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1.
Adv Exp Med Biol ; 1131: 537-545, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646525

RESUMO

Calcium signaling plays an important role in gene expression. At the transcriptional level, this may underpin mammalian neuronal synaptic plasticity. Calcium influx into the postsynaptic neuron via: N-methyl-D-aspartate (NMDA) receptors activates small GTPase Rac1 and other Rac guanine nucleotide exchange factors, and stimulates calmodulin-dependent kinase kinase (CaMKK) and CaMKI; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors that are not impermeable to calcium ions, that is, those lacking the glutamate receptor-2 subunits, leads to activation of Ras guanine nucleotide-releasing factor proteins, which is coupled with activation of the mitogen-activated protein kinases/extracellular signal-regulated kinases signaling cascade; L-type voltage-gated calcium channels activates signaling pathways involving CaMKII, downstream responsive element antagonist modulator and distinct microdomains. Key members of these signaling cascades then translocate into the nucleus, where they alter the expression of genes involved in neuronal synaptic plasticity. At the post-transcriptional level, intracellular calcium level changes can change alternative splicing patterns; in the mammalian brain, alterations in calcium signaling via NMDA receptors is associated with exon silencing of the CI cassette of the NMDA R1 receptor (GRIN1) transcript by UAGG motifs in response to neuronal excitation. Regulation also occurs at the translational level; transglutaminase-2 (TG2) mediates calcium ion-regulated crosslinking of Y-box binding protein-1 (YB-1) translation-regulatory protein in TGFß1-activated myofibroblasts; YB-1 binds smooth muscle α-actin mRNA and regulates its translational activity. Calcium signaling is also important in epigenetic regulation, for example in respect of changes in cytosine bases. Targeting calcium signaling may provide therapeutically useful options, for example to induce epigenetic reactivation of tumor suppressor genes in cancer patients.


Assuntos
Sinalização do Cálcio , Regulação da Expressão Gênica , Animais , Cálcio/metabolismo , Epigênese Genética , Regulação da Expressão Gênica/fisiologia , Humanos , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo
2.
Nat Commun ; 10(1): 3042, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31316054

RESUMO

Stress resistance and longevity are positively correlated but emerging evidence indicates that they are physiologically distinct. Identifying factors with distinctive roles in these processes is challenging because pro-longevity genes often enhance stress resistance. We demonstrate that TCER-1, the Caenorhabditis elegans homolog of human transcription elongation and splicing factor, TCERG1, has opposite effects on lifespan and stress resistance. We previously showed that tcer-1 promotes longevity in germline-less C. elegans and reproductive fitness in wild-type animals. Surprisingly, tcer-1 mutants exhibit exceptional resistance against multiple stressors, including infection by human opportunistic pathogens, whereas, TCER-1 overexpression confers immuno-susceptibility. TCER-1 inhibits immunity only during fertile stages of life. Elevating its levels ameliorates the fertility loss caused by infection, suggesting that TCER-1 represses immunity to augment fecundity. TCER-1 acts through repression of PMK-1 as well as PMK-1-independent factors critical for innate immunity. Our data establish key roles for TCER-1 in coordinating immunity, longevity and fertility, and reveal mechanisms that distinguish length of life from functional aspects of aging.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica/fisiologia , Imunidade Inata/genética , Longevidade/genética , Fatores de Alongamento de Peptídeos/metabolismo , Estresse Fisiológico/imunologia , Envelhecimento/genética , Envelhecimento/imunologia , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/imunologia , Suscetibilidade a Doenças/imunologia , Fertilidade/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Modelos Animais , Mutação , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/imunologia , Estresse Fisiológico/genética
3.
J Dairy Sci ; 102(9): 7684-7696, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31255276

