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1.
Braz. j. biol ; 84: e250151, 2024. tab, graf
Artigo em Inglês | MEDLINE, LILACS, VETINDEX | ID: biblio-1350306

RESUMO

Abstract Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.


Resumo Os mamíferos têm uma capacidade limitada de regenerar seus tecidos e órgãos. Um dos mecanismos associados à regeneração natural é a desdiferenciação. Várias moléculas pequenas, como vitamina C e fatores de crescimento, podem melhorar a eficiência da reprogramação. Neste estudo, as células NTERA2-D1 (NT2) foram induzidas à diferenciação (NT2-RA) com ácido retinóico (RA) 10-5 M por três dias e depois submetidas a várias quantidades de humor vítreo (VH). Os resultados mostram que a taxa de crescimento dessas células foi reduzida, enquanto essa taxa foi parcialmente restaurada após o tratamento com VH (NT2-RA-VH). A análise do ciclo celular com o método PI também mostrou que o número de células na fase S do ciclo celular nessas células estava aumentado. Os níveis de antígenos SSEA3 e TRA-1-81 em NT2-RA diminuíram, mas aumentaram em NT2-RA-VH a um nível semelhante ao das células NT2. O nível de SSEA1 teve um padrão oposto. A expressão do gene OCT4 diminuiu após o tratamento com AR, mas foi recuperado em células NT2-RA-VH. Em conclusão, sugerimos o VH como uma mistura potente para melhorar a reprogramação celular levando à desdiferenciação.


Assuntos
Humanos , Corpo Vítreo , Proliferação de Células , Desdiferenciação Celular , Tretinoína , Células Tumorais Cultivadas , Diferenciação Celular , Divisão Celular , Linhagem Celular
2.
In Vitro Cell Dev Biol Anim ; 58(1): 14-20, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35075604

RESUMO

Extensive usage of synthetic chemical pesticides has collateral effects in harming human and animal health and the environment and promoting the development of resistance in pests. The potential of plant compounds as bio insecticides has been described as a promising field of agricultural development. The present study involved the use of Artemisia annua essential oils to evaluate their cytotoxic activities against an established cell line of lesser mulberry pyralid. Five types of hemocytes were recognized (prohaemocytes, plasmatocytes, granulocytes, oenocytoids, and spherulocytes) in the primary cultures maintained in Ex-Cell media with 10% fetal bovine serum (FBS). Artemisia annua essential oils produced noticeable cytotoxicity against the insect cell lines. Applied at a concentration 500 ppm, oils extracted from the vegetative or flowering stages of A. annua produced 71% and 80% cell death, respectively. Nanoemulsions of EOs from the vegetative or flowering stages of A. annua killed 67 and 60% of the cells, respectively. This study has clearly shown significant bioactivities of A. annua secondary metabolites to insect cell lines.


Assuntos
Artemisia annua , Asteraceae , Mariposas , Óleos Voláteis , Animais , Artemisia annua/química , Linhagem Celular , Hemócitos , Óleos Voláteis/química , Óleos Voláteis/farmacologia
3.
PLoS Genet ; 18(1): e1009666, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35061661

RESUMO

Dynamic and temporally specific gene regulatory changes may underlie unexplained genetic associations with complex disease. During a dynamic process such as cellular differentiation, the overall cell type composition of a tissue (or an in vitro culture) and the gene regulatory profile of each cell can both experience significant changes over time. To identify these dynamic effects in high resolution, we collected single-cell RNA-sequencing data over a differentiation time course from induced pluripotent stem cells to cardiomyocytes, sampled at 7 unique time points in 19 human cell lines. We employed a flexible approach to map dynamic eQTLs whose effects vary significantly over the course of bifurcating differentiation trajectories, including many whose effects are specific to one of these two lineages. Our study design allowed us to distinguish true dynamic eQTLs affecting a specific cell lineage from expression changes driven by potentially non-genetic differences between cell lines such as cell composition. Additionally, we used the cell type profiles learned from single-cell data to deconvolve and re-analyze data from matched bulk RNA-seq samples. Using this approach, we were able to identify a large number of novel dynamic eQTLs in single cell data while also attributing dynamic effects in bulk to a particular lineage. Overall, we found that using single cell data to uncover dynamic eQTLs can provide new insight into the gene regulatory changes that occur among heterogeneous cell types during cardiomyocyte differentiation.


