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1.
Nat Commun ; 11(1): 5417, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33110091

RESUMO

De novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. While the paternal genome undergoes widespread DNAme loss before the first S-phase following fertilization, recent mass spectrometry analysis revealed that the zygotic paternal genome is paradoxically also subject to a low level of de novo DNAme. However, the loci involved, and impact on transcription were not addressed. Here, we employ allele-specific analysis of whole-genome bisulphite sequencing data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome by the 2-cell stage. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with paternal DNAme acquisition, many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Paternal DNAme acquisition is lost following maternal deletion of Dnmt3a, with a subset of promoters, which are normally transcribed from the paternal allele in blastocysts, being prematurely transcribed at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover a role for maternal DNMT3A activity in post-fertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.


Assuntos
Blastocisto/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Genoma , Herança Materna , Herança Paterna , Alelos , Animais , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Epigenômica , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos Endogâmicos DBA , Oócitos/metabolismo , Espermatozoides/metabolismo
2.
Med Mycol J ; 61(3): 33-48, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32863327

RESUMO

Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances (CADS) such as the hot water extract of C. albicans and Candida water-soluble fractions (CAWS) induce coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the mannoprotein fractions (MN fractions) of clinically isolated Candida species induce vasculitis in mice. We prepared MN fractions from 26 strains of Candida species by conventional hot water extraction and compared vasculitis in DBA/2 mice. The results obtained revealed that the induction of vasculitis and resulting heart failure were significantly dependent on the species; namely, death rates on day 200 were as follows: Candida krusei (100%), Candida albicans (84%), Candida dubliniensis (47%), Candida parapsilosis (44%), Candida glabrata (32%), Candida guilliermondii (20%), and Candida tropicalis (20%). Even for C. albicans, some strains did not induce vasculitis. The present results suggest that MN-induced vasculitis is strongly dependent on the species and strains of Candida, and also that the MN fractions of some non-albicans Candida induce similar toxicity to those of C. albicans.


Assuntos
Candida albicans/química , Candida albicans/patogenicidade , Candidíase , Vasos Coronários/microbiologia , Proteínas Fúngicas/efeitos adversos , Vasculite/microbiologia , Animais , Candida albicans/classificação , Fracionamento Celular , Proteínas Fúngicas/isolamento & purificação , Camundongos Endogâmicos DBA , Especificidade da Espécie
3.
PLoS One ; 15(9): e0233072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32911495

RESUMO

In mammals, the fusion of two gametes, an oocyte and a spermatozoon, during fertilization forms a totipotent zygote. There has been no reported case of adult mammal development by natural parthenogenesis, in which embryos develop from unfertilized oocytes. The genome and epigenetic information of haploid gametes are crucial for mammalian development. Haploid embryonic stem cells (haESCs) can be established from uniparental blastocysts and possess only one set of chromosomes. Previous studies have shown that sperm or oocyte genome can be replaced by haESCs with or without manipulation of genomic imprinting for generation of mice. Recently, these remarkable semi-cloning methods have been applied for screening of key factors of mouse embryonic development. While haESCs have been applied as substitutes of gametic genomes, the fundamental mechanism how haESCs contribute to the genome of totipotent embryos is unclear. Here, we show the generation of fertile semi-cloned mice by injection of parthenogenetic haESCs (phaESCs) into oocytes after deletion of two differentially methylated regions (DMRs), the IG-DMR and H19-DMR. For characterizing the genome of semi-cloned embryos further, we establish ESC lines from semi-cloned blastocysts. We report that polyploid karyotypes are observed in semi-cloned ESCs (scESCs). Our results confirm that mitotically arrested phaESCs yield semi-cloned embryos and mice when the IG-DMR and H19-DMR are deleted. In addition, we highlight the occurrence of polyploidy that needs to be considered for further improving the development of semi-cloned embryos derived by haESC injection.