RESUMO

Oxidative stress is the basic reason for aging and age-related diseases. In this study, we investigated the protective effect of 2 strains of lactic acid bacteria (LAB), Lactobacillus rhamnosus GG and L. plantarum J26, against oxidative stress in Caco-2 cells, and gave an overview of the mechanisms of lactic acid bacteria antioxidant activity using digital gene expression profiling. The 2 LAB strains provided significant protection against hydrogen peroxide (H2O2)-induced reduction in superoxide dismutase activity and increase in glutathione peroxidase activity in Caco-2 cells. However, inactive bacteria had little effect on alleviating oxidation stress in Caco-2 cells. Eight genes related to oxidative stress-FOSB, TNF, PPP1R15A, NUAK2, ATF3, TNFAIP3, EGR2, and FBN2-were significantly upregulated in H2O2-induced Caco-2 cells compared with untreated Caco-2 cells. After incubation of the H2O2-induced Caco-2 cells with L. rhamnosus GG and L. plantarum J26, 5 genes (TNF, EGR2, NUAK2, FBN2, and TNFAIP3) and 2 genes (NUAK2 and FBN2) were downregulated, respectively. In addition, the Kyoto Encyclopedia of Genes and Genomes indicated that some signaling pathways associated with inflammation, immune response, and apoptosis, such as Janus kinase/signal transducers and activators of transcription (Jak-STAT), mitogen-activated protein kinase (MAPK), nuclear factor-κB, and tumor necrosis factor, were all negatively modulated by the 2 strains, especially L. rhamnosus GG. In this paper, we reveal the mechanism of LAB in relieving oxidative stress and provide a theoretical basis for the rapid screening and evaluation of new LAB resources.


Assuntos
Enterócitos/metabolismo , Lactobacillus plantarum/fisiologia , Lactobacillus rhamnosus/fisiologia , Estresse Oxidativo/genética , Transcriptoma/genética , Animais , Apoptose/genética , Células CACO-2 , Enterócitos/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Humanos , Peróxido de Hidrogênio/farmacologia , Imunidade/genética , Inflamação/genética , Probióticos/farmacologia
4.
Nat Commun ; 10(1): 2837, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253775

RESUMO

The diagnostic yield of exome and genome sequencing remains low (8-70%), due to incomplete knowledge on the genes that cause disease. To improve this, we use RNA-seq data from 31,499 samples to predict which genes cause specific disease phenotypes, and develop GeneNetwork Assisted Diagnostic Optimization (GADO). We show that this unbiased method, which does not rely upon specific knowledge on individual genes, is effective in both identifying previously unknown disease gene associations, and flagging genes that have previously been incorrectly implicated in disease. GADO can be run on www.genenetwork.nl by supplying HPO-terms and a list of genes that contain candidate variants. Finally, applying GADO to a cohort of 61 patients for whom exome-sequencing analysis had not resulted in a genetic diagnosis, yields likely causative genes for ten cases.


Assuntos
Regulação da Expressão Gênica/fisiologia , Predisposição Genética para Doença , Análise de Sequência de RNA/métodos , Transcriptoma , Bases de Dados de Ácidos Nucleicos , Humanos , Modelos Genéticos , Análise de Componente Principal , Software , Interface Usuário-Computador
5.
Nat Commun ; 10(1): 2854, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253781

RESUMO

SETD1A, a Set1/COMPASS family member maintaining histone-H3-lysine-4 (H3K4) methylation on transcriptionally active promoters, is overexpressed in breast cancer. Here, we show that SETD1A supports mitotic processes and consequentially, its knockdown induces senescence. SETD1A, through promoter H3K4 methylation, regulates several genes orchestrating mitosis and DNA-damage responses, and its depletion causes chromosome misalignment and segregation defects. Cell cycle arrest in SETD1A knockdown senescent cells is independent of mutations in p53, RB and p16, known senescence mediators; instead, it is sustained through transcriptional suppression of SKP2, which degrades p27 and p21. Rare cells escaping senescence by restoring SKP2 expression display genomic instability. In > 200 cancer cell lines and in primary circulating tumor cells, SETD1A expression correlates with genes promoting mitosis and cell cycle suggesting a broad role in suppressing senescence induced by aberrant mitosis. Thus, SETD1A is essential to maintain mitosis and proliferation and its suppression unleashes the tumor suppressive effects of senescence.


Assuntos
Senescência Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Mitose/fisiologia , Linhagem Celular Tumoral , Histona-Lisina N-Metiltransferase/genética , Histonas , Humanos , Metilação , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
6.
Nat Commun ; 10(1): 2851, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253786

RESUMO

Male and female brains differ significantly in both health and disease, and yet the female brain has been understudied. Sex-hormone fluctuations make the female brain particularly dynamic and are likely to confer female-specific risks for neuropsychiatric disorders. The molecular mechanisms underlying the dynamic nature of the female brain structure and function are unknown. Here we show that neuronal chromatin organization in the female ventral hippocampus of mouse fluctuates with the oestrous cycle. We find chromatin organizational changes associated with the transcriptional activity of genes important for neuronal function and behaviour. We link these chromatin dynamics to variation in anxiety-related behaviour and brain structure. Our findings implicate an immediate-early gene product, Egr1, as part of the mechanism mediating oestrous cycle-dependent chromatin and transcriptional changes. This study reveals extreme, sex-specific dynamism of the neuronal epigenome, and establishes a foundation for the development of sex-specific treatments for disorders such as anxiety and depression.


