RESUMO
BACKGROUND: The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR-Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency. METHOD: Here we report the development of a multicolour fluorescence assay for studying CRISPR-Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR-Cas9 strategies. RESULT: We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration. CONCLUSION: Our results highlight the need for a more stringent assessment of CRISPR-Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Edição de Genes/métodos , Quebras de DNA de Cadeia Dupla , Transgenes , DNARESUMO
KEY POINTS: Alpha-melanocyte stimulating hormone (α-MSH) is an anorexigenic peptide. Injection of the α-MSH analog MTII into the ventral tegmental area (VTA) decreases food and sucrose intake and food reward. Melanocortin-3 receptors (MC3R) are highly expressed in the VTA, suggesting that the effects of intra-VTA α-MSH may be mediated by α-MSH changing the activity of MC3R-expressing VTA neurons. α-MSH increased the firing rate of MC3R VTA neurons in acute brain slices from mice, although it did not affect the firing rate of non-MC3R VTA neurons. The α-MSH induced increase in MC3R neuron firing rate is probably activity-dependent, and was independent of fast synaptic transmission and intracellular Ca2+ levels. These results help us to better understand how α-MSH acts in the VTA to affect feeding and other dopamine-dependent behaviours. ABSTRACT: The mesocorticolimbic dopamine system, the brain's reward system, regulates multiple behaviours, including food intake and food reward. There is substantial evidence that the melanocortin system of the hypothalamus, an important neural circuit controlling feeding and body weight, interacts with the mesocorticolimbic dopamine system to affect feeding, food reward and body weight. For example, melanocortin-3 receptors (MC3Rs) are expressed in the ventral tegmental area (VTA) and our laboratory previously showed that intra-VTA injection of the MC3R agonist, MTII, decreases home-cage food intake and operant responding for sucrose pellets. However, the cellular mechanisms underlying the effects of intra-VTA alpha-melanocyte stimulating hormone (α-MSH) on feeding and food reward are unknown. To determine how α-MSH acts in the VTA to affect feeding, we performed electrophysiological recordings in acute brain slices from mice expressing enhanced yellow fluorescent protein in MC3R neurons to test how α-MSH affects the activity of VTA MC3R neurons. α-MSH significantly increased the firing rate of VTA MC3R neurons without altering the activity of non-MC3R expressing VTA neurons. In addition, the α-MSH-induced increase in MC3R neuron activity was independent of fast synaptic transmission and intracellular Ca2+ levels. Finally, we show that the effect of α-MSH on MC3R neuron firing rate is probably activity-dependent. Overall, these studies provide an important advancement in the understanding of how α-MSH acts in the VTA to affect feeding and food reward.
Assuntos
Receptor Tipo 3 de Melanocortina/fisiologia , Área Tegmentar Ventral/fisiologia , alfa-MSH/fisiologia , Animais , Feminino , Técnicas In Vitro , Masculino , Camundongos Transgênicos , Neurônios/fisiologiaRESUMO
The mesocorticolimbic dopamine system, the brain's reward system, regulates many different behaviors including food intake, food reward, and feeding-related behaviors, and there is increasing evidence that hypothalamic feeding-related neuropeptides alter dopamine neuron activity to affect feeding. For example, neuropeptide-Y (NPY), a strong orexigenic hypothalamic neuropeptide, increases motivation for food when injected into the ventral tegmental area (VTA). How NPY affects the activity of VTA dopamine neurons to regulate feeding behavior is unknown, however. In these studies we have used whole cell patch-clamp electrophysiology in acute brain slices from mice to examine how NPY affects VTA dopamine neuron activity. NPY activated an outward current that exhibited characteristics of a G protein-coupled inwardly rectifying potassium channel current in ~60% of dopamine neurons tested. In addition to its direct effects on VTA dopamine neurons, NPY also decreased the amplitude and increased paired-pulse ratios of evoked excitatory postsynaptic currents in a subset of dopamine neurons, suggesting that NPY decreases glutamatergic transmission through a presynaptic mechanism. Interestingly, NPY also strongly inhibited evoked inhibitory postsynaptic currents onto dopamine neurons by a presynaptic mechanism. Overall these studies demonstrate that NPY utilizes multiple mechanisms to affect VTA dopamine neuron activity, and they provide an important advancement in our understanding of how NPY acts in the VTA to control feeding behavior.NEW & NOTEWORTHY Neuropeptide-Y (NPY) has been shown to act on mesolimbic dopamine circuits to increase motivated behaviors toward food, but it is unclear exactly how NPY causes these responses. Here, we demonstrate that NPY directly inhibited a subset of ventral tegmental area (VTA) dopamine neurons through the activation of G protein-coupled inwardly rectifying potassium currents, and it inhibited both excitatory postsynaptic currents and inhibitory postsynaptic currents onto subsets of dopamine neurons through a presynaptic mechanism. Thus NPY uses multiple mechanisms to dynamically control VTA dopamine neuron activity.