Assuntos
Perfilação da Expressão Gênica/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Análise de Célula Única/métodos , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/química , Miócitos Cardíacos/química , RNA-Seq
4.
Biochem J ; 479(11): 1121-1126, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35647902

RESUMO

Numerous studies, published over many years, have established the key role that the IκB kinase (IKK) subunits, α and ß, play in regulating the Nuclear Factor κB (NF-κB) pathway. This research generally concluded that their functions can be separated, with IKKß being the critical regulator of the canonical NF-κB pathway, while IKKα functions as the key activating kinase for the non-canonical pathway. However, other roles for these kinases have been described and several reports concluded that this separation of their functions may not always be the case. This commentary discusses the recent report by Biochem J. 479, 305-325, who elegantly demonstrate that in KRAS driven colorectal cancer cell lines, IKKα is an important regulator of the canonical NF-κB pathway. As is so often the case with trying to understand the complexity of NF-κB signalling, cellular context is everything.


Assuntos
Quinase I-kappa B , NF-kappa B , Linhagem Celular , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais
5.
Molecules ; 27(11)2022 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-35684505

RESUMO

The in vitro antiproliferative activity of a phenolic-rich extract from Lycium barbarum fruits against head and neck HPV16 squamous cell carcinoma (OSCC) has been demonstrated, indicating for the first time that L. barbarum extract inhibits human papillomavirus (HPV) type 16 cell lines. Ethanol extract of L. barbarum was used for cell viability evaluation on SCC090, CAL27, and HGnF cell lines. After 24 and 48 h, the cell cycle effect of L. barbarum extract (at 1.0, 10, and 100 µg/mL) was measured via flow cytometry. In addition, the mRNA expression on E6/E7 and p53 via RT-PCR and the expression of p16, p53, Ki-67, and Bcl-2 via immunohistochemistry were also determined. Untreated cells, 20 µM cisplatin, and a Camellia sinensis-derived extract were used as negative and positive controls, respectively. We demonstrated that the studied L. barbarum extract resulted in G0/G1 arrest and S phase accumulation in SCC090 at 1.0 and 10 µg/mL. A reduction in mRNA levels of E6/E7 oncogenes (p < 0.05) with p53 overexpression was also observed through PCR, while immunohistochemical analyses indicated p16 overexpression (p > 0.05) and a decrease in p53 overexpression. The observed effects were associated with anticancer and immunomodulatory phenolics, such as flavonols/flavan-3-ols and tyramine-conjugated hydroxycinnamic acid amides, identified in the studied extract. These findings revealed that the phenolic-rich extract of L. barbarum fruits has promising properties to be considered further for developing new therapies against oral and oropharyngeal HPV lesions.


Assuntos
Neoplasias de Cabeça e Pescoço , Lycium , Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Linhagem Celular , Frutas/química , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Papillomavirus Humano 16/genética , Humanos , Lycium/química , Proteínas E7 de Papillomavirus/análise , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/complicações , Fenóis/análise , Fenóis/farmacologia , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , RNA Mensageiro , Proteínas Repressoras/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
6.
Biotechniques ; 72(6): 279-286, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35703314

RESUMO

Although several genome editing options are available, CRISPR/Cas9 is one of the most commonly used systems for protein and advanced therapies. There are some long-term data regarding genomic and phenotypic stability, however, information is sparse. Flow cytometry can offer a method to characterize these edited cells for longitudinal studies. The objective of this work is to describe a protocol for using flow cytometry to measure the edits from CRISPR/Cas9 on a well-characterized B-lymphoblast cell line, GM24385, with the goal of supporting safe and effective CRISPR/Cas9-engineered therapies.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Linhagem Celular , Citometria de Fluxo , Edição de Genes/métodos
7.
Stem Cell Res ; 62: 102824, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35679757

RESUMO

We have established the footprint-free Vietnamese human induced pluripotent stem cell (hiPSC) line, VRISGi002-A, from CD71 + CD235a + erythroid progenitor cells (EPCs) of a 27-year-old healthy donor. The EPCs were enriched from isolated peripheral blood and reprogrammed using Sendai viruses which carried the reprogramming factors c-MYC, SOX2, KLF4, and OCT4 under a feeder-free culture system. The established VRISGi002-A cell line expressed typical pluripotency markers, displayed a normal karyotype, and demonstrated the potential to differentiate into the three germ layers. This hiPSC line could serve as a Vietnamese healthy control model for physiological processes and drug screening.