Assuntos
Encéfalo/fisiologia , Cromatina/fisiologia , Ciclo Estral/fisiologia , Neurônios/fisiologia , Animais , Comportamento Animal , Encéfalo/citologia , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Epigenômica , Estradiol/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Progesterona/metabolismo , Ligação Proteica , RNA/genética , RNA/metabolismo
7.
Nat Commun ; 10(1): 2865, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253771

RESUMO

The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, ß-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar ß-propeller proteins.


Assuntos
Chaperonina com TCP-1/fisiologia , Homólogo LST8 da Proteína Associada a MTOR/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Células Hep G2 , Humanos , Homólogo LST8 da Proteína Associada a MTOR/genética , Espectrometria de Massas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Proteína Regulatória Associada a mTOR/genética , Serina-Treonina Quinases TOR/genética
8.
Nat Commun ; 10(1): 2418, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160574

RESUMO

In transcriptional regulatory networks (TRNs), a canonical 3-node feed-forward loop (FFL) is hypothesized to evolve to filter out short spurious signals. We test this adaptive hypothesis against a novel null evolutionary model. Our mutational model captures the intrinsically high prevalence of weak affinity transcription factor binding sites. We also capture stochasticity and delays in gene expression that distort external signals and intrinsically generate noise. Functional FFLs evolve readily under selection for the hypothesized function but not in negative controls. Interestingly, a 4-node "diamond" motif also emerges as a short spurious signal filter. The diamond uses expression dynamics rather than path length to provide fast and slow pathways. When there is no idealized external spurious signal to filter out, but only internally generated noise, only the diamond and not the FFL evolves. While our results support the adaptive hypothesis, we also show that non-adaptive factors, including the intrinsic expression dynamics, matter.


Assuntos
Regulação da Expressão Gênica/fisiologia , Redes Reguladoras de Genes/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Adaptação Fisiológica , Retroalimentação Fisiológica , Modelos Genéticos , Modelos Teóricos , Saccharomyces cerevisiae
9.
Nat Commun ; 10(1): 2849, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253762

RESUMO

Fanconi anemia (FA) is a multigenic disease of bone marrow failure and cancer susceptibility stemming from a failure to remove DNA crosslinks and other chromosomal lesions. Within the FA DNA damage response pathway, DNA-dependent monoubiquitinaton of FANCD2 licenses downstream events, while timely FANCD2 deubiquitination serves to extinguish the response. Here, we show with reconstituted biochemical systems, which we developed, that efficient FANCD2 deubiquitination by the USP1-UAF1 complex is dependent on DNA and DNA binding by UAF1. Surprisingly, we find that the DNA binding activity of the UAF1-associated protein RAD51AP1 can substitute for that of UAF1 in FANCD2 deubiquitination in our biochemical system. We also reveal the importance of DNA binding by UAF1 and RAD51AP1 in FANCD2 deubiquitination in the cellular setting. Our results provide insights into a key step in the FA pathway and help define the multifaceted role of the USP1-UAF1-RAD51AP1 complex in DNA damage tolerance and genome repair.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Anemia de Fanconi/genética , Proteínas Nucleares/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Mutação , Proteínas Nucleares/genética , Ligação Proteica , Proteases Específicas de Ubiquitina/genética , Ubiquitinação
10.
Nat Commun ; 10(1): 2889, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253831

RESUMO

The sinus node is a collection of highly specialised cells constituting the heart's pacemaker. The molecular underpinnings of its pacemaking abilities are debated. Using high-resolution mass spectrometry, we here quantify >7,000 proteins from sinus node and neighbouring atrial muscle. Abundances of 575 proteins differ between the two tissues. By performing single-nucleus RNA sequencing of sinus node biopsies, we attribute measured protein abundances to specific cell types. The data reveal significant differences in ion channels responsible for the membrane clock, but not in Ca2+ clock proteins, suggesting that the membrane clock underpins pacemaking. Consistently, incorporation of ion channel expression differences into a biophysically-detailed atrial action potential model result in pacemaking and a sinus node-like action potential. Combining our quantitative proteomics data with computational modeling, we estimate ion channel copy numbers for sinus node myocytes. Our findings provide detailed insights into the unique molecular make-up of the cardiac pacemaker.