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Neuropeptídeo Y/metabolismo , Terminações Pré-Sinápticas/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Cálcio/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Ácido Glutâmico/metabolismo , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/efeitos dos fármacos , Receptores de Neuropeptídeo Y/metabolismo , Técnicas de Cultura de Tecidos , Área Tegmentar Ventral/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismoRESUMO
Reprogramming of mouse fibroblasts toward a myocardial cell fate by forced expression of cardiac transcription factors or microRNAs has recently been demonstrated. The potential clinical applicability of these findings is based on the minimal regenerative potential of the adult human heart and the limited availability of human heart tissue. An initial but mandatory step toward clinical application of this approach is to establish conditions for conversion of adult human fibroblasts to a cardiac phenotype. Toward this goal, we sought to determine the optimal combination of factors necessary and sufficient for direct myocardial reprogramming of human fibroblasts. Here we show that four human cardiac transcription factors, including GATA binding protein 4, Hand2, T-box5, and myocardin, and two microRNAs, miR-1 and miR-133, activated cardiac marker expression in neonatal and adult human fibroblasts. After maintenance in culture for 4-11 wk, human fibroblasts reprogrammed with these proteins and microRNAs displayed sarcomere-like structures and calcium transients, and a small subset of such cells exhibited spontaneous contractility. These phenotypic changes were accompanied by expression of a broad range of cardiac genes and suppression of nonmyocyte genes. These findings indicate that human fibroblasts can be reprogrammed to cardiac-like myocytes by forced expression of cardiac transcription factors with muscle-specific microRNAs and represent a step toward possible therapeutic application of this reprogramming approach.
Assuntos
Transdiferenciação Celular/fisiologia , Fibroblastos/citologia , Regulação da Expressão Gênica/fisiologia , Miócitos Cardíacos/citologia , Fenótipo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citometria de Fluxo , Fator de Transcrição GATA4/metabolismo , Humanos , Imuno-Histoquímica , MicroRNAs/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full-length and HMGN3b which lacks the C-terminal RD (regulatory domain). In the present study, we have used the Glyt1 (glycine transporter 1) gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 (trimethylated Lys(4) of histone H3) and H3K9ac (acetylated Lys(9) of histone H3) at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 (Lys(14) of histone H3) acetylation and stimulates Glyt1a expression, but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b-stimulated H3K14 acetylation across the bodies of large genes such as Glyt1 can lead to more efficient transcription elongation and increased mRNA production.
Assuntos
Cromatina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/genética , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Proteínas HMGN/metabolismo , Histonas/metabolismo , Acetilação , Animais , Sítios de Ligação , Linhagem Celular , Proteínas HMGN/genética , Histonas/genética , Camundongos , TransfecçãoRESUMO
Cyanobacteria produce many neurotoxins including beta-methylamino-L-alanine (BMAA) that has been liked to amyotrophic lateral sclerosis (ALS) and neurodegenerative disease. A number of ALS cases have been diagnosed among residents of Enfield, NH, a town encompassing a lake with a history of cyanobacteria algal blooms. To investigate an association between toxic cyanobacterial blooms in New Hampshire and development of ALS, we reviewed records from our institution and other community databases to obtain demographic information on patients diagnosed with ALS within New England. We identified nine ALS patients who lived near Lake Mascoma in Enfield, NH, an incidence of sporadic ALS that is 10 to 25 times the expected incidence of 2/100,000/year. We suggest that the high incidence of ALS in this potential cluster could be directly related to chronic exposure to cyanobacterial neurotoxins such as BMAA. Possible routes of toxin exposure include inhalation of aerosolized toxins, consuming fish, or ingestion of lake water. Further investigation, including analysis of brain tissue for cyanobacterial toxins, will be helpful to test for an association between BMAA and ALS.
