RESUMO
Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.
Assuntos
Metilação de DNA , Epigênese Genética , Gástrula/citologia , Gástrula/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA/genética , Análise de Célula Única , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/genética , Cromatina/metabolismo , Desmetilação , Corpos Embrioides/citologia , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Elementos Facilitadores Genéticos/genética , Epigenoma/genética , Eritropoese , Análise Fatorial , Gástrula/embriologia , Gastrulação/fisiologia , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA/análise , Fatores de Tempo , Dedos de ZincoRESUMO
The integrity of chromatin, which provides a dynamic template for all DNA-related processes in eukaryotes, is maintained through replication-dependent and -independent assembly pathways. To address the role of histone deposition in the absence of DNA replication, we deleted the H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is dynamic and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. Our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non-replicative system in vivo.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Oogênese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Metilação de DNA , Feminino , Fertilização , Regulação da Expressão Gênica , Camundongos , Oócitos/metabolismo , Transcrição GênicaRESUMO
The origin of the jaw is a long-standing problem in vertebrate evolutionary biology. Classical hypotheses of serial homology propose that the upper and lower jaw evolved through modifications of dorsal and ventral gill arch skeletal elements, respectively. If the jaw and gill arches are derived members of a primitive branchial series, we predict that they would share common developmental patterning mechanisms. Using candidate and RNAseq/differential gene expression analyses, we find broad conservation of dorsoventral (DV) patterning mechanisms within the developing mandibular, hyoid, and gill arches of a cartilaginous fish, the skate (Leucoraja erinacea). Shared features include expression of genes encoding members of the ventralizing BMP and endothelin signaling pathways and their effectors, the joint markers nkx3.2 and gdf5 and prochondrogenic transcription factor barx1, and the dorsal territory marker pou3f3. Additionally, we find that mesenchymal expression of eya1/six1 is an ancestral feature of the mandibular arch of jawed vertebrates, whereas differences in notch signaling distinguish the mandibular and gill arches in skate. Comparative transcriptomic analyses of mandibular and gill arch tissues reveal additional genes differentially expressed along the DV axis of the pharyngeal arches, including scamp5 as a novel marker of the dorsal mandibular arch, as well as distinct transcriptional features of mandibular and gill arch muscle progenitors and developing gill buds. Taken together, our findings reveal conserved patterning mechanisms in the pharyngeal arches of jawed vertebrates, consistent with serial homology of their skeletal derivatives, as well as unique transcriptional features that may underpin distinct jaw and gill arch morphologies.
Assuntos
Região Branquial , Rajidae , Animais , Brânquias , Arcada Osseodentária , Rajidae/genética , Vertebrados/genéticaRESUMO
We report scM&T-seq, a method for parallel single-cell genome-wide methylome and transcriptome sequencing that allows for the discovery of associations between transcriptional and epigenetic variation. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription. Notably, the method revealed previously unrecognized associations between heterogeneously methylated distal regulatory elements and transcription of key pluripotency genes.
Assuntos
Células-Tronco Embrionárias/fisiologia , Epigênese Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Elementos Reguladores de Transcrição/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Células Cultivadas , Camundongos , Dados de Sequência MolecularRESUMO
Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7 -/-) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.
Assuntos
Desmetilação do DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA , Células Germinativas , Animais , Feminino , Humanos , Masculino , Camundongos , Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Epigênese Genética , Células Germinativas/metabolismo , Meiose/genética , Camundongos Knockout , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Síntese de DNA TranslesãoRESUMO
The gill skeleton of cartilaginous fishes (sharks, skates, rays, and holocephalans) exhibits a striking anterior-posterior polarity, with a series of fine appendages called branchial rays projecting from the posterior margin of the gill arch cartilages. We previously demonstrated in the skate (Leucoraja erinacea) that branchial rays derive from a posterior domain of pharyngeal arch mesenchyme that is responsive to Sonic hedgehog (Shh) signaling from a distal gill arch epithelial ridge (GAER) signaling centre. However, how branchial ray progenitors are specified exclusively within posterior gill arch mesenchyme is not known. Here, we show that genes encoding several Wnt ligands are expressed in the ectoderm immediately adjacent to the skate GAER, and that these Wnt signals are transduced largely in the anterior arch environment. Using pharmacological manipulation, we show that inhibition of Wnt signalling results in an anterior expansion of Shh signal transduction in developing skate gill arches, and in the formation of ectopic anterior branchial ray cartilages. Our findings demonstrate that ectodermal Wnt signalling contributes to gill arch skeletal polarity in skate by restricting Shh signal transduction and chondrogenesis to the posterior arch environment and highlights the importance of signalling interactions at embryonic tissue boundaries for cell fate determination in vertebrate pharyngeal arches.