Assuntos
Relógios Biológicos/fisiologia , Peptídeos/química , Peptídeos/metabolismo , Proteômica , Nó Sinoatrial/metabolismo , Transcriptoma , Potenciais de Ação , Animais , Cromatografia Líquida , Regulação da Expressão Gênica/fisiologia , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/genética , Espectrometria de Massas em Tandem
11.
Nat Commun ; 10(1): 2568, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189923

RESUMO

Activation of the p16Ink4a-associated senescence pathway during aging breaks muscle homeostasis and causes degenerative muscle disease by irreversibly dampening satellite cell (SC) self-renewal capacity. Here, we report that the zinc-finger transcription factor Slug is highly expressed in quiescent SCs of mice and functions as a direct transcriptional repressor of p16Ink4a. Loss of Slug promotes derepression of p16Ink4a in SCs and accelerates the entry of SCs into a fully senescent state upon damage-induced stress. p16Ink4a depletion partially rescues defects in Slug-deficient SCs. Furthermore, reduced Slug expression is accompanied by p16Ink4a accumulation in aged SCs. Slug overexpression ameliorates aged muscle regeneration by enhancing SC self-renewal through active repression of p16Ink4a transcription. Our results identify a cell-autonomous mechanism underlying functional defects of SCs at advanced age. As p16Ink4a dysregulation is the chief cause for regenerative defects of human geriatric SCs, these findings highlight Slug as a potential therapeutic target for aging-associated degenerative muscle disease.


Assuntos
Autorrenovação Celular/genética , Senescência Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Células Satélites de Músculo Esquelético/fisiologia , Fatores de Transcrição da Família Snail/metabolismo , Envelhecimento/fisiologia , Animais , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Fatores de Transcrição da Família Snail/genética
12.
Nat Commun ; 10(1): 2771, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235807

RESUMO

Diverse antibody repertoires are generated through remote genomic interactions involving immunoglobulin variable (VH), diversity (DH) and joining (JH) gene segments. How such interactions are orchestrated remains unknown. Here we develop a strategy to track VH-DHJH motion in B-lymphocytes. We find that VH and DHJH segments are trapped in configurations that allow only local motion, such that spatially proximal segments remain in proximity, while spatially remote segments remain remote. Within a subset of cells, however, abrupt changes in VH-DHJH motion are observed, plausibly caused by temporal alterations in chromatin configurations. Comparison of experimental and simulated data suggests that constrained motion is imposed by a network of cross-linked chromatin chains characteristic of a gel phase, yet poised near the sol phase, a solution of independent chromatin chains. These results suggest that chromosome organization near the sol-gel phase transition dictates the timing of genomic interactions to orchestrate gene expression and somatic recombination.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Regulação da Expressão Gênica/fisiologia , Genes de Imunoglobulinas/genética , Recombinação V(D)J/fisiologia , Animais , Linfócitos B/metabolismo , Linhagem Celular , Cromossomos/genética , Proteínas de Ligação a DNA/deficiência , Genômica , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Transição de Fase
13.
Zhonghua Yan Ke Za Zhi ; 55(5): 355-360, 2019 May 11.
Artigo em Chinês | MEDLINE | ID: mdl-31137147