Assuntos
Diamino Aminoácidos/toxicidade , Esclerose Lateral Amiotrófica/epidemiologia , Esclerose Lateral Amiotrófica/etiologia , Cianobactérias/fisiologia , Diamino Aminoácidos/análise , Esclerose Lateral Amiotrófica/diagnóstico , Cromatografia Líquida de Alta Pressão/métodos , Análise por Conglomerados , Cianobactérias/química , Toxinas de Cianobactérias , Ecossistema , Eucariotos/microbiologia , Humanos , Incidência , New Hampshire/epidemiologia , Ficocianina/análise , Estudos Retrospectivos , Espectrometria de Massas em Tandem/métodos , Microbiologia da ÁguaRESUMO
OBJECTIVE: Amphetamine was formerly used as a treatment to combat obesity, but amphetamine's use as an appetite suppressant was discontinued because of its significant abuse potential. Most of the rewarding and reinforcing effects of amphetamine differ by sex, with females showing higher levels of drug intake and amphetamine-induced motivation, relapse, and locomotion, but it is unknown whether amphetamine's effects on feeding also differ by sex. Furthermore, previous research on the anorectic effects of amphetamine has been focused primarily on its effects on baseline homeostatic feeding, but it is unknown whether amphetamine also affects hedonic, reward-related feeding, which is an important factor driving the rise in obesity levels. METHODS: This study tested whether amphetamine alters food intake in a sex-dependent manner in two reward-related feeding paradigms: a sucrose two-bottle choice test and a high-fat/high-sugar binge intake model. RESULTS: Amphetamine altered food intake equally in males and females in both paradigms, with higher doses significantly inhibiting feeding and low doses of amphetamine increasing feeding at later time points. CONCLUSIONS: Amphetamine's effects on feeding and drug reward may be mediated by distinct mechanisms, which could allow for the development of new approaches to combat obesity with limited abuse and addiction-related side effects.
Assuntos
Anfetamina/farmacologia , Bulimia , Carboidratos da Dieta/administração & dosagem , Gorduras na Dieta/administração & dosagem , Ingestão de Energia/efeitos dos fármacos , Sacarose/administração & dosagem , Animais , Depressores do Apetite/farmacologia , Bulimia/induzido quimicamente , Bulimia/metabolismo , Bulimia/prevenção & controle , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores SexuaisRESUMO
BACKGROUND: Members of the HMGN protein family modulate chromatin structure and influence epigenetic modifications. HMGN1 and HMGN2 are highly expressed during early development and in the neural stem/progenitor cells of the developing and adult brain. Here, we investigate whether HMGN proteins contribute to the chromatin plasticity and epigenetic regulation that is essential for maintaining pluripotency in stem cells. RESULTS: We show that loss of Hmgn1 or Hmgn2 in pluripotent embryonal carcinoma cells leads to increased levels of spontaneous neuronal differentiation. This is accompanied by the loss of pluripotency markers Nanog and Ssea1, and increased expression of the pro-neural transcription factors Neurog1 and Ascl1. Neural stem cells derived from these Hmgn-knockout lines also show increased spontaneous neuronal differentiation and Neurog1 expression. The loss of HMGN2 leads to a global reduction in H3K9 acetylation, and disrupts the profile of H3K4me3, H3K9ac, H3K27ac and H3K122ac at the Nanog and Oct4 loci. At endodermal/mesodermal genes, Hmgn2-knockout cells show a switch from a bivalent to a repressive chromatin configuration. However, at neuronal lineage genes whose expression is increased, no epigenetic changes are observed and their bivalent states are retained following the loss of HMGN2. CONCLUSIONS: We conclude that HMGN1 and HMGN2 maintain the identity of pluripotent embryonal carcinoma cells by optimising the pluripotency transcription factor network and protecting the cells from precocious differentiation. Our evidence suggests that HMGN2 regulates active and bivalent genes by promoting an epigenetic landscape of active histone modifications at promoters and enhancers.
Assuntos
Cromatina/metabolismo , Proteína HMGN2/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Autorrenovação Celular , Proteína HMGN1/genética , Proteína HMGN1/metabolismo , Proteína HMGN2/genética , Histonas/metabolismo , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: The Ebola virus disease outbreak highlighted the lack of consistent guidelines and training for workers outside of hospital settings. Specifically, emergency medical services (EMS) workers, who are frequently the first professionals to evaluate patients, often do not have advanced notice of patient diagnosis, and have limited time in their national curricula devoted to highly infectious disease (HID) identification and containment. All of these can place them at increased risk. To explore the depth of US EMS practitioners' HID training and education, a pilot gap analysis survey was distributed to determine where the aforementioned can be bolstered to increase occupational safety. METHODS: Electronic surveys were distributed to EMS organization members. The survey collected respondent willingness to encounter HID scenarios; current policies and procedures; and levels of knowledge, training, and available resources to address HIDs. RESULTS: A total of 2,165 surveys were initiated and collected. Eighty percent of frontline personnel were aware that their agency had an HID standard operating guideline. Almost 85% of respondents correctly marked routes of exposure for select HIDs. More than half of respondents indicated no maximum shift times in personal protective equipment. DISCUSSION: This research suggests EMS practitioners could benefit from enhanced industry-specific education, training, and planning on HID mitigation and management. CONCLUSION: Strengthening EMS preparedness in response to suspected or confirmed HID cases may not only improve patient outcomes, but also worker and community safety.
