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1.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064452

RESUMO

Polycystic Kidney Disease (PKD) is a disorder that affects the kidneys and other organs, and its major forms are encoded by polycystin-1 (PC1) and polycystin-2 (PC2), as PKD1 and PKD2. It is located sandwiched inside and outside cell membranes and interacts with other cells. This protein is most active in kidney cells before birth, and PC1 and PC2 work together to help regulate cell proliferation, cell migration, and interactions with other cells. The molecular relationship and the function between PKD1 and cancer is well known, such as increased or decreased cell proliferation and promoting or suppressing cell migration depending on the cancer cell type specifically. However, its function in stem cells has not been revealed. Therefore, in this study, we investigated the biological function of PC1 and umbilical cord blood-derived mesenchymal stem cell (UCB-MSC). Furthermore, we assessed how it affects cell migration, proliferation, and the viability of cells when expressed in the PKD1 gene. In addition, we confirmed in an ex vivo artificial tooth model generated by the three-dimension printing technique that the ability to differentiate into osteocytes improved according to the expression level of the stemness markers when PKD1 was expressed. This study is the first report to examine the biological function of PKD1 in UCB-MSC. This gene may be capable of enhancing differentiation ability and maintaining long-term stemness for the therapeutic use of stem cells.


Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Osteócitos/metabolismo , Canais de Cátion TRPP/genética , Células A549 , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos/métodos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Células-Tronco Mesenquimais/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Osteócitos/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Canais de Cátion TRPP/metabolismo , Transfecção , Transgenes
2.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068728

RESUMO

To mimic more realistic lung tissue conditions, co-cultures of epithelial and immune cells are one comparatively easy-to-use option. To reveal the impact of immune cells on the mode of action (MoA) of CuO nanoparticles (NP) on epithelial cells, A549 cells as a model for epithelial cells have been cultured with or without differentiated THP-1 cells, as a model for macrophages. After 24 h of submerged incubation, cytotoxicity and transcriptional toxicity profiles were obtained and compared between the cell culture systems. Dose-dependent cytotoxicity was apparent starting from 8.0 µg/cm2 CuO NP. With regard to gene expression profiles, no differences between the cell models were observed concerning metal homeostasis, oxidative stress, and DNA damage, confirming the known MoA of CuO NP, i.e., endocytotic particle uptake, intracellular particle dissolution within lysosomes with subsequent metal ion deliberation, increased oxidative stress, and genotoxicity. However, applying a co-culture of epithelial and macrophage-like cells, CuO NP additionally provoked a pro-inflammatory response involving NLRP3 inflammasome and pro-inflammatory transcription factor activation. This study demonstrates that the application of this easy-to-use advanced in vitro model is able to extend the detection of cellular effects provoked by nanomaterials by an immunological response and emphasizes the use of such models to address a more comprehensive MoA.


Assuntos
Epitélio/efeitos dos fármacos , Nanopartículas Metálicas/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Estresse Oxidativo/genética , Transcrição Genética/efeitos dos fármacos , Células A549 , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Cobre/química , Cobre/farmacologia , Dano ao DNA/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
3.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073774

RESUMO

Predicting in vivo protein-DNA binding sites is a challenging but pressing task in a variety of fields like drug design and development. Most promoters contain a number of transcription factor (TF) binding sites, but only a small minority has been identified by biochemical experiments that are time-consuming and laborious. To tackle this challenge, many computational methods have been proposed to predict TF binding sites from DNA sequence. Although previous methods have achieved remarkable performance in the prediction of protein-DNA interactions, there is still considerable room for improvement. In this paper, we present a hybrid deep learning framework, termed DeepD2V, for transcription factor binding sites prediction. First, we construct the input matrix with an original DNA sequence and its three kinds of variant sequences, including its inverse, complementary, and complementary inverse sequence. A sliding window of size k with a specific stride is used to obtain its k-mer representation of input sequences. Next, we use word2vec to obtain a pre-trained k-mer word distributed representation model. Finally, the probability of protein-DNA binding is predicted by using the recurrent and convolutional neural network. The experiment results on 50 public ChIP-seq benchmark datasets demonstrate the superior performance and robustness of DeepD2V. Moreover, we verify that the performance of DeepD2V using word2vec-based k-mer distributed representation is better than one-hot encoding, and the integrated framework of both convolutional neural network (CNN) and bidirectional LSTM (bi-LSTM) outperforms CNN or the bi-LSTM model when used alone. The source code of DeepD2V is available at the github repository.