RESUMO

Objective: To investigate the expression of microRNA-1 (miR-1) and its regulatory function on fibronectin (FN) in human trabecular meshwork cells (HTMC) under oxidative stress. Methods: Experimental study. After HTMC were treated with 0, 60, 100, 200, 400 µmol/L hydrogen peroxide (H(2)O(2)) for 6 h, respectively, the cells were placed in culture medium for 24 h. The expression of miR-1 and FN mRNA in these cells were detected by real-time quantitative PCR. According to bioinformatics analysis, the target gene of miR-1 is predicted to be FN; pcDNA3/pri-miR-1 vectors, pcDNA3/enhanced green fluorescent protein (EGFP)-FN-3'UTR vectors and pcDNA3/EGFP-FN-3'UTRmut vectors were constructed. pcDNA3/pri-miR-1 were co-transfected with pcDNA3/EGFP-FN-3'UTR or pcDNA3/EGFP-FN-3'UTRmut respectively into HTMC. pDsRed2-N1 was taken as internal reference. After 48 h transfection, the absorbance of EGFP and red fluorescent protein (REP) was detected with fluorescence spectrophotometer to explore the effect of miR-1 on FN expression. HTMC was stimulated with 200 µmol/L H(2)O(2) for 24 h after overexpression plasmid of miR-1 was transfected into it, and then FN mRNA and protein levels were detected via real time PCR, Western blotting and immunofluorescence. Data were analyzed via one-way analysis of variance or t test. Results: With the increase of H(2)O(2) concentration, miR-1 decreased (F=390.80, P<0.01) while FN increased (F=13.16, P<0.01). The level of miR-1 in HTMC stimulated by 200 µmol/L and 400 µmol/L H(2)O(2) decreased to 0.608±0.014 (t=21.67, P<0.01) and 0.409±0.020 (t=29.91, P<0.01), respectively, compared with untreated control cells (1.000); whereas, the mRNA levels of FN increased to 1.630±0.233 (t=4.47, P=0.011) and 1.903±0.246 (t=6.15, P=0.003), respectively, compared with untreated control cells(1.000). Through bioinformatics analysis, miR-1 might have candidate binding site in FN mRNA 3'-UTR. Meanwhile, these cells co-transfected with pcDNA3/pri-miR-1 and pcDNA3/EGFP-FN-3'UTRmut (0.562±0.018) had higher EGFP expression than cells co-transfected with pcDNA3/pri-miR-1 and pcDNA3/EGFP-FN-3'UTR (0.329±0.015) (t=17.39, P<0.01). Compared with the control (1.000), after overexpressing miR-1 the mRNA expression and the protein level of FN decreased to 0.294±0.081 (t=11.01, P<0.01) and 0.584±0.022 (t=5.57, P<0.01), respectively. Conclusions: MiR-1 decreases while FN increased in HTMC under oxidative stress. MiR-1 inhibits FN expression through targeting FN 3'-UTR. (Chin J Ophthalmol, 2019, 55: 355-360).


Assuntos
Fibronectinas/genética , Regulação da Expressão Gênica/fisiologia , MicroRNAs/genética , Estresse Oxidativo/fisiologia , Malha Trabecular/metabolismo , Fibronectinas/metabolismo , Humanos , Peróxido de Hidrogênio , Reação em Cadeia da Polimerase em Tempo Real , Transfecção
14.
Can J Vet Res ; 83(2): 133-141, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31097875

RESUMO

Osteoarthritis, the leading cause of chronic joint pain, is studied through different animal models, but none of them is ideal in terms of reliability and translational value. In this pilot study of female rats, 3 surgical models of osteoarthritic pain, i.e., destabilization of the medial meniscus (DMM), cranial cruciate ligament transection (CCLT), and the combination of both surgical models (COMBO) and 1 chemical model [intra-articular injection of monosodium iodoacetate (MIA)] were compared for their impact on functional pain outcomes [static weight-bearing (SWB) and punctate tactile paw withdrawal threshold (PWT)] and spinal neuropeptides [substance P (SP), calcitonin gene-related peptide (CGRP), bradykinin (BK), and somatostatin (SST)]. Six rats were assigned to each model group and a sham group. Both the chemical model (MIA) and surgical COMBO model induced functional alterations in SWB and PWT, with the changes being more persistent in the surgical combination group. Both models also produced an increase in levels of pro-nociceptive and anti-nociceptive neuropeptides at different timepoints. Pain comparison with the MIA model showed the advantage of a surgical model, especially the combination of the DMM and CCLT models, whereas each surgical model alone only led to temporary functional alterations and no change in neuropeptidomics.