Assuntos
Clonagem de Organismos/métodos , Desenvolvimento Embrionário , Haploidia , Partenogênese , Poliploidia , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA
4.
Adv Clin Exp Med ; 29(8): 929-936, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32820872

RESUMO

BACKGROUND: Recurrent spontaneous abortion (RSA), presenting as one of the difficult clinical diseases, has a high incidence rate among women of reproductive age, with a rising trend in recent years. OBJECTIVES: To confirm a target relationship between miR-24 and CDX1. This study aimed to explore miR-24 expression in decidual tissue under recurrent spontaneous abortion (RSA) and its mechanism of regulating downstream gene CDX1. MATERIAL AND METHODS: Female CBA/J mice were mated with male BALB/C mice to establish normal pregnancy models, and mated with male DBA/2 mice to establish RSA models. Recurrent spontaneous abortion model mice were randomized into 5 groups: a model group, a NC group, a miR-24 mimic group, a CDX1 vector group, and a miR-24 mimic+CDX1 vector group. Expressions of miR-24, CDX1, VEGF, cleaved caspase-3, Fas, and FasL, as well as apoptosis in decidual tissues, embryonic development and embryo loss rate were compared. RESULTS: Compared with the normal group, the embryo loss rate, apoptosis rate, and the expressions of cleaved caspase-3, Fas and CDX1 in decidual tissue in other groups were significantly increased, and the expressions of miR-24, VEGF, and FasL were significantly decreased (all p < 0.05). The miR-24 mimic group showed the opposite changes when compared with the model group (all p < 0.05). However, CDX1 overexpression can significantly block the protective effect of miR-24 overexpression on embryonic development (p < 0.05). CONCLUSIONS: MiR-24 can inhibit CDX1 expression in decidual tissue of RSA mice, thus improving the embryonic development of the mice and reducing the RSA risk.


Assuntos
Aborto Habitual , Animais , Feminino , Proteínas de Homeodomínio , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , MicroRNAs , Gravidez , Fatores de Risco
5.
Nat Commun ; 11(1): 4289, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855397

RESUMO

Older organs represent an untapped potential to close the gap between demand and supply in organ transplantation but are associated with age-specific responses to injury and increased immunogenicity, thereby aggravating transplant outcomes. Here we show that cell-free mitochondrial DNA (cf-mt-DNA) released by senescent cells accumulates with aging and augments immunogenicity. Ischemia reperfusion injury induces a systemic increase of cf-mt-DNA that promotes dendritic cell-mediated, age-specific inflammatory responses. Comparable events are observed clinically, with the levels of cf-mt-DNA elevated in older deceased organ donors, and with the isolated cf-mt-DNA capable of activating human dendritic cells. In experimental models, treatment of old donor animals with senolytics clear senescent cells and diminish cf-mt-DNA release, thereby dampening age-specific immune responses and prolonging the survival of old cardiac allografts comparable to young donor organs. Collectively, we identify accumulating cf-mt-DNA as a key factor in inflamm-aging and present senolytics as a potential approach to improve transplant outcomes and availability.


Assuntos
DNA Mitocondrial/efeitos adversos , Dasatinibe/farmacologia , Inflamação/prevenção & controle , Transplante de Órgãos/métodos , Quercetina/farmacologia , Adulto , Envelhecimento/fisiologia , Animais , Diferenciação Celular , Ácidos Nucleicos Livres , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Citocinas/metabolismo , DNA Mitocondrial/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Transplante de Coração/efeitos adversos , Transplante de Coração/métodos , Humanos , Inflamação/etiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Transplante de Órgãos/efeitos adversos , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/imunologia , Doadores de Tecidos
6.
Gene ; 758: 144975, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32707302