Assuntos
Biologia Computacional/métodos , DNA/metabolismo , Aprendizado Profundo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Humanos , Células K562 , Software
4.
Proc Natl Acad Sci U S A ; 118(25)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34099577

RESUMO

Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV-infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Zoonoses/virologia , África , Animais , Arábia , Linhagem Celular , Dipeptidil Peptidase 4/metabolismo , Técnicas de Introdução de Genes , Humanos , Cinética , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Fenótipo , Filogenia , Glicoproteína da Espícula de Coronavírus/metabolismo , Replicação Viral/fisiologia
5.
Nat Commun ; 12(1): 3413, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099701

RESUMO

Bottom-up approaches using building blocks of modules to fabricate scaffolds for tissue engineering applications have enabled the fabrication of structurally complex and multifunctional materials allowing for physical and chemical flexibility to better mimic the native extracellular matrix. Here we report a vapor-phased fabrication process for constructing three-dimensional modulated scaffold materials via simple steps based on controlling mass transport of vapor sublimation and deposition. We demonstrate the fabrication of scaffolds comprised of multiple biomolecules and living cells with built-in boundaries separating the distinct compartments containing defined biological configurations and functions. We show that the fabricated scaffolds have mass production potential. We demonstrate overall >80% cell viability of encapsulated cells and that modulated scaffolds exhibit enhanced cell proliferation, osteogenesis, and neurogenesis, which can be assembled into various geometric configurations. We perform cell co-culture experiments to show independent osteogenesis and angiogenesis activities from separate compartments in one scaffold construct.


Assuntos
Materiais Biomiméticos/química , Vapor , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Técnicas de Cultura de Células , Linhagem Celular , Proliferação de Células , Técnicas de Cocultura , Matriz Extracelular , Humanos , Hidrogéis/química , Camundongos , Neovascularização Fisiológica , Neurogênese , Osteogênese , Ratos
6.
Nat Commun ; 12(1): 3450, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103504

RESUMO

The epigenetic mechanisms coordinating the maintenance of adult cellular lineages and the inhibition of alternative cell fates remain poorly understood. Here we show that targeted ablation of the histone chaperone HIRA in myogenic cells leads to extensive transcriptional modifications, consistent with a role in maintaining skeletal muscle cellular identity. We demonstrate that conditional ablation of HIRA in muscle stem cells of adult mice compromises their capacity to regenerate and self-renew, leading to tissue repair failure. Chromatin analysis of Hira-deficient cells show a significant reduction of histone variant H3.3 deposition and H3K27ac modification at regulatory regions of muscle genes. Additionally, we find that genes from alternative lineages are ectopically expressed in Hira-mutant cells via MLL1/MLL2-mediated increase of H3K4me3 mark at silent promoter regions. Therefore, we conclude that HIRA sustains the chromatin landscape governing muscle cell lineage identity via incorporation of H3.3 at muscle gene regulatory regions, while preventing the expression of alternative lineage genes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Chaperonas de Histonas/metabolismo , Músculo Esquelético/patologia , Fatores de Transcrição/metabolismo , Acetilação , Animais , Proteínas de Ciclo Celular/deficiência , Linhagem Celular , Linhagem da Célula/genética , Loci Gênicos , Chaperonas de Histonas/deficiência , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Desenvolvimento Muscular/genética , Músculo Esquelético/lesões , Músculo Esquelético/fisiopatologia , Regeneração , Sequências Reguladoras de Ácido Nucleico/genética , Células Satélites de Músculo Esquelético/metabolismo , Fatores de Transcrição/deficiência
7.
Int J Med Sci ; 18(12): 2561-2569, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34104087

