Contração Miocárdica , Miocárdio , Organoides/citologia , Organoides/crescimento & desenvolvimento , Organoides/ultraestrutura
15.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(6): 828-836, 2020 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895198

RESUMO

OBJECTIVE: To investigate enterovirus 71 (EV71)-induced of autophagy, apoptosis and the related signaling pathways in THP-1 macrophages. METHODS: THP-1 macrophages were infected with EV71 at the multiplicity of infection (MOI) of 0.1 for 2, 8 or 16 h, and the cell proliferation and toxicity were analyzed using CCK-8 kit. The intracellular viral nucleic acid in THP-1 macrophages were detected by fluorescence quantitative PCR, and the ultrastructural changes of the cells were observed using transmission electron microscopy. Cell apoptosis induced by EV71 infection was detected using Hoechst 33342 staining and AnnexinV/PI double staining. Western blotting was performed for analysis of changes in autophagy and apoptosis of the cells and in the expressions of the related proteins. The effect of EV71 infection on apoptosis of THP-1 macrophages incubated with 3-MA and Ac-DEVD-CHO inhibitor for 2 h was assessed using Western blotting. RESULTS: EV71 infection significantly lowered the cell survival rate of THP-1 macrophages at 2, 8 h and 16 h after the infection (P < 0.05). The total copy number of viral nucleic acid in THP-1 macrophages incubated with EV71 increased significantly and progressively over time (P < 0.01). Intracellular autophagosomes and virions could be seen in EV71-infected THP-1 macrophages. The total apoptotic rate of the infected cell also increased significantly over time (P < 0.01). EV71 infection significantly increased LC3 conversion (LC3-Ⅱ/ LC3-I) and the expression of cleaved caspase 3 protein and decreased the protein expressions of p62, Bcl-2 and caspase-3 (P < 0.01) without causing obvious changes in cleaved caspase-8 (P>0.05). 3-MA significantly inhibited the EV71-induced autophagy of THP-1 macrophages and reduced LC3 conversion (LC3-Ⅱ/LC3-I) and p62 protein expression at 8 h after EV71 infection (P < 0.01). Compared with DMSO, Ac-DEVD-CHO significantly inhibited EV71-induced apoptosis of THP-1 macrophages (15.5% vs 7.7%, P < 0.01). CONCLUSIONS: EV71 not only can infect and replicate in THP-1 macrophages, but also induces autophagy and cell apoptosis possibly by activating LC3/p62 autophagy pathway and caspase apoptosis pathway.


Assuntos
Autofagia , Enterovirus Humano A , Apoptose , Linhagem Celular , Humanos , Macrófagos
16.
Anticancer Res ; 40(9): 4885-4894, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878776

RESUMO

AIM: The aim of this study was to investigate the antitumor potential of guaiazulene-3-carboxylate derivatives against oral malignant cells. MATERIALS AND METHODS: Twelve guaiazulene-3-carboxylate derivatives were synthesized by introduction of either with alkyl group [1-5], alkoxy group [6, 7], hydroxyl group [8, 9] or primary amine [10-12] at the end of sidechains. Tumor-specificity (TS) was calculated by the ratio of mean 50% cytotoxic concentration (CC50) against 3 human oral mesenchymal cell lines to that against 4 human oral squamous cell carcinoma (OSCC) cell lines. Potency-selectivity expression (PSE) was calculated by dividing TS value by CC50value against OSCC cell lines. Cell cycle analysis was performed by cell sorter. RESULTS: [6, 7] showed the highest TS and PSE values, and induced the accumulation of both subG1 and G2/M cell populations in HSC-2 OSCC cells. Quantitative structure-activity relationship analysis demonstrated that their tumor-specificity was correlated with chemical descriptors that explain the 3D shape, electric state and ionization potential. CONCLUSION: Alkoxyl guaiazulene-3-carboxylates [6, 7] can be potential candidates of lead compound for developing novel anticancer drugs.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Azulenos/química , Azulenos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Sesquiterpenos de Guaiano/química , Sesquiterpenos de Guaiano/farmacologia , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Azulenos/síntese química , Carcinoma de Células Escamosas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Estrutura Molecular , Neoplasias Bucais/patologia , Relação Quantitativa Estrutura-Atividade , Sesquiterpenos de Guaiano/síntese química
17.
PLoS Pathog ; 16(9): e1008844, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886716

RESUMO

The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with specific suppression of CpG motifs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. We show that acute virus replication is mutually exclusive with high levels of cellular ZAP, potentially explaining the higher levels of CpG in viral genes expressed subsequent to IE1 due to the loss of pressure from ZAP in infected cells. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early 1 transcript. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.