RESUMO

Dip2C is highly expressed in brain and many other tissues but its biological functions are still not clear. Genes regulated by Dip2C in brain have never been studied. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, adaptive immune systems of bacteria and archaea, have been recently developed and broadly used in genome editing. Here, we describe targeted gene deletions of Dip2c gene in mice via CRISPR/Cas9 system and study of brain transcriptome under Dip2C regulation. The CRISPR/Cas9 system effectively generated targeted deletions of Dip2c by pronuclei injection of plasmids that express Cas9 protein and two sgRNAs. We achieved targeted large fragment deletion with efficiencies at 14.3% (1/7), 66.7% (2/3) and 20% (1/5) respectively in 3 independent experiments, averaging 26.7%. The large deletion DNA segments are 160.4 kb (Dip2CΔ160kb), spanning from end of exon 4 to mid of exon 38. A mouse with two base pair deletion was generated from a single sgRNA targeting in exon 4 (Dip2cΔ2bp) by non-homologous end joining (NHEJ). Loss of gene expression for Dip2c mRNA was confirmed by quantitative real-time PCR (qPCR). Dip2C-regulated genes and pathways in brain were investigated through RNAseq of Dip2cΔ2bp. In total, 838 genes were found differentially regulated, with 252 up and 586 down. Gene ontology (GO) analysis indicated that DEGs in brain are enriched in neurological functions including 'memory', 'neuropeptide signaling pathway', and 'response to amphetamine' while KEGG analysis shows that 'neuroactive ligand-receptor interaction pathway' is the most significantly enriched. DEGs Grid2ip, Grin2a, Grin2c, Grm4, Gabbr2, Gabra5, Gabre, Gabrq, Gabra6 and Gabrr2 are among the highly regulated genes by Dip2C. Results confirm Dip2C may play important roles in brain development and function.


Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Neoplasias/genética , Transcriptoma/genética , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Feminino , Deleção de Genes , Edição de Genes/métodos , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , RNA Guia/genética
7.
Toxicol Lett ; 332: 130-139, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32645461

RESUMO

Cadmium (Cd) is an environmental contaminant that triggers toxic effects in various tissues such as the kidney, liver, and lung. Cd can also cause abnormal iron metabolism, leading to anemia. Iron homeostasis is regulated by intestinal absorption. However, whether Cd affects the iron absorption pathway is unclear. We aimed to elucidate the relationship between the intestinal iron transporter system and Cd-induced iron deficiency anemia. C57BL/6J female and male mice, 129/Sv female mice, and DBA/2 female mice were given a single oral dose of CdCl2 by gavage. After 3 or 24 h, Cd decreased serum iron concentrations and inhibited the expression of iron transport-related genes in the duodenum. In particular, Cd decreased the levels of divalent metal transporter 1 and ferroportin 1 in the duodenum. In addition, human colon carcinoma Caco-2 cells were treated with CdCl2. After 72 h, Cd decreased the expression of iron transport-related factors in Caco-2 cells with a pattern similar to that seen in the murine duodenum. These findings suggest that Cd inhibits iron absorption through direct suppression of iron transport in duodenal enterocytes and contributes to abnormal iron metabolism.


Assuntos
Anemia Ferropriva/induzido quimicamente , Cádmio/toxicidade , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Ferro/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Células CACO-2 , Cádmio/farmacocinética , Cloreto de Cádmio/toxicidade , Proteínas de Transporte de Cátions/metabolismo , Feminino , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA
8.
Mol Cell ; 79(4): 689-701.e10, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32610038

RESUMO

Meiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo, with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Recombinação Homóloga , Meiose , Proteínas de Ligação a Fosfato/metabolismo , Rad51 Recombinase/metabolismo , Proteína de Replicação A/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Cromossomos/genética , Cromossomos/metabolismo , Troca Genética , DNA de Cadeia Simples/metabolismo , Genoma , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas de Ligação a Fosfato/genética , Rad51 Recombinase/genética , Proteína de Replicação A/genética , Testículo
9.
Emerg Top Life Sci ; 4(2): 207-227, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32691841