Assuntos
Região Branquial , Rajidae , Animais , Via de Sinalização Wnt , Proteínas Hedgehog/genética , Ectoderma , Brânquias , EsqueletoRESUMO
BACKGROUND: Perturbation of DNA methyltransferases (DNMTs) and of the active DNA demethylation pathway via ten-eleven translocation (TET) methylcytosine dioxygenases results in severe developmental defects and embryonic lethality. Dynamic control of DNA methylation is therefore vital for embryogenesis, yet the underlying mechanisms remain poorly understood. RESULTS: Here we report a single-cell transcriptomic atlas from Dnmt and Tet mutant mouse embryos during early organogenesis. We show that both the maintenance and de novo methyltransferase enzymes are dispensable for the formation of all major cell types at E8.5. However, DNA methyltransferases are required for silencing of prior or alternative cell fates such as pluripotency and extraembryonic programmes. Deletion of all three TET enzymes produces substantial lineage biases, in particular, a failure to generate primitive erythrocytes. Single-cell multi-omics profiling moreover reveals that this is linked to a failure to demethylate distal regulatory elements in Tet triple-knockout embryos. CONCLUSIONS: This study provides a detailed analysis of the effects of perturbing DNA methylation on mouse organogenesis at a whole organism scale and affords new insights into the regulatory mechanisms of cell fate decisions.
Assuntos
Metilação de DNA , Dioxigenases , Animais , DNA/metabolismo , Dioxigenases/genética , Metiltransferases/metabolismo , Camundongos , Organogênese/genéticaRESUMO
Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, displays impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and ample phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. Here, we generate a single-cell epigenomics and transcriptomics census of naïve-to-memory B cell differentiation in a CVID-discordant MZ twin pair. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B-cells mirroring defective cell-cell communication upon activation. These findings are validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naïve-to-memory B-cell transition in CVID and indicate links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, gives insight into future diagnosis and treatments of CVID patients.
Assuntos
Imunodeficiência de Variável Comum , Linfócitos B , Imunodeficiência de Variável Comum/diagnóstico , Imunodeficiência de Variável Comum/genética , Epigênese Genética , Epigenômica , Centro Germinativo , HumanosRESUMO
Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.
Assuntos
Elementos Facilitadores Genéticos , Células Germinativas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animais , Diferenciação Celular , Cromatina/metabolismo , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica , Células Germinativas/citologia , Camadas Germinativas/citologia , Masculino , Metilação , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Mutação/genética , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , RNA-Seq , Análise de Célula Única , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
INTRODUCTION: This case report describes the collaborative management of an extensive combined endodontic-periodontal lesion related to a long palato-radicular groove (PRG) on a maxillary lateral incisor. Cases with similar severity have been reported minimally in the endodontic journals but even less in the periodontal journals. This case report illustrates the result of multidisciplinary treatment of the combined lesions associated with PRG. CASE PRESENTATION: A 63-year-old patient presented with a periapical radiolucency on tooth #10. After evaluation, the patient was diagnosed with an endodontic-periodontal lesion associated with PRG. After being informed of a guarded prognosis, the patient consented to a surgical procedure in an effort to retain the tooth. Management of the case involved a combination of endodontic therapy, odontoplasty under dental operating microscopy to attempt to eliminate the root anomaly, and periodontal regenerative procedures with allografts and a resorbable barrier membrane. Clinical examination and the cone-beam computed tomography scan at a 2-year postoperative visit revealed a substantial reduction in probing depth and significant bone fill of the defect. CONCLUSIONS: In the past, a long PRG in combination with a periapical lesion often resulted in extraction of the tooth. With accurate assessment of the etiology of the defect, patient education, and a multidisciplinary approach, teeth with a PRG may be retained with a stable outcome for years.