Assuntos
Células-Tronco Pluripotentes Induzidas , Adulto , Diferenciação Celular/fisiologia , Linhagem Celular , Reprogramação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/metabolismo , Vírus Sendai
8.
Proc Natl Acad Sci U S A ; 119(25): e2205536119, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35700360

RESUMO

Dystrophin is an essential muscle protein that contributes to cell membrane stability by mechanically linking the actin cytoskeleton to the extracellular matrix via an adhesion complex called the dystrophin-glycoprotein complex. The absence or impaired function of dystrophin causes muscular dystrophy. Focal adhesions (FAs) are also mechanosensitive adhesion complexes that connect the cytoskeleton to the extracellular matrix. However, the interplay between dystrophin and FA force transmission has not been investigated. Using a vinculin-based bioluminescent tension sensor, we measured FA tension in transgenic C2C12 myoblasts expressing wild-type (WT) dystrophin, a nonpathogenic single nucleotide polymorphism (SNP) (I232M), or two missense mutations associated with Duchenne (L54R), or Becker muscular dystrophy (L172H). Our data revealed cross talk between dystrophin and FAs, as the expression of WT or I232M dystrophin increased FA tension compared to dystrophin-less nontransgenic myoblasts. In contrast, the expression of L54R or L172H did not increase FA tension, indicating that these disease-causing mutations compromise the mechanical function of dystrophin as an FA allosteric regulator. Decreased FA tension caused by these mutations manifests as defective migration, as well as decreased Yes-associated protein 1 (YAP) activation, possibly by the disruption of the ability of FAs to transmit forces between the extracellular matrix and cytoskeleton. Our results indicate that dystrophin influences FA tension and suggest that dystrophin disease-causing missense mutations may disrupt a cellular tension-sensing pathway in dystrophic skeletal muscle.


Assuntos
Distrofina , Adesões Focais , Mecanotransdução Celular , Distrofia Muscular de Duchenne , Animais , Linhagem Celular , Distrofina/genética , Adesões Focais/genética , Mecanotransdução Celular/genética , Camundongos , Células Musculares , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único
9.
Cell Rep ; 39(11): 110954, 2022 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-35671758

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1's shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1's explicit role in blocking the IFN response.


Assuntos
COVID-19 , Proteínas não Estruturais Virais , Linhagem Celular , Humanos , Estabilidade de RNA , SARS-CoV-2 , Proteínas não Estruturais Virais/metabolismo
10.
Curr Microbiol ; 79(8): 225, 2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35704105

RESUMO

The present study aimed to isolate and identify the potential probiotic, pathobiont, and pathogenic microorganisms in the stool samples of 12 healthy individuals and evaluate their in vitro effects on cancer formation. A total of 83 strains were isolated from the stool samples and identified using MALDI-Biotyper. Fourteen of the isolates were identified as Candida spp., three isolates were identified as Cryptococcus neoformans, 55 isolates were identified as lactic acid bacteria, and the remaining isolates belonged to different 11 bacterial genera. Important microbial properties for cancer prevention and some probiotic properties were examined. All strains maintained their viability under acidic conditions and in media containing bile salts. Of the bacterial strains, 62.5% were resistant to ampicillin, chloramphenicol, gentamicin, erythromycin, kanamycin, penicillin, streptomycin, tetracycline, and vancomycin. All yeast strains were resistant to ketoconazole and susceptible to nystatin. The susceptibility of the strains to fluconazole, voriconazole, amphotericin B, and itraconazole varied. Fifty-nine percent of the strains produced EPS and 21.7% showed proteolytic activity (PA). Of the strains, 15.7% both produced exopolysaccharides (EPS) and had PA. The antioxidant activity (AOA) varied depending on the strains. The pathobiont and pathogenic microorganisms promoted tumor formation, while potential probiotic microorganisms had a suppressive effect on tumor formation (P > 0.01). One yeast (Candida kefyr MK17) and three lactic acid bacteria strains (Lacticaseibacillus paracasei MK73, Lactiplantibacillus plantarum MK55, Limosilactobacillus mucosae MK45) have superior potential thanks to their anticarcinogenic properties as well as tolerance to gastrointestinal tract conditions. Stool samples of each individual contain various potential probiotic, pathobiont, and pathogenic microorganisms.