RESUMO

Human pregnancy, critical for our species survival, is inefficient and prone to complications such as infertility, spontaneous miscarriages and preeclampsia (PE). Immunological factors may be important as the embryo is 50% paternal and foreign to the mother. Mouse pregnancy models, and in particular the murine CBA/J x DBA/2 mating combination, has been widely used to investigate mechanisms causing and preventing partner-specific recurrent miscarriages (RM) and PE. Occult losses can represent T cell-mediated rejection, and antigen-specific regulatory T cells (Tregs) with classical αß T cell receptors (TcR) activated by semen antigens at the time of mating are protective. If there is no occult loss, an inadequate Treg response can also predispose to RM. In RM, proinflammatory cytokines from natural killer (NK)-type cells and macrophages of the innate immune system are responsible and cells with γδ TcR protect via release of TGF-ß-type molecules. Immunization of abortion-prone female CBA/J mice or administration of cell-associated or soluble CD200, an immune check point inhibitor, can prevent abortions by augmenting uterine decidual suppressor cell activity. Human studies suggest that is also true in couples with RM. Environmental activators of the innate immune system, such as bacterial LPS and stress, can cause abortions as well as occult losses. The endogenous level of Tregs and activation of Tregs specific for the male H-Y antigen may determine success rates and alter the male:female birth ratio. Intralipid alters LPS clearance, prevents abortions in the CBAxDBA/2 model, and is effective in increasing live birth rates in couples undergoing IVF treatment.


Assuntos
Aborto Habitual/imunologia , Modelos Animais de Doenças , Eclampsia/imunologia , Infertilidade/imunologia , Aborto Habitual/prevenção & controle , Aborto Habitual/terapia , Animais , Antígenos CD/metabolismo , Citocinas/metabolismo , Eclampsia/prevenção & controle , Eclampsia/terapia , Implantação do Embrião , Feminino , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Humanos , Infertilidade/prevenção & controle , Infertilidade/terapia , Células Matadoras Naturais/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Gravidez , Complicações na Gravidez , Resultado da Gravidez , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Razão de Masculinidade , Linfócitos T Reguladores/metabolismo
10.
Cell ; 182(3): 744-753.e4, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32553273

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of human infections. One limitation to the evaluation of potential therapies and vaccines to inhibit SARS-CoV-2 infection and ameliorate disease is the lack of susceptible small animals in large numbers. Commercially available laboratory strains of mice are not readily infected by SARS-CoV-2 because of species-specific differences in their angiotensin-converting enzyme 2 (ACE2) receptors. Here, we transduced replication-defective adenoviruses encoding human ACE2 via intranasal administration into BALB/c mice and established receptor expression in lung tissues. hACE2-transduced mice were productively infected with SARS-CoV-2, and this resulted in high viral titers in the lung, lung pathology, and weight loss. Passive transfer of a neutralizing monoclonal antibody reduced viral burden in the lung and mitigated inflammation and weight loss. The development of an accessible mouse model of SARS-CoV-2 infection and pathogenesis will expedite the testing and deployment of therapeutics and vaccines.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Betacoronavirus/imunologia , Infecções por Coronavirus/terapia , Modelos Animais de Doenças , Pneumonia Viral/terapia , Animais , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Feminino , Células HEK293 , Humanos , Imunização Passiva/métodos , Pulmão/metabolismo , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Transdução Genética , Células Vero , Carga Viral/imunologia
11.
Int J Exp Pathol ; 101(1-2): 55-64, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32459025

RESUMO

Rheumatoid arthritis is a disabling autoimmune disease with a high global prevalence. Treatment with disease-modifying anti-arthritic drugs (DIMARDs) has been routinely used with beneficial effects but with adverse long-term consequences; novel targeted biologics and small-molecule inhibitors are promising options. In this study, we investigated whether purified omega unsaturated fatty acids (ω-UFAs) and dialysable leukocyte extracts (DLEs) prevented the development of arthritis in a model of collagen-induced arthritis (CIA) in mice. We also investigated whether the transcription factor NF-κB and the NLRP3 inflammasome were involved in the process and whether their activity was modulated by treatment. The development of arthritis was evaluated for 84 days following treatment with nothing, dexamethasone, DLEs, docosahexaenoic acid, arachidonic acid, and oleic acid. Progression of CIA was monitored by evaluating clinical manifestations, inflammatory changes, and histological alterations in the pads' articular tissues. Both DLEs and ω-UFAs led to an almost complete inhibition of the inflammatory histopathology of CIA and this was concomitant with the inhibition of NF-kB and the inhibition of the activation of NLRP3. These data suggest that ω-UFAs and DLEs might have NF-κB as a common target and that they might be used as ancillary medicines in the treatment of arthritis.