Assuntos
Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Neuropeptídeos/metabolismo , Osteoartrite/etiologia , Dor/metabolismo , Animais , Feminino , Injeções Intra-Articulares , Medição da Dor , Projetos Piloto , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Suporte de Carga
15.
Zoolog Sci ; 36(2): 167-171, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31120653

RESUMO

The two-pore domain potassium ion (K + ) channel-related K + (TREK) channel and melatonin receptors play roles in the regulation of reproduction in zebrafish. Since reproduction is regulated by diurnal rhythms, the TREK family and melatonin receptors may exhibit diurnal rhythms in expression. In this study, we aimed to investigate diurnal variations of the gene expressions of TREK family and melatonin receptors and their associations with kisspeptin and gonadotrophin-releasing hormone (GnRH). Diurnal variations of trek1b, trek2a, trek2b, mt1, mt2, mel1a, kiss2 and gnrh3 expressions were examined by real-time PCR. For reproduction-related genes, kiss2 and gnrh3 exhibited diurnal rhythms. trek2a revealed a diurnal rhythm in the TREK family. mt2 and mel1c exhibited diurnal rhythms in the melatonin receptors. Since Trek2a regulates gnrh3 expression, the diurnal rhythm of gnrh3 expression suggests to be regulated by the diurnal rhythm of trek2a expression.


Assuntos
Ritmo Circadiano/genética , Hormônio Liberador de Gonadotropina/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Ácido Pirrolidonocarboxílico/análogos & derivados , Peixe-Zebra/metabolismo , Animais , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica/fisiologia , Hormônio Liberador de Gonadotropina/genética , Kisspeptinas/metabolismo , Masculino , Canais de Potássio de Domínios Poros em Tandem/genética , Ácido Pirrolidonocarboxílico/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Melatonina/genética , Receptores de Melatonina/metabolismo , Peixe-Zebra/genética
16.
Nat Commun ; 10(1): 2278, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31123254

RESUMO

Mammalian spermatogenesis is sustained by mitotic germ cells with self-renewal potential known as undifferentiated spermatogonia. Maintenance of undifferentiated spermatogonia and spermatogenesis is dependent on tightly co-ordinated transcriptional and post-transcriptional mechanisms. The RNA helicase DDX5 is expressed by spermatogonia but roles in spermatogenesis are unexplored. Using an inducible knockout mouse model, we characterise an essential role for DDX5 in spermatogonial maintenance and show that Ddx5 is indispensable for male fertility. We demonstrate that DDX5 regulates appropriate splicing of key genes necessary for spermatogenesis. Moreover, DDX5 regulates expression of cell cycle genes in undifferentiated spermatogonia post-transcriptionally and is required for cell proliferation and survival. DDX5 can also act as a transcriptional co-activator and we demonstrate that DDX5 interacts with PLZF, a transcription factor required for germline maintenance, to co-regulate select target genes. Combined, our data reveal a critical multifunctional role for DDX5 in regulating gene expression programmes and activity of undifferentiated spermatogonia.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Processamento de RNA/fisiologia , Espermatogênese/genética , Espermatogônias/metabolismo , Animais , Ciclo Celular/genética , Proliferação de Células/genética , Técnicas de Cocultura , RNA Helicases DEAD-box/genética , Embrião de Mamíferos , Fertilidade/genética , Fibroblastos , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Cultura Primária de Células , Testículo/citologia
17.
Nat Commun ; 10(1): 2232, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110186

RESUMO

Sparse populations of neurons in the dentate gyrus (DG) of the hippocampus are causally implicated in the encoding of contextual fear memories. However, engram-specific molecular mechanisms underlying memory consolidation remain largely unknown. Here we perform unbiased RNA sequencing of DG engram neurons 24 h after contextual fear conditioning to identify transcriptome changes specific to memory consolidation. DG engram neurons exhibit a highly distinct pattern of gene expression, in which CREB-dependent transcription features prominently (P = 6.2 × 10-13), including Atf3 (P = 2.4 × 10-41), Penk (P = 1.3 × 10-15), and Kcnq3 (P = 3.1 × 10-12). Moreover, we validate the functional relevance of the RNAseq findings by establishing the causal requirement of intact CREB function specifically within the DG engram during memory consolidation, and identify a novel group of CREB target genes involved in the encoding of long-term memory.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/metabolismo , Giro Denteado/fisiologia , Consolidação da Memória/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Animais , Condicionamento (Psicologia)/fisiologia , Giro Denteado/citologia , Encefalinas/genética , Encefalinas/metabolismo , Medo/fisiologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/fisiologia , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Neurônios/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Análise de Sequência de RNA , Técnicas Estereotáxicas
18.
Nat Commun ; 10(1): 2266, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118463

RESUMO

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We establish an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, allows the prediction of IMP3 targets, and provides a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.