Assuntos
Neoplasias Colorretais , Lactobacillales , Probióticos , Linhagem Celular , Humanos , Testes de Sensibilidade Microbiana , Probióticos/farmacologia , Saccharomyces cerevisiae
11.
Molecules ; 27(11)2022 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-35684552

RESUMO

Natural cytokinines are a promising group of cytoprotective and anti-tumor agents. In this research, we synthesized a set of aryl carbamate, pyridyl urea, and aryl urea cytokinine analogs with alkyl and chlorine substitutions and tested their antiproliferative activity in MDA-MB-231, A-375, and U-87 MG cell lines, and cytoprotective properties in H2O2 and CoCl2 models. Aryl carbamates with the oxamate moiety were selectively anti-proliferative for the cancer cell lines tested, while the aryl ureas were inactive. In the cytoprotection studies, the same aryl carbamates were able to counteract the CoCl2 cytotoxicity by 3-8%. The possible molecular targets of the aryl carbamates during the anti-proliferative action were the adenosine A2 receptor and CDK2. The obtained results are promising for the development of novel anti-cancer therapeutics.


Assuntos
Carbamatos , Ureia , Carbamatos/farmacologia , Linhagem Celular , Cloro/química , Halogênios/química , Peróxido de Hidrogênio/química , Relação Estrutura-Atividade , Ureia/farmacologia
12.
Methods Mol Biol ; 2495: 49-66, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35696027

RESUMO

The piggyBac transposon system has been adapted to be a highly efficient genome engineering tool for transgenesis of eukaryotic cells and organisms. As with other methods of transgenesis, incorporation of an inducible promoter, such as a tetracycline-responsive element, enables inducible transgene expression. Here, we describe an efficient method of using the piggyBac system to create stably transfected mammalian cell lines, including inducible transgene expression. Gibson assembly is used to construct the required vectors as it enables multiple DNA fragments to be seamlessly assembled in a single isothermal reaction. We demonstrate an application of this approach to generate a stably transfected pluripotent stem cell line that can be induced to express a transcription factor transgene and rapidly differentiate into neurons in a single step.


Assuntos
Elementos de DNA Transponíveis , Vetores Genéticos , Animais , Linhagem Celular , Elementos de DNA Transponíveis/genética , Vetores Genéticos/genética , Mamíferos/genética , Neurônios , Transgenes
13.
Methods Mol Biol ; 2495: 91-97, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35696029

RESUMO

The ability of modifying the genome of multiple species, precisely and without or minimal off-targeted effects, have opened numerous opportunities for the biotechnology industry. In this chapter, we describe an easy to establish, robust, and practical pipeline that can be used to generate immortalized cell lines, from different tissues, to capture cell linage context and validate the tools required for genome editing and genetic modification. This pipeline serves as a reference for similar approaches for gene interrogation in other species.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Linhagem Celular , Genoma
14.
Methods Mol Biol ; 2495: 129-148, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35696032

RESUMO

The CRISPR-on system is a programmable, simple, and versatile gene activator that has proven to be efficient in cultured cells from several species and in bovine embryos. This technology allows for the precise and specific activation of single endogenous gene expression and also multiplexed gene expression in a simple fashion. Therefore, CRISPR-on has unique advantages over other activator systems and a wide adaptability for studies in basic and applied science, such as cell reprogramming and cell fate differentiation for regenerative medicine.In this chapter, we describe the materials and methods of the CRISPR-on system for activation of the endogenous SMARCA4 expression in bovine embryos.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Animais , Bovinos , Diferenciação Celular , Linhagem Celular , Reprogramação Celular/genética
15.
Molecules ; 27(12)2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35744786