RESUMO

SARS-CoV-2 infection poses a global challenge to human health. Upon viral infection, host cells initiate the innate antiviral response, which primarily involves type I interferons (I-IFNs), to enable rapid elimination of the invading virus. Previous studies revealed that SARS-CoV-2 infection limits the expression of I-IFNs in vitro and in vivo, but the underlying mechanism remains incompletely elucidated. In the present study, we performed data mining and longitudinal data analysis using SARS-CoV-2-infected normal human bronchial epithelial (NHBE) cells and ferrets, and the results confirmed the strong inhibitory effect of SARS-CoV-2 on the induction of I-IFNs. Moreover, we identified genes that are negatively correlated with IFNB1 expression in vitro and in vivo based on Pearson correlation analysis. We found that SARS-CoV-2 activates numerous intrinsic pathways, such as the circadian rhythm, phosphatidylinositol signaling system, peroxisome, and TNF signaling pathways, to inhibit I-IFNs. These intrinsic inhibitory pathways jointly facilitate the successful immune evasion of SARS-CoV-2. Our study elucidates the underlying mechanism by which SARS-CoV-2 evades the host innate antiviral response in vitro and in vivo, providing theoretical evidence for targeting these immune evasion-associated pathways to combat SARS-CoV-2 infection.


Assuntos
COVID-19/imunologia , Interações Hospedeiro-Patógeno/imunologia , Interferon gama/metabolismo , SARS-CoV-2/imunologia , Animais , Brônquios/citologia , COVID-19/virologia , Linhagem Celular , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Células Epiteliais , Furões , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/genética , Humanos , Imunidade Inata , Interferon gama/imunologia , RNA-Seq , Mucosa Respiratória/citologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia
8.
Viruses ; 13(6)2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070524

RESUMO

SARS-CoV-2 emerged in 2019 as a devastating viral pathogen with no available preventative or treatment to control what led to the current global pandemic. The continued spread of the virus and increasing death toll necessitate the development of effective antiviral treatments to combat this virus. To this end, we evaluated a new class of organometallic complexes as potential antivirals. Our findings demonstrate that two pentamethylcyclopentadienyl (Cp*) rhodium piano stool complexes, Cp*Rh(1,3-dicyclohexylimidazol-2-ylidene)Cl2 (complex 2) and Cp*Rh(dipivaloylmethanato)Cl (complex 4), have direct virucidal activity against SARS-CoV-2. Subsequent in vitro testing suggests that complex 4 is the more stable and effective complex and demonstrates that both 2 and 4 have low toxicity in Vero E6 and Calu-3 cells. The results presented here highlight the potential application of organometallic complexes as antivirals and support further investigation into their activity.


Assuntos
Antivirais/farmacologia , Compostos Organometálicos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/química , COVID-19/virologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Compostos Organometálicos/química , SARS-CoV-2/fisiologia , Células Vero , Replicação Viral/efeitos dos fármacos
9.
Nat Commun ; 12(1): 3266, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075032

RESUMO

The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and combat these pathogens. Here, we present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. We use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 Å, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. We anticipate the chimeric platform to be widely applicable for investigating flavivirus biology.


Assuntos
Infecções por Flavivirus/terapia , Flavivirus/ultraestrutura , Proteínas do Envelope Viral/ultraestrutura , Vírion/ultraestrutura , Aedes/virologia , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Linhagem Celular , Chlorocebus aethiops , Microscopia Crioeletrônica , Dengue/terapia , Dengue/virologia , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/farmacologia , Desenho de Fármacos , Flavivirus/efeitos dos fármacos , Flavivirus/imunologia , Flavivirus/patogenicidade , Infecções por Flavivirus/virologia , Humanos , Mesocricetus , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Células Vero , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/farmacologia , Vacinas Virais/uso terapêutico , Vírion/efeitos dos fármacos , Vírion/metabolismo
10.
Nat Commun ; 12(1): 3309, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083527

RESUMO

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Reposicionamento de Medicamentos/métodos , Pandemias , SARS-CoV-2 , Animais , COVID-19/prevenção & controle , COVID-19/virologia , Linhagem Celular , Citidina/administração & dosagem , Citidina/análogos & derivados , Citidina/farmacologia , Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Hidroxilaminas/administração & dosagem , Hidroxilaminas/farmacologia , Mesocricetus , Nelfinavir/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos
11.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(3): 1007-1010, 2021 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-34105509