Assuntos
Controle de Doenças Transmissíveis/métodos , Controle de Doenças Transmissíveis/normas , Serviços Médicos de Emergência/métodos , Serviços Médicos de Emergência/normas , Pessoal de Saúde/educação , Doenças Transmissíveis/transmissão , Coleta de Dados , Serviço Hospitalar de Emergência , Tratamento de Emergência , Guias como Assunto , Humanos , Estados UnidosRESUMO
HMGN proteins promote chromatin unfolding, enhance access to nucleosomes, and modulate transcription from chromatin templates. It is not known whether they act indiscriminately as general modulators of transcription or whether they regulate specific gene expression. Here, we investigated the role of HMGN3, a recently discovered HMGN family member, in transcription in vivo. We created cell lines overexpressing HMGN3a or its splice variant, HMGN3b, and analyzed their gene expression profiles using microarrays and reverse transcriptase PCR. We found that ectopic expression of HMGN3a alters the expression of approximately 0.8% of genes. Both HMGN3a and HMGN3b upregulate the expression of the glycine transporter 1 gene (Glyt1). Glyt1 encodes a membrane transporter that regulates the glycine concentration in synaptic junctions. Both GLYT1 and HMGN3 are highly expressed in glia cells and the eye, and we show that both proteins are coexpressed in the retina. Chromatin immunoprecipitation assays showed that HMGN3 protein is recruited to a region of the Glyt1 gene encompassing the Glyt1a transcriptional start site. These results suggest that HMGN3 regulates Glyt1 expression and demonstrate that members of the HMGN family can regulate the transcription of specific genes.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Regulação da Expressão Gênica , Proteínas HMGN/metabolismo , Isoformas de Proteínas/metabolismo , Transcrição Gênica , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Proteínas da Membrana Plasmática de Transporte de Glicina , Proteínas HMGN/genética , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas/genética , Retina/citologia , Retina/metabolismoRESUMO
We report that loss of HMGN1, a nucleosome-binding protein that alters the compaction of the chromatin fiber, increases the cellular sensitivity to ionizing radiation and the tumor burden of mice. The mortality and tumor burden of ionizing radiation-treated Hmgn1-/- mice is higher than that of their Hmgn1+/+ littermates. Hmgn1-/- fibroblasts have an altered G2-M checkpoint activation and are hypersensitive to ionizing radiation. The ionizing radiation hypersensitivity and the aberrant G2-M checkpoint activation of Hmgn1-/- fibroblasts can be reverted by transfections with plasmids expressing wild-type HMGN1, but not with plasmids expressing mutant HMGN proteins that do not bind to chromatin. Transformed Hmgn1-/- fibroblasts grow in soft agar and produce tumors in nude mice with a significantly higher efficiency than Hmgn1+/+ fibroblasts, suggesting that loss of HMGN1 protein disrupts cellular events controlling proliferation and growth. Hmgn1-/- mice have a higher incidence of multiple malignant tumors and metastases than their Hmgn1+/+ littermates. We suggest that HMGN1 optimizes the cellular response to ionizing radiation and to other tumorigenic events; therefore, loss of this protein increases the tumor burden in mice.
Assuntos
Transformação Celular Neoplásica/efeitos da radiação , Proteína HMGN1/deficiência , Neoplasias Induzidas por Radiação/metabolismo , Tolerância a Radiação/fisiologia , Animais , Divisão Celular/efeitos da radiação , Transformação Celular Neoplásica/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Fase G2/efeitos da radiação , Proteína HMGN1/metabolismo , Proteína HMGN1/fisiologia , Masculino , Camundongos , Camundongos Nus , Neoplasias Induzidas por Radiação/patologiaRESUMO
Sympathetic neurons require NGF from their target fields for survival, axonal target innervation, dendritic growth and formation, and maintenance of synaptic inputs from preganglionic neurons. Target-derived NGF signals are propagated retrogradely, from distal axons to somata of sympathetic neurons via TrkA signaling endosomes. We report that a subset of TrkA endosomes that are transported from distal axons to cell bodies translocate into dendrites, where they are signaling competent and move bidirectionally, in close proximity to synaptic protein clusters. Using a strategy for spatially confined inhibition of TrkA kinase activity, we found that distal-axon-derived TrkA signaling endosomes are necessary within sympathetic neuron dendrites for maintenance of synapses. Thus, TrkA signaling endosomes have unique functions in different cellular compartments. Moreover, target-derived NGF mediates circuit formation and synapse maintenance through TrkA endosome signaling within dendrites to promote aggregation of postsynaptic protein complexes.