RESUMO

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, having a poor prognosis and rapid metastases. TNBC is characterized by the absence of estrogen, progesterone, and human epidermal growth receptor-2 (HER2) expressions and has a five-year survival rate. Compared to other breast cancer subtypes, TNBC patients only respond to conventional chemotherapies, and even then, with limited success. Shortages of chemotherapeutic medication can lead to resistance, pressured index therapy, non-selectivity, and severe adverse effects. Finding targeted treatments for TNBC is difficult owing to the various features of cancer. Hence, identifying the most effective molecular targets in TNBC pathogenesis is essential for predicting response to targeted therapies and preventing TNBC cell metastases. Nowadays, natural compounds have gained attention as TNBC treatments, and have offered new strategies for solving drug resistance. Here, we report a systematic review using the database from Pubmed, Science Direct, MDPI, BioScince, Springer, and Nature for articles screening from 2003 to 2022. This review analyzes relevant signaling pathways and the prospect of utilizing natural compounds as a therapeutic agent to improve TNBC treatments in the future.


Assuntos
Neoplasias de Mama Triplo Negativas , Linhagem Celular , Humanos , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/metabolismo
16.
Viruses ; 14(6)2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35746595

RESUMO

Chikungunya virus (CHIKV) is a re-emerging arbovirus in the alphavirus genus. Upon infection, it can cause severe joint pain that can last years in some patients, significantly affecting their quality of life. Currently, there are no vaccines or anti-viral therapies available against CHIKV. Its spread to the Americas from the eastern continents has substantially increased the count of the infected by millions. Thus, there is an urgent need to identify therapeutic targets for CHIKV treatment. A potential point of intervention is the sphingosine-1-phosphate (S1P) pathway. Conversion of sphingosine to S1P is catalyzed by Sphingosine kinases (SKs), which we previously showed to be crucial pro-viral host factor during CHIKV infection. In this study, we screened inhibitors of SKs and identified a novel potent inhibitor of CHIKV infection-SLL3071511. We showed that the pre-treatment of cells with SLL3071511 in vitro effectively inhibited CHIKV infection with an EC50 value of 2.91 µM under both prophylactic and therapeutic modes, significantly decreasing the viral gene expression and release of viral particles. Our studies suggest that targeting SKs is a viable approach for controlling CHIKV replication.


Assuntos
Febre de Chikungunya , Vírus Chikungunya , Antivirais/uso terapêutico , Linhagem Celular , Vírus Chikungunya/genética , Humanos , Fosfotransferases (Aceptor do Grupo Álcool) , Inibidores de Proteínas Quinases/farmacologia , Qualidade de Vida , Esfingosina/metabolismo , Replicação Viral
17.
Viruses ; 14(6)2022 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-35746619

RESUMO

MicroRNAs (miRNAs) have been identified as a class of crucial regulators of virus-host crosstalk, modulating such processes as viral replication, antiviral immune response, viral latency, and pathogenesis. Pseudorabies virus (PRV), a model for the study of alphaherpesvirus biology, codes for 11 distinct miRNAs mapped to the ~4.6 kb intron of Large Latency Transcript (LLT). Recent studies have revealed the role of clusters consisting of nine and eleven miRNA genes in the replication and virulence of PRV. The function of separate miRNA species in regulating PRV biology has not been thoroughly investigated. To analyze the regulatory potential of three PRV miRNAs located in the frontal cluster of the LLT intron, we generated a research model based on the constitutive expression of viral miRNAs in swine testis cells (ST_LLT [1-3] cell line). Using a cell culture system providing a stable production of individual miRNAs at high levels, we demonstrated that the LLT [1-3] miRNA cluster significantly downregulated IE180, EP0, and gE at the early stages of PRV infection. It was further determined that LLT [1-3] miRNAs could regulate the infection process, leading to a slight distortion in transmission and proliferation ability. Collectively, our findings indicate the potential of LLT [1-3] miRNAs to retard the host responses by reducing viral antigenic load and suppressing the expansion of progeny viruses at the early stages of infection.