RESUMO

Cancer cell lines are an indispensable tool in the cancer research. Since the first human cell line, HeLa was established in the 1950s, thousands of cancer cell lines have been established, including 637 characterized leukemia-lymphoma cell lines. The probability to successfully establish cancer cell lines is a low by traditional methods, and the addition of regulatory factors is often required. However, a novel "conditional reprogramming" technology can improve this situction. The establishment and description of a new cell line should be consistent with international guidelines. Cancer cell lines are mainly used in the research of tumor pathogenesis and drug development. Scientists have developed many kinds of cell line panels which can be used for the high-throughput screening of anticancer drugs. Mycoplasma contamination and/or cross-contamination from other cells should be avoided during the use of cell lines. The establishment of a cell model passport database can prevent those misidentifications. In this review, the types, establishment and usage of leukemia-lymphoma cell lines as well as points of attention when using them are summarized briefly.


Assuntos
Leucemia , Linfoma , Linhagem Celular , Humanos , Linfócitos
12.
Biomolecules ; 11(5)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067685

RESUMO

Cm-p5 is a snail-derived antimicrobial peptide, which demonstrated antifungal activity against the pathogenic strains of Candida albicans. Previously we synthetized a cyclic monomer as well as a parallel and an antiparallel dimer of Cm-p5 with improved antifungal activity. Considering the alarming increase of microbial resistance to conventional antibiotics, here we evaluated the antimicrobial activity of these derivatives against multiresistant and problematic bacteria and against important viral agents. The three peptides showed a moderate activity against Pseudomonas aeruginosa, Klebsiella pneumoniae Extended Spectrum ß-Lactamase (ESBL), and Streptococcus agalactiae, with MIC values > 100 µg/mL. They exerted a considerable activity with MIC values between 25-50 µg/mL against Acinetobacter baumanii and Enterococcus faecium. In addition, the two dimers showed a moderate activity against Pseudomonas aeruginosa PA14. The three Cm-p5 derivatives inhibited a virulent extracellular strain of Mycobacterium tuberculosis, in a dose-dependent manner. Moreover, they inhibited Herpes Simplex Virus 2 (HSV-2) infection in a concentration-dependent manner, but had no effect on infection by the Zika Virus (ZIKV) or pseudoparticles of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). At concentrations of >100 µg/mL, the three new Cm-p5 derivatives showed toxicity on different eukaryotic cells tested. Considering a certain cell toxicity but a potential interesting activity against the multiresistant strains of bacteria and HSV-2, our compounds require future structural optimization.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Antivirais/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antivirais/química , Candida albicans/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dimerização , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , SARS-CoV-2/efeitos dos fármacos
13.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069441

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel emerging pathogen causing an unprecedented pandemic in 21st century medicine. Due to the significant health and economic burden of the current SARS-CoV-2 outbreak, there is a huge unmet medical need for novel interventions effectively blocking SARS-CoV-2 infection. Unknown details of SARS-CoV-2 cellular biology hamper the development of potent and highly specific SARS-CoV-2 therapeutics. Angiotensin-converting enzyme-2 (ACE2) has been reported to be the primary receptor for SARS-CoV-2 cellular entry. However, emerging scientific evidence suggests the involvement of additional membrane proteins, such as heparan sulfate proteoglycans, in SARS-CoV-2 internalization. Here, we report that syndecans, the evolutionarily conserved family of transmembrane proteoglycans, facilitate the cellular entry of SARS-CoV-2. Among syndecans, the lung abundant syndecan-4 was the most efficient in mediating SARS-CoV-2 uptake. The S1 subunit of the SARS-CoV-2 spike protein plays a dominant role in the virus's interactions with syndecans. Besides the polyanionic heparan sulfate chains, other parts of the syndecan ectodomain, such as the cell-binding domain, also contribute to the interaction with SARS-CoV-2. During virus internalization, syndecans colocalize with ACE2, suggesting a jointly shared internalization pathway. Both ACE2 and syndecan inhibitors exhibited significant efficacy in reducing the cellular entry of SARS-CoV-2, thus supporting the complex nature of internalization. Data obtained on syndecan specific in vitro assays present syndecans as novel cellular targets of SARS-CoV-2 and offer molecularly precise yet simple strategies to overcome the complex nature of SARS-CoV-2 infection.