Assuntos
Anti-Inflamatórios/farmacologia , Antirreumáticos/farmacologia , Artrite Experimental/prevenção & controle , Cartilagem Articular/efeitos dos fármacos , Extratos Celulares/farmacologia , Ácidos Graxos Insaturados/farmacologia , Leucócitos , Animais , Ácido Araquidônico/farmacologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Colágeno Tipo II , Diálise , Ácidos Docosa-Hexaenoicos/farmacologia , Feminino , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ácido Oleico/farmacologia
12.
Arthritis Rheumatol ; 72(6): 943-956, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32362074

RESUMO

OBJECTIVE: This study was undertaken to uncover the pathophysiologic role of discoidin domain receptor 2 (DDR-2), a putative fibrillar collagen receptor, in inflammation promotion and joint destruction in rheumatoid arthritis (RA). METHODS: In synovial tissue from patients with RA and from mice with collagen antibody-induced arthritis (CAIA) (using Ddr2-/- and DBA/1 mice), gene and protein expression levels of DDR-2, interleukin-15 (IL-15), and Dkk-1 were measured by quantitative reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. Gene knockdown of DDR2 in human RA fibroblast-like synoviocytes (FLS) was conducted via small interfering RNA. Interaction between the long noncoding RNA H19 and microRNA 103a (miR-103a) was assessed in RA FLS using RNA pulldown assays. Cellular localization of H19 was examined using fluorescence in situ hybridization assays. Chromatin immunoprecipitation and dual luciferase reporter assays were applied to verify H19 transcriptional and posttranscriptional regulation by miR-103a. RESULTS: DDR2 messenger RNA (mRNA) expression was significantly associated with the levels of IL-15 and Dkk-1 mRNA in the synovial tissue of RA patients (r2 = 0.2022-0.3293, all P < 0.05; n = 33) and with the serum levels of IL-15 and Dkk-1 in mice with CAIA (P < 0.05). In human RA FLS, activated DDR-2 induced the expression of H19 through c-Myc. Moreover, H19 directly interacted with and promoted the degradation of miR-103a. CONCLUSION: These results indicate a novel role for activated DDR-2 in RA FLS, showing that DDR-2 is responsible for regulating the expression of IL-15 and Dkk-1 in RA FLS and is involved in the promotion of inflammation and joint destruction during pathophysiologic development of RA. Moreover, DDR-2 inhibition, acting through the H19-miR-103a axis, leads to reductions in the inflammatory reaction and severity of joint destruction in mice with CAIA, suggesting that inhibition of DDR-2 may be a potential therapeutic strategy for RA.


Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/genética , Receptor com Domínio Discoidina 2/metabolismo , Interleucina-15/metabolismo , Transdução de Sinais/genética , Animais , Regulação da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Inflamação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos DBA , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Membrana Sinovial/metabolismo , Sinoviócitos/metabolismo
13.
Mol Cell ; 78(6): 1252-1263.e3, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32362315

RESUMO

Crossover recombination is critical for meiotic chromosome segregation, but how mammalian crossing over is accomplished is poorly understood. Here, we illuminate how strands exchange during meiotic recombination in male mice by analyzing patterns of heteroduplex DNA in recombinant molecules preserved by the mismatch correction deficiency of Msh2-/- mutants. Surprisingly, MSH2-dependent recombination suppression was not evident. However, a substantial fraction of crossover products retained heteroduplex DNA, and some provided evidence of MSH2-independent correction. Biased crossover resolution was observed, consistent with asymmetry between DNA ends in earlier intermediates. Many crossover products yielded no heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these simple modifications of the original double-strand break repair model-asymmetry in recombination intermediates and D-loop migration-may be sufficient to explain most meiotic crossing over in mice while also addressing long-standing questions related to Holliday junction resolution.