Assuntos
Processamento Alternativo , Ilhas de CpG , Infecções por Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Regulação Viral da Expressão Gênica , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Replicação Viral , Linhagem Celular , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/patologia , Humanos , Proteínas Imediatamente Precoces , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
18.
Mol Cell ; 80(1): 72-86.e7, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32910895

RESUMO

Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion.


Assuntos
Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Complexos Multiproteicos/metabolismo , Linhagem Celular , Sequência Conservada , Evolução Molecular , Humanos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fosfatidilinositóis/metabolismo , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Emerg Microbes Infect ; 9(1): 2076-2090, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32897177

RESUMO

The current coronavirus disease 2019 (COVID-19) pandemic was the result of the rapid transmission of a highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there is no efficacious vaccine or therapeutic. Toward the development of a vaccine, here we expressed and evaluated as potential candidates four versions of the spike (S) protein using an insect cell expression system: receptor binding domain (RBD), S1 subunit, the wild-type S ectodomain (S-WT), and the prefusion trimer-stabilized form (S-2P). We showed that RBD appears as a monomer in solution, whereas S1, S-WT, and S-2P associate as homotrimers with substantial glycosylation. Cryo-electron microscopy analyses suggested that S-2P assumes an identical trimer conformation as the similarly engineered S protein expressed in 293 mammalian cells but with reduced glycosylation. Overall, the four proteins confer excellent antigenicity with convalescent COVID-19 patient sera in enzyme-linked immunosorbent assay (ELISA), yet show distinct reactivities in immunoblotting. RBD, S-WT and S-2P, but not S1, induce high neutralization titres (>3-log) in mice after a three-round immunization regimen. The high immunogenicity of S-2P could be maintained at the lowest dose (1 µg) with the inclusion of an aluminium adjuvant. Higher doses (20 µg) of S-2P can elicit high neutralization titres in non-human primates that exceed 40-times the mean titres measured in convalescent COVID-19 subjects. Our results suggest that the prefusion trimer-stabilized SARS-CoV-2 S-protein from insect cells may offer a potential candidate strategy for the development of a recombinant COVID-19 vaccine.


Assuntos
Antígenos Virais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Imunogenicidade da Vacina/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular , Infecções por Coronavirus/imunologia , Microscopia Crioeletrônica , Ensaio de Imunoadsorção Enzimática , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Peptidil Dipeptidase A/metabolismo , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Células Sf9 , Glicoproteína da Espícula de Coronavírus/genética , Spodoptera , Vacinação , Proteínas do Envelope Viral/imunologia
20.
Life Sci ; 259: 118397, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32896557

RESUMO

There is increasing evidence that Bazedoxifene, as an FDA-approved selective estrogen inhibitor, approved by FDA, not only inhibits estrogen receptors, but also has other pharmacological effects. The purpose of this study was to investigate the effects of Bazedoxifene on the functional changes of vascular smooth muscle cells (VSMCs) after PDGF-BB stimulation. VSMCs were divided into control group, PDGF-BB treatment group, and PDGF-BB treatment group with different concentrations of Bazedoxifene. CCK-8 and EdU staining were used to determine the VSMCs viability and proliferation. Western blot was used to detect the expressions of vimentin, SMA, ERK, p-ERK, STAT3, p-STAT3, AKT, p-AKT, and LC3 I/II. Wound healing method was used to detect the migration of VSMCs. PDGF-BB treatment significantly enhanced the viability and proliferation of VSMCs as indicated by CCK-8 and EdU assays (P < 0.01), while Bazedoxifene pretreatment could reduce the increased viability and proliferation of VSMCs caused by PDGF-BB (P < 0.05). Wound healing test also showed Bazedoxifene significantly attenuated the migration in the PDGF-BB stimulated VSMCs (P < 0.01). PDGF-BB also induced the phenotypic switch and decreased the autophagy level in VSMCs, manifested as a reduction in vimentin, SMA, and LC3 II (P < 0.01). These effects of PDGF-BB were partially reversed by Bazedoxifene (P < 0.05). Bazedoxifene may inhibit the proliferation and migration of VSMCs through up-regulate the autophagy level after PDGF-BB stimulation.


Assuntos
Autofagia/efeitos dos fármacos , Becaplermina/farmacologia , Indóis/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Becaplermina/antagonistas & inibidores , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Músculo Liso Vascular/citologia , Fenótipo
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