Assuntos
COVID-19/metabolismo , Receptores de Coronavírus/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Sindecanas/metabolismo , Internalização do Vírus , Amilorida/farmacologia , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , COVID-19/virologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Humanos , Peptídeos/farmacologia , Domínios Proteicos , SARS-CoV-2/metabolismo , Sindecana-4/antagonistas & inibidores , Sindecana-4/metabolismo , Sindecanas/antagonistas & inibidores
14.
Biomolecules ; 11(5)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069869

RESUMO

Several RNA viruses, including SARS-CoV-2, can infect or use the eye as an entry portal to cause ocular or systemic diseases. Povidone-Iodine (PVP-I) is routinely used during ocular surgeries and eye banking as a cost-effective disinfectant due to its broad-spectrum antimicrobial activity, including against viruses. However, whether PVP-I can exert antiviral activities in virus-infected cells remains elusive. In this study, using Zika (ZIKV) and Chikungunya (CHIKV) virus infection of human corneal and retinal pigment epithelial cells, we report antiviral mechanisms of PVP-I. Our data showed that PVP-I, even at the lowest concentration (0.01%), drastically reduced viral replication in corneal and retinal cells without causing cellular toxicity. Antiviral effects of PVP-I against ZIKV and CHIKV were mediated by direct viral inactivation, thus attenuating the ability of the virus to infect host cells. Moreover, one-minute PVP-I exposure of infected ocular cells drastically reduced viral replication and the production of infectious progeny virions. Furthermore, viral-induced (CHIKV) expression of inflammatory genes (TNF-α, IL-6, IL-8, and IL1ß) were markedly reduced in PVP-I treated corneal epithelial cells. Together, our results demonstrate potent antiviral effects of PVP-I against ZIKV and CHIKV infection of ocular cells. Thus, a low dose of PVP-I can be used during tissue harvesting for corneal transplants to prevent potential transmission of RNA viruses via infected cells.


Assuntos
Antivirais/farmacologia , Povidona-Iodo/farmacologia , Vírus de RNA/fisiologia , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Vírus Chikungunya/fisiologia , Chlorocebus aethiops , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/virologia , SARS-CoV-2/fisiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Células Vero , Zika virus/fisiologia
15.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070125

RESUMO

The neuropeptide substance P (SP) mediates neurogenic inflammation and pain and contributes to atopic dermatitis in mice through the activation of mast cells (MCs) via Mas-related G protein-coupled receptor (GPCR)-B2 (MrgprB2, human ortholog MRGPRX2). In addition to G proteins, certain MRGPRX2 agonists activate an additional signaling pathway that involves the recruitment of ß-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused ß-arrestin recruitment, MRGPRX2 internalization, and desensitization. These responses were independent of G proteins, indicating that SP serves as a balanced agonist for MRGPRX2. A tyrosine residue in the highly conserved NPxxY motif contributes to the activation and internalization of many GPCRs. We have previously shown that Tyr279 of MRGPRX2 is essential for G protein-mediated signaling and degranulation. To assess its role in ß-arrestin-mediated MRGPRX2 regulation, we replaced Tyr279 in the NPxxY motif of MRGPRX2 with Ala (Y279A). Surprisingly, we found that, unlike the wild-type receptor, Y279A mutant of MRGPRX2 was resistant to SP-induced ß-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by ß-arrestins and that a highly conserved tyrosine residue within MRGPRX2's NPxxY motif contributes to both G protein- and ß-arrestin-mediated responses.