Assuntos
Troca Genética/fisiologia , Recombinação Homóloga/fisiologia , Meiose/fisiologia , Animais , Segregação de Cromossomos/genética , Troca Genética/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Recombinação Homóloga/genética , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos DBA , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Ácidos Nucleicos Heteroduplexes/genética
14.
PLoS One ; 15(4): e0231108, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251418

RESUMO

Clinical applications of oocytes cryopreservation include preservation of future fertility of young cancer patients, substitution of embryo freezing to avoid associated legal and ethical issues, and delaying childbearing years. While the outcome of oocyte cryopreservation has recently been improved, currently used vitrification method still suffer from increased biosafety risk and handling issues while slow freezing techniques yield overall low success. Understanding better the mechanism of cryopreservation-induced injuries may lead to development of more reliable and safe methods for oocyte cryopreservation. Using the mouse model, a microarray study was conducted on oocyte cryopreservation to identify cryoinjuries to transcriptionally active genome. To this end, metaphase II (MII) oocytes were subjected to standard slow freezing, and then analyzed at the four-cell stage after embryonic genome activation. Non-frozen four-cell embryos served as controls. Differentially expressed genes were identified and validated using RT-PCR. Embryos produced from the cryopreserved oocytes displayed 200 upregulated and 105 downregulated genes, associated with the regulation of mitochondrial function, protein ubiquitination and maintenance, cellular response to stress and oxidative states, fatty acid and lipid regulation/metabolism, and cell cycle maintenance. These findings reveal previously unrecognized effects of standard slow oocyte freezing on embryonic gene expression, which can be used to guide improvement of oocyte cryopreservation methods.


Assuntos
Criopreservação/normas , Embrião de Mamíferos/fisiologia , Congelamento/efeitos adversos , Oócitos/fisiologia , Transcriptoma/genética , Animais , Desenvolvimento Embrionário/genética , Feminino , Fertilização In Vitro/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Metáfase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Mapas de Interação de Proteínas/genética , Reação em Cadeia da Polimerase em Tempo Real
15.
PLoS Pathog ; 16(4): e1008409, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32287326

RESUMO

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned.


Assuntos
Doenças Transmissíveis Emergentes/veterinária , Doenças do Cão/virologia , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Zoonoses/virologia , Animais , Doenças Transmissíveis Emergentes/transmissão , Doenças Transmissíveis Emergentes/virologia , Doenças do Cão/transmissão , Cães , Furões , Cobaias , Humanos , Vírus da Influenza A Subtipo H3N2/classificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N8/classificação , Vírus da Influenza A Subtipo H3N8/genética , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Influenza Humana/transmissão , Influenza Humana/virologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Estados Unidos , Zoonoses/transmissão
16.
PLoS One ; 15(3): e0229445, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160239

RESUMO

The Wnt/ß-catenin signaling pathway has been implicated in human proliferative diseases such as cancer and fibrosis. The functions of ß-catenin and several other components of this pathway have been investigated in fibrosis. However, the potential role of R-spondin proteins (RSPOs), enhancers of the Wnt/ß-catenin signaling, has not been described. A specific interventional strategy targeting this pathway for fibrosis remains to be defined. We developed monoclonal antibodies against members of the RSPO family (RSPO1, 2, and 3) and probed their potential function in fibrosis in vivo. We demonstrated that RSPO3 plays a critical role in the development of fibrosis in multiple organs. Specifically, an anti-RSPO3 antibody, OMP-131R10, when dosed therapeutically, attenuated fibrosis in carbon tetrachloride (CCl4)-induced liver fibrosis, bleomycin-induced pulmonary and skin fibrosis models. Mechanistically, we showed that RSPO3 induces multiple pro-fibrotic chemokines and cytokines in Kupffer cells and hepatocytes. We found that the anti-fibrotic activity of OMP-131R10 is associated with its inhibition of ß-catenin activation in vivo. Finally, RSPO3 was found to be highly elevated in the active lesions of fibrotic tissues in mouse models of fibrosis and in patients with idiopathic pulmonary fibrosis (IPF) and nonalcoholic steatohepatitis (NASH). Together these data provide an anti-fibrotic strategy for targeting the Wnt/ß-catenin pathway through RSPO3 blockade and support that OMP-131R10 could be an important therapeutic agent for fibrosis.