Assuntos
Incisivo , Raiz Dentária , Tomografia Computadorizada de Feixe Cônico , Humanos , Pessoa de Meia-Idade , Raiz Dentária/diagnóstico por imagem , Raiz Dentária/cirurgiaRESUMO
Advancing maternal age causes a progressive reduction in fertility. The decline in developmental competence of the oocyte with age is likely to be a consequence of multiple contributory factors. Loss of epigenetic quality of the oocyte could impair early developmental events or programme adverse outcomes in offspring that manifest only later in life. Here, we undertake joint profiling of the transcriptome and DNA methylome of individual oocytes from reproductively young and old mice undergoing natural ovulation. We find reduced complexity as well as increased variance in the transcriptome of oocytes from aged females. This transcriptome heterogeneity is reflected in the identification of discrete sub-populations. Oocytes with a transcriptome characteristic of immature chromatin configuration (NSN) clustered into two groups: one with reduced developmental competence, as indicated by lower expression of maternal effect genes, and one with a young-like transcriptome. Oocytes from older females had on average reduced CpG methylation, but the characteristic bimodal methylation landscape of the oocyte was preserved. Germline differentially methylated regions of imprinted genes were appropriately methylated irrespective of age. For the majority of differentially expressed transcripts, the absence of correlated methylation changes suggests a post-transcriptional basis for most age-related effects on the transcriptome. However, we did find differences in gene body methylation at which there were corresponding changes in gene expression, indicating age-related effects on transcription that translate into methylation differences. Interestingly, oocytes varied in expression and methylation of these genes, which could contribute to variable competence of oocytes or penetrance of maternal age-related phenotypes in offspring.
Assuntos
Envelhecimento/genética , Envelhecimento/metabolismo , Metilação de DNA , Oócitos/metabolismo , Transcriptoma , Envelhecimento/patologia , Animais , Senescência Celular/genética , Senescência Celular/fisiologia , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Feminino , Idade Materna , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/crescimento & desenvolvimento , Oócitos/patologia , RNA-Seq , Análise de Célula ÚnicaRESUMO
BACKGROUND: Alternative splicing is a key regulatory mechanism in eukaryotic cells and increases the effective number of functionally distinct gene products. Using bulk RNA sequencing, splicing variation has been studied across human tissues and in genetically diverse populations. This has identified disease-relevant splicing events, as well as associations between splicing and genomic features, including sequence composition and conservation. However, variability in splicing between single cells from the same tissue or cell type and its determinants remains poorly understood. RESULTS: We applied parallel DNA methylation and transcriptome sequencing to differentiating human induced pluripotent stem cells to characterize splicing variation (exon skipping) and its determinants. Our results show that variation in single-cell splicing can be accurately predicted based on local sequence composition and genomic features. We observe moderate but consistent contributions from local DNA methylation profiles to splicing variation across cells. A combined model that is built based on genomic features as well as DNA methylation information accurately predicts different splicing modes of individual cassette exons. These categories include the conventional inclusion and exclusion patterns, but also more subtle modes of cell-to-cell variation in splicing. Finally, we identified and characterized associations between DNA methylation and splicing changes during cell differentiation. CONCLUSIONS: Our study yields new insights into alternative splicing at the single-cell level and reveals a previously underappreciated link between DNA methylation variation and splicing.