Assuntos
Proteínas do Tecido Nervoso/agonistas , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neuropeptídeos/agonistas , Substância P/metabolismo , beta-Arrestinas/metabolismo , Substituição de Aminoácidos , Animais , Degranulação Celular , Linhagem Celular , Feminino , Humanos , Masculino , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neuroimunomodulação/fisiologia , Ratos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/genética , Tirosina/química , beta-Arrestina 2/deficiência , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
16.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073283

RESUMO

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and IL-6. Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, we investigated the roles of the integrated stress response (ISR) and activator protein-1 (AP-1) family proteins in regulating coronavirus-induced IL-8 and IL-6 upregulation. The mRNA expression of IL-8 and IL-6 was significantly induced in cells infected with infectious bronchitis virus (IBV), a gammacoronavirus, and porcine epidemic diarrhea virus, an alphacoronavirus. Overexpression of a constitutively active phosphomimetic mutant of eukaryotic translation initiation factor 2α (eIF2α), chemical inhibition of its dephosphorylation, or overexpression of its upstream double-stranded RNA-dependent protein kinase (PKR) significantly enhanced IL-8 mRNA expression in IBV-infected cells. Overexpression of the AP-1 protein cJUN or its upstream kinase also increased the IBV-induced IL-8 mRNA expression, which was synergistically enhanced by overexpression of cFOS. Taken together, this study demonstrated the important regulatory roles of ISR and AP-1 proteins in IL-8 production during coronavirus infection, highlighting the complex interactions between cellular stress pathways and the innate immune response.


Assuntos
Infecções por Coronavirus/metabolismo , Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Interleucina-8/metabolismo , Resposta a Proteínas não Dobradas/genética , Alphacoronavirus/metabolismo , Alphacoronavirus/patogenicidade , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/genética , Gammacoronavirus/metabolismo , Gammacoronavirus/patogenicidade , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Vírus da Bronquite Infecciosa/metabolismo , Vírus da Bronquite Infecciosa/patogenicidade , Interleucina-8/genética , Fosforilação , Vírus da Diarreia Epidêmica Suína/metabolismo , Vírus da Diarreia Epidêmica Suína/patogenicidade , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Regulação para Cima , Células Vero , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
17.
Nat Commun ; 12(1): 3292, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078910

RESUMO

Autophagy regulates primary cilia formation, but the underlying mechanism is not fully understood. In this study, we identify NIMA-related kinase 9 (NEK9) as a GABARAPs-interacting protein and find that NEK9 and its LC3-interacting region (LIR) are required for primary cilia formation. Mutation in the LIR of NEK9 in mice also impairs in vivo cilia formation in the kidneys. Mechanistically, NEK9 interacts with MYH9 (also known as myosin IIA), which has been implicated in inhibiting ciliogenesis through stabilization of the actin network. MYH9 accumulates in NEK9 LIR mutant cells and mice, and depletion of MYH9 restores ciliogenesis in NEK9 LIR mutant cells. These results suggest that NEK9 regulates ciliogenesis by acting as an autophagy adaptor for MYH9. Given that the LIR in NEK9 is conserved only in land vertebrates, the acquisition of the autophagic regulation of the NEK9-MYH9 axis in ciliogenesis may have possible adaptive implications for terrestrial life.


Assuntos
Autofagia/genética , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Cadeias Pesadas de Miosina/genética , Quinases Relacionadas a NIMA/genética , Sequência de Aminoácidos , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular , Cílios/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Rim/citologia , Rim/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Quinases Relacionadas a NIMA/deficiência , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais
18.
Int J Nanomedicine ; 16: 3649-3660, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34079255