Assuntos
Modelos Moleculares , RNA Mensageiro/metabolismo , Motivos de Ligação ao RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ligação Proteica/fisiologia , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Técnica de Seleção de Aptâmeros , Análise de Sequência de DNA/métodos
19.
Artigo em Inglês | MEDLINE | ID: mdl-31078703

RESUMO

Doublesex and Mab-3 related transcription factor (Dmrt) genes play important roles in the process of sex determination and differentiation. In this study, a Dmrt1 gene open reading frame sequence was obtained from the gonadal transcriptome data of largemouth bass (Micropterus salmoides), and identified by cloning and sequencing. The ORF of Dmrt1 is 900 bp long, encodes 298 amino acids, and contains the DM region which is characteristic of Dmrt1. Full gDNA sequence of Dmrt1 was composed of five exons and four introns. RT-PCR and Q-PCR analysis of Dmrt1 were conducted in eight tissues and three developmental stages of mature male and female individuals. In situ hybridization was used to locate the expression of Dmrt1 in the testis and ovary of largemouth bass. The results showed that Dmrt1 was highly expressed in the testis of mature fish, but only weakly expressed in other tissues such as heart, liver, and brain, and exhibited gender dimorphism in the gonads of male and female fish at different stages. Furthermore, the expression level in female fish was very low and decreased gradually with ovary maturation. In situ hybridization indicated positive signals were found in early oocytes, but not in mature oocytes, while strong positive signals were found in all types of mature testis cells. The study showed that the sequence and structure of Dmrt1 were highly conserved and exhibited significant gender dimorphism in largemouth bass, as in other fish species. It is suggested that Dmrt1 plays an important role in sex determination and differentiation in largemouth bass.


Assuntos
Bass/metabolismo , Proteínas de Peixes/biossíntese , Regulação da Expressão Gênica/fisiologia , Caracteres Sexuais , Processos de Determinação Sexual/fisiologia , Fatores de Transcrição/biossíntese , Animais , Feminino , Masculino , Especificidade de Órgãos/fisiologia
20.
Invest Ophthalmol Vis Sci ; 60(5): 1571-1580, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30995314

RESUMO

Purpose: To understand the role and further dissect pathways downstream of tissue plasminogen activator (tPA) and the fibrinolytic pathway in modulating outflow facility. Methods: Outflow facility of tissue plasminogen activator (Plat) knockout (KO) mice was determined and compared to that of wild-type (WT) littermates. Gene expression of urokinase plasminogen activator (Plau), plasminogen activator inhibitor (Pai-1), plasminogen (Plg), and matrix metalloproteinases (Mmp-2, -9, and -13) was measured in angle tissues. Expression of the same genes and outflow facility were measured in KO and WT mice treated with triamcinolone acetonide (TA). Amiloride was used to inhibit urokinase plasminogen activator (uPA) in Plat KO mice, and outflow facility was measured. Results: Plat deletion resulted in outflow facility reduction and decreased Mmp-9 expression in angle tissues. Plasminogen expression was undetectable in both KO and WT mice. TA led to further reduction in outflow facility and decreases in expression of Plau and Mmp-13 in plat KO mice. Amiloride inhibition of uPA activity prevented the TA-induced outflow facility reduction in Plat KO mice. Conclusions: tPA deficiency reduced outflow facility in mice and was associated with reduced MMP expression. The mechanism of action of tPA is unlikely to involve plasminogen activation. tPA is not the only mediator of TA-induced outflow facility change, as TA caused reduction in outflow facility of Plat KO mice. uPA did not substitute for tPA in outflow facility regulation but abrogated the effect of TA in the absence of tPA, suggesting a complex role of components of the fibrinolytic system in outflow regulation.


Assuntos
Fibrinólise/fisiologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Plasminogênio/fisiologia , Ativador de Plasminogênio Tecidual/fisiologia , Malha Trabecular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Amilorida/farmacologia , Animais , Diuréticos/farmacologia , Regulação da Expressão Gênica/fisiologia , Glucocorticoides/farmacologia , Injeções Intraoculares , Pressão Intraocular/fisiologia , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Malha Trabecular/efeitos dos fármacos , Triancinolona Acetonida/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores
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