Assuntos
Anticorpos/uso terapêutico , Fibrose Pulmonar Idiopática , Hepatopatia Gordurosa não Alcoólica , Trombospondinas/fisiologia , Animais , Células Cultivadas , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos
17.
Phytomedicine ; 69: 153195, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32200293

RESUMO

BACKGROUND: Areca nut has anti-inflammatory, antiparasitic, antihypertensive, and antidepressant properties. The pathological hallmarks of inflammatory joint diseases are an increased number of osteoclasts and impaired differentiation of osteoblasts, which may disrupt the bone remodeling balance and eventually lead to bone loss. PURPOSE: The present study assessed the effects of arecoline, the main alkaloid found in areca nut, on osteoclast and osteoblast differentiation and function. METHOD: M-CSF/RANKL-stimulated murine bone marrow-derived macrophages (BMMs) were incubated with several concentrations of arecoline, and TRAP staining and pit formation were assessed to monitor osteoclast formation. Quantitative real-time RT-PCR and western blot analyses were used to analyze the expression of osteoclast-associated genes and signaling pathways. The effects of arecoline on bone were investigated in an in vivo mouse model of lipopolysaccharide (LPS)-induced trabecular bone loss after oral administration of arecoline. Alizarin red S staining and assays to measure ALP activity and the transcription level of osteoblast-related genes were used to evaluate the effects of arecoline on osteoblast differentiation and bone mineralization. RESULTS: In a dose-dependent manner, arecoline at concentrations of 50-100 µM reduced both the development of TRAP-positive multinucleated osteoclasts and the formation of resorption pits in M-CSF/RANKL-stimulated BMMs. In M-CSF/RANKL-stimulated BMMs, arecoline also suppressed the expression and translocation of c-Fos and NFATcl, and osteoclast differentiated-related genes via interference with the AKT, MAPK, and NF-kB activation pathways. Femur bone loss and microcomputed tomography parameters were recovered by oral administration of arecoline in the mouse LPS-induced bone loss model. Lastly, arecoline increased ALP activity, bone mineralization, and the expression of osteoblast differentiation-related genes, such as ALP and Runx2, in MC3T3-E1 cells. CONCLUSION: Our data suggest that arecoline may attenuate or prevent bone loss by suppressing osteoclastogenesis and promoting osteoblastogenesis. These findings provide evidence supporting arecoline's use as a potential therapeutic agent in bone-loss disorders and diseases.


Assuntos
Arecolina/farmacologia , Reabsorção Óssea/tratamento farmacológico , Osteoclastos/efeitos dos fármacos , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos DBA , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteoclastos/citologia , Osteoclastos/fisiologia , Osteogênese/efeitos dos fármacos , Osteoporose/induzido quimicamente , Osteoporose/tratamento farmacológico , Ligante RANK/metabolismo , Ligante RANK/farmacologia , Microtomografia por Raio-X
18.
Exp Anim ; 69(3): 287-294, 2020 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-32051391

RESUMO

The pronuclear injection (PI)-based targeted transgenesis (PITT) method allows the generation of targeted transgenic (Tg) mice wherein a single copy of a transgene is integrated into the Rosa26 locus following PI. The Rosa26 locus allows unbiased ubiquitous expression of integrated transgenes; however, it remains little known whether tissue-specific promoters retain their functional properties when placed at the Rosa26 locus. We evaluated tissue-specific activity and reproducibility of exogenous tissue-specific promoters targeted to the Rosa26 locus by generating Thy1-Dre/Dre reporter mice using PITT and assessed spatial expression patterns of the transgenes. The Thy1 promoter targeted to the Rosa26 locus appeared active in virtually all Purkinje cells in the cerebellum and hippocampus. However, mosaic expression of the transgene under the Thy1 promoter was observed in many other organs. This phenomenon was consistent in all the Tg lines generated by PITT, indicating a high degree of reproducibility for this experiment.