RESUMO

Introduction: Semiconductor nanoplatelets (NPLs) are promising materials for nonlinear optical microscopy since they feature good two-photon absorption (TPA) properties, narrow photoluminescence spectra and high quantum yields of luminescence. Nevertheless, the use of semiconductor NPLs is inevitably connected with concerns about heavy metal ion toxicity and their intrinsically hydrophobic character. Methods: Our contribution focuses on the design and engineering of coloidal bionanomaterial consisting of two-dimensional highly luminescent CdSe semiconductor NPLs loaded into spherical and homogeneous polymeric nanocarriers (NCs) based on poly(ethylene oxide) and poly(propylene oxide) block co-polymer. The biocompatibility and usefulness of the NPLs-loaded polymeric NCs in two-photon induced bioimaging was demonstrated in vitroby cytotoxicity and two-photon microscopic studies using eukaryotic (normal fibroblasts and cancer ovarian) cells. Results: The encapsulated NPLs maintain their intensive and spectrally narrow photoluminescence, as well as preserve good TPA properties, while the surrounding polymer shell imparts hydrophilic character and non-toxicity towards eukaryotic cells. Specifically, TPA cross-sections of the colloidal NCs loaded with NPLs show large values reaching up to 2.0 × 108 GM, with simultaneously two-photon brightness reaching 2.2 × 107 GM at 870 nm. MTT proliferation assay performed on cell lines treated with encapsulated NPLs revealed at least 70% viability of normal human gingival fibroblast (HGF) and cancer ovarian (MDAH-2774) cells, while the results of multiphoton imaging of murine (L-929) fibroblasts suggest that the encapsulated NPLs are capable of labelling the target cells enabling their visualization. Conclusion: As a result, we obtained water dispersible and temporally stable hydrophilic NPLs-loaded NCs that offer excellent, both one- and two-photon excited fluorescence preserving optical properties of the raw hydrophobic and colloidal NPLs. The biological responses upon eukaryotic cells indicate that the encapsulation process protects cells from the toxic influence of cadmium simultaneously preserving the unique multiphoton properties of the active cargo which opens a promising perspective for its application in multiphoton cancer bioimaging excited at the "optical transmission window" of biological tissues in near-infrared range.


Assuntos
Interações Hidrofóbicas e Hidrofílicas , Substâncias Luminescentes/química , Microscopia/métodos , Nanoestruturas/química , Fótons , Polietilenos/química , Polipropilenos/química , Animais , Linhagem Celular , Coloides , Camundongos , Semicondutores , Água/química
19.
Nat Commun ; 12(1): 3308, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083519

RESUMO

The spatial partitioning of the transcriptome in the cell is an important form of gene-expression regulation. Here, we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH reveals that nuclear TERT transcripts uniformly and robustly retain specific introns. Our data suggest that the splicing of TERT retained introns occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution has significant effects on cell viability. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of these RNAs within the cell. This process reveals that modulating RNA localization via targeted intron retention can be utilized for RNA-based therapies.


Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/genética , Animais , Compartimento Celular , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons , Camundongos , Mitose , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Estabilidade de RNA , Especificidade da Espécie
20.
Zhonghua Gan Zang Bing Za Zhi ; 29(5): 427-432, 2021 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-34107579

RESUMO

Objective: To study LIM kinase 1 (LIMK1) expressional condition, and its regulatory effects on the proliferation and metastasis of hepatocellular carcinoma cells and tissues. Methods: The online database starBase v3.0 and GEPIA were used to analyze the LIMK1 expression in hepatocellular carcinoma cells and normal liver tissues, and then the relevant survival analysis was performed. LIMK1 expression in hepatocellular carcinoma cell line was analyzed by Western blot. Hep3B and Huh7 cells were transiently transfected after LIMK1 protein expression was down-regulated by small interfering RNA (siRNA). LIMK1 effects on the proliferation of Hep3B and Huh7 cells were observed by MTT assay and colony formation assay. Transwell assay was used to detect the change in metastatic ability of hepatocellular carcinoma cell after the down-regulation of LIMK1 expression. Western blot was used to detect the changes of related indexes in the process of epithelial mesenchymal transition after the down-regulation of LIMK1 expression. Data were analyzed by one-way ANOVA. Results: The expression level of LIMK1 in liver cancer tissues was significantly higher than that of normal liver tissues, and was related with prognosis (P ​< 0.01). Furthermore, LIMK1 expression in HCC cell lines was significantly higher than that of immortalized liver L02 cells (P < 0.05). Functional correlated experiment showed that the proliferation and metastatic ability of liver cancer cells were significantly inhibited after LIMK1 expression down-regulation (P < 0.05). Simultaneously, LIMK1 was also involved in the process of epithelial-mesenchymal transition. Conclusion: LIMK1 was overexpressed in HCC tissues and cells, and may regulate the proliferation and metastasis of HCC cells and participate in epithelial-mesenchymal transition process.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Quinases Lim/genética , Quinases Lim/metabolismo , Neoplasias Hepáticas/genética , Invasividade Neoplásica
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