Assuntos
Herpesvirus Suídeo 1 , MicroRNAs , Animais , Linhagem Celular , MicroRNAs/genética , MicroRNAs/metabolismo , Latência Viral/genética , Replicação Viral
18.
Viruses ; 14(6)2022 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-35746641

RESUMO

We recently published an article about myelin oligodendrocyte glycoprotein-independent rubella infection of keratinocytes in vitro, in which first-trimester trophoblast cells were shown as rubella virus (RuV)-resistant. Given an incident rate as high as 90% of congenital rubella syndrome in the first eight weeks of pregnancy, the RuV infection of first-trimester trophoblasts is considered key to opening the gate to transplacental transmission mechanisms. Therefore, with this study, we aimed to verify the susceptibility/resistance of first-trimester trophoblast cell lines, HTR-8/SVneo and Swan.71, against RuV. Cells cultured on multi-well plates were challenged with a RuV clinical strain at a multiplicity of infection from 5 to 10 for 3 h. The infectivity was investigated by immunofluorescence (IF) assay and flow cytometry (FCM) analysis. Supernatants collected during the post-infection period were used to determine virus-progeny production. The scattered signaling of RuV infection of these cells was noted by IF assay, and the FCM analysis showed an average of 4-5% of gated cells infected with RuV. In addition, a small but significant production of virus progeny was also observed. In conclusion, by employing appropriate approaches, we determined the low infectivity of RuV in first-trimester trophoblast cell lines but not resistance as in our previous report.


Assuntos
Vírus da Rubéola , Rubéola (Sarampo Alemão) , Linhagem Celular , Feminino , Humanos , Gravidez , Primeiro Trimestre da Gravidez , Rubéola (Sarampo Alemão)/metabolismo , Trofoblastos/metabolismo
19.
Viruses ; 14(6)2022 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-35746674

RESUMO

Paraoxonase-1 (PON1), an esterase with specifically paraoxonase activity, has been proven to be involved in inflammation and infection. Porcine reproductive and respiratory syndrome virus (PRRSV) is still a major concern in pigs and causes severe economic losses to the swine industry worldwide. In this study, the role of PON1 was investigated in porcine alveolar macrophages (PAMs) during PRRSV infection. The results showed that PRRSV replication downregulated PON1, and the knockdown of PON1 significantly decreased PRRSV replication. Similarly, PON1 overexpression could enhance PRRSV replication. Interestingly, we observed that PON1 interacted with PRRSV nonstructural protein 9 (Nsp9), the RNA-dependent RNA polymerase, and the knockdown of PON1 lowered the RNA binding ability of Nsp9, suggesting that PON1 can facilitate Nsp9 function in viral replication. In addition, the knockdown of PON1 expression led to the amplification of type I interferon (IFN) genes and vice versa. In summary, our data demonstrate that PON1 facilitates PRRSV replication by interacting with Nsp9 and inhibiting the type I IFN signaling pathway. Hence, PON1 may be an additional component of the anti-PRRSV defenses.


Assuntos
Interferon Tipo I , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Animais , Arildialquilfosfatase/genética , Arildialquilfosfatase/metabolismo , Linhagem Celular , Interferon Tipo I/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Ligação Proteica , Suínos , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
20.
Viruses ; 14(6)2022 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-35746762

RESUMO

Canid herpesvirus 1 (CHV-1) infects polarized canine epithelia. Herein, we present our initial work characterizing CHV-1 infection of Madin-Darby canine kidney (MDCK) cells that were polarized on trans-wells. We previously showed that infection of these cells in non-polarized cultures stimulated the formation of extensive lamellipodial membrane protrusions. Uninfected polarized MDCK cells already form extensive lamellipodial membrane protrusions on the apical surface in the absence of virus. Using scanning electron microscopy, we found that CHV-1 infection does not lead to a change in the form of the lamellipodial membrane protrusions on the apical surface of polarized MDCK cells. We found that CHV-1 was able to infect polarized cultures from either the apical or basolateral side; however, higher viral titers were produced upon infection of the basolateral side. Regardless of the side infected, titers of virus were higher in the apical compartment compared to the basal compartment; however, these differences were not statistically significant. In addition to cell-free virus that was recovered in the media, the highest amount of virus produced remained cell-associated over the course of the experiment. The efficiency of CHV-1 infection of the basolateral side of polarized epithelial cells is consistent with the pathobiology of this varicellovirus.


Assuntos
Herpesvirus Canídeo 1 , Animais , Linhagem Celular , Membrana Celular , Cães , Epitélio , Rim , Células Madin Darby de Rim Canino
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