Assuntos
Dendritos/metabolismo , Endossomos/metabolismo , Neurônios/metabolismo , Receptor trkA/metabolismo , Sistema Nervoso Simpático/metabolismo , Sinapses/metabolismo , Animais , Axônios/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microesferas , Fator de Crescimento Neural/metabolismo , Imagem Óptica , Fosforilação , Polissacarídeos/metabolismo , Transdução de Sinais/fisiologia , Imagem com Lapso de TempoRESUMO
We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIbeta. We show that topo IIbeta is a DNA-dependent ATPase that appears to fit Michaelis-Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k(cat) for ATP hydrolysis by human DNA topo IIbeta in the presence of DNA is 2.25 s(-1). We have characterised a topo IIbeta derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the kcat for ATP hydrolysis by 7-fold, to 0.32 s(-1), while not significantly altering the apparent K(m). The specificity constant for the interaction between ATP and topo IIbeta (kcat/K(mapp)) showed a 90% reduction for betaS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIbeta. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , DNA/genética , DNA/farmacologia , DNA Helicases/química , DNA Helicases/genética , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Teste de Complementação Genética , Humanos , Hidrólise/efeitos dos fármacos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Serina/genéticaRESUMO
HMGN1 is a nuclear protein that binds to nucleosomes and alters the accessibility of regulatory factors to their chromatin targets. To elucidate its biological function and identify specific HMGN1 target genes, we generated Hmgn1-/- mice. DNA microarray analysis of Hmgn1+/+ and Hmgn1-/- embryonic fibroblasts identified N-cadherin as a potential HMGN1 gene target. RT-PCR and western blot analysis confirmed a linkage between HMGN1 expression and N-cadherin levels. In both transformed and primary mouse embryonic fibroblasts (MEFs), HMGN1 acted as negative regulator of N-cadherin expression. Likewise, the N-cadherin levels in early embryos of Hmgn1-/- mice were higher than those of their Hmgn1+/+ littermates. Loss of HMGN1 increased the adhesiveness, motility and aggregation potential of Hmgn1-/- MEFs, a phenotype consistent with increased levels of N-cadherin protein. Re-expression of wild-type HMGN1, but not of the mutant HMGN1 protein that does not bind to chromatin, in Hmgn1-/- MEFs, decreased the levels of N-cadherin and restored the Hmgn1+/+ phenotype. These studies demonstrate a role for HMGN1 in the regulation of specific gene expression. We suggest that in MEFs, and during early mouse development, the interaction of HMGN1 with chromatin down-regulates the expression of N-cadherin.
Assuntos
Caderinas/metabolismo , Cromossomos/química , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína HMGN1/metabolismo , Animais , Western Blotting , Adesão Celular , Moléculas de Adesão Celular , Linhagem Celular Transformada , Movimento Celular , Células Cultivadas , Cromatina/metabolismo , Regulação para Baixo , Embrião de Mamíferos , Fibroblastos/citologia , Marcação de Genes , Proteína HMGN1/genética , Camundongos , Camundongos Knockout , Mutação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta Catenina/metabolismoRESUMO
Infection-control principles and practices based on science and common sense have been around for thousands of years. They've proven effective. If you know how a disease is transmitted, then you'll know what PPE to use to block transmission. Viruses don't multiply or survive for long outside a living host, so they may be present on a given surface, but in low numbers--and most often not numbers large enough to cause infection. However, hepatitis B virus can survive up to seven days in the presence of dried blood; that's why contaminated equipment must be cleaned as soon as possible after use. If it's not possible to use PPE in a given situation or it fails, then post-exposure medical follow-up and treatment can protect you. Ensuring there's an effective communication network in place for reporting an exposure to your DO is essential. Your agency should verify that hospitals are aware of their responsibilities under the Ryan White Law to share patient testing information if an exposure event occurs. The DO's role includes working with hospitals to provide proper medical follow-up, rapid patient testing and counseling for the exposed provider. Your DO is your advocate. Each and every component of your department's exposure control plan assists in reducing the risk for acquiring an infection on the job. Risk reduction and risk management are both vital. Ultimately, the responsibility lies with you--the provider--to know infection-control principles and practice good habits.