Assuntos
Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , RNA não Traduzido/genética , Animais , Encéfalo/metabolismo , Genótipo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , RNA não Traduzido/metabolismo , Transgenes/genética
19.
Cell Immunol ; 349: 104049, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32057353

RESUMO

Pathogenic microorganisms utilize multiple approaches to break down host immunity in favor of their invasion, of which, cystatin C is one of the soluble factors secreted by parasites reported to affect host immunity in vivo. The cellular targets and mechanisms of action in vivo of cystatin C, however, are far from clear. As professional antigen-presenting cells, dendritic cells (DCs) are first immune cells that contact foreign pathogenic agents or their products to initiate immune responses. We previously reported that cystatin C can regulate the functions of DCs in terms of suppressed CD4+ T cell activation but enhanced Th1/Th17 differentiation via different mechanisms. Here, we further verified these regulatory effects of cystatin C on DCs in vivo. We found that the suppressive role of DC-mediated CD4+ T cell proliferation by cystatin C was partly cell-contact independent and extended to CD8+ T cells in vivo. Although cystatin C-overexpressing DCs trafficked equally as their mock-transduced counterparts, their adoptive transfer suppressed CD8+ T cell immunity and resulted in compromised tumor rejection in both vaccination and treatment regimes. Compared with their role in promoting Th17 differentiation in vivo, cystatin C-transduced DCs had far greater ability to induce T regulatory cells (Tregs), leading to collectively a higher Treg/Th17 ratio in an adoptively transferred disease model, and thus relieved Th17-dependent autoimmunity. Collectively, these data demonstrated strong in vivo evidences for immune regulatory roles of cystatin C in DCs and provided theoretical basis for the application of cystatin C-transduced cell therapy in the treatment or remission of certain autoimmune diseases. (246).


Assuntos
Transferência Adotiva , Artrite Experimental/terapia , Doenças Autoimunes/terapia , Cistatina C/fisiologia , Células Dendríticas/imunologia , Evasão Tumoral/imunologia , Transferência Adotiva/efeitos adversos , Animais , Comunicação Celular , Células Cultivadas , Cistatina C/genética , Células Dendríticas/transplante , Regulação para Baixo , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Granzimas/biossíntese , Granzimas/genética , Imunoterapia Adotiva , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Ativação Linfocitária , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Ovalbumina/imunologia , Proteínas Citotóxicas Formadoras de Poros/biossíntese , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Recombinantes/metabolismo , Organismos Livres de Patógenos Específicos , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Transdução Genética
20.
Immunohorizons ; 4(2): 93-107, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32086319

RESUMO

TLR7 and TLR8 are pattern recognition receptors that reside in the endosome and are activated by ssRNA molecules. TLR7 and TLR8 are normally part of the antiviral defense response, but they have also been implicated as drivers of autoimmune diseases such as lupus. The receptors have slightly different ligand-binding specificities and cellular expression patterns that suggest they have nonredundant specialized roles. How the roles of TLR7 and TLR8 differ may be determined by which cell types express each TLR and how the cells respond to activation of each receptor. To provide a better understanding of the effects of TLR7/8 activation, we have characterized changes induced by TLR-specific agonists in different human immune cell types and defined which responses are a direct consequence of TLR7 or TLR8 activation and which are secondary responses driven by type I IFN or cytokines produced subsequent to the primary response. Using cell sorting, gene expression analysis, and intracellular cytokine staining, we have found that the IFN regulatory factor (IRF) and NF-κB pathways are differentially activated downstream of the TLRs in various cell types. Studies with an anti-IFNAR Ab in human cells and lupus mice showed that inhibiting IFN activity can block secondary IFN-induced gene expression changes downstream of TLR7/8 activation, but not NF-κB-regulated genes induced directly by TLR7/8 activation at earlier timepoints. In summary, these results elucidate the different roles TLR7 and TLR8 play in immunity and inform strategies for potential treatment of autoimmune diseases driven by TLR7/8 activation.


Assuntos
Fatores Reguladores de Interferon/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , NF-kappa B/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Animais , Autoanticorpos/sangue , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação , Interferon-alfa/farmacologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/genética , Camundongos , Camundongos Endogâmicos DBA , Modelos Biológicos , Células Mieloides/classificação , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas
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