Assuntos
Processamento Alternativo , Metilação de DNA , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas , Análise de Célula ÚnicaRESUMO
BACKGROUND: Neoadjuvant therapy is increasingly recognized as an effective strategy prior to pancreatoduodenectomy. We investigate the role of neoadjuvant chemotherapy (NAC) followed by surgery and the predictive role of viable residual tumour cells histopathologically on outcomes. METHODS: The study population comprised of 195 consecutive patients with pancreatic adenocarcinoma who were treated with either NAC or a surgery-first (SF) strategy. Histopathological viable tumour cells were examined in the NAC patients and clinicopathological factors were correlated with overall survival. RESULTS: Forty-two patients (22%) were treated with NAC and 153 patients (78%) underwent SF. NAC was associated with higher estimated blood loss during surgery (928 mL versus 615 mL; P = 0.004), fewer (<15) excised lymph nodes (37% versus 17%; P = 0.015) and lower rates of lymphovascular invasion (65% versus 45%; P = 0.044) when compared with SF. Two-year survival of patients undergoing NAC was 63% and 51% in patients undergoing SF (P = 0.048). The 2-year survival of patients who had >65% residual tumour cells was 45% and 90% in patients who had <65% residual tumour cells (P = 0.022). Favourable responders (<65% viable tumour cells) were observed to have shorter operation time (<420 min) (55% versus 13%; P = 0.038), trend towards negative lymph node status (38% versus 10%; P = 0.067) and greater lymph node harvest in node positive patients (≥4 positive lymph nodes) (77% versus 37%; P = 0.045). CONCLUSION: The improved survival of patients undergoing NAC indicates effective management of micrometastatic disease and is an effective option requiring further investigation. Histopathological viable tumour cells after NAC was a surrogate marker for survival.
Assuntos
Adenocarcinoma/patologia , Adenocarcinoma/terapia , Terapia Neoadjuvante , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimioterapia Adjuvante , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Pancreatectomia , Neoplasias Pancreáticas/mortalidade , Estudos Retrospectivos , Taxa de Sobrevida , Resultado do TratamentoRESUMO
Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.
Assuntos
Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Nucleossomos/metabolismo , Análise de Sequência de DNA/métodos , Transcrição Gênica , Animais , Diferenciação Celular , Metilação de DNA , Feminino , Histonas/metabolismo , Masculino , Camundongos , Nucleossomos/genética , Análise de Célula ÚnicaRESUMO
Pluripotency is accompanied by the erasure of parental epigenetic memory, with naïve pluripotent cells exhibiting global DNA hypomethylation both in vitro and in vivo. Exit from pluripotency and priming for differentiation into somatic lineages is associated with genome-wide de novo DNA methylation. We show that during this phase, co-expression of enzymes required for DNA methylation turnover, DNMT3s and TETs, promotes cell-to-cell variability in this epigenetic mark. Using a combination of single-cell sequencing and quantitative biophysical modeling, we show that this variability is associated with coherent, genome-scale oscillations in DNA methylation with an amplitude dependent on CpG density. Analysis of parallel single-cell transcriptional and epigenetic profiling provides evidence for oscillatory dynamics both in vitro and in vivo. These observations provide insights into the emergence of epigenetic heterogeneity during early embryo development, indicating that dynamic changes in DNA methylation might influence early cell fate decisions.
Assuntos
Metilação de DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Reprogramação Celular , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Embrião de Mamíferos/citologia , Epigênese Genética/genética , Epigenômica , Genoma , Impressão Genômica , Células Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologiaRESUMO
DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.
Assuntos
Metilação de DNA/efeitos dos fármacos , Genômica/métodos , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , Sulfitos/farmacologia , Animais , Sequência de Bases , Ilhas de CpG/genética , CamundongosRESUMO
The sexual abuse of boys by priests was at the center of the 2002 scandal in the Roman Catholic Church. This scandal has resurrected the misconception that a link exists between having a homosexual orientation and being at increased risk for being a pedophile or child molester. This paper reviews and debunks the arguments in support of this misconception. Central to these arguments is what might be called the "proportionality argument": that the ratio of homosexuals to heterosexuals among child molesters is higher than the ratio of homosexuals to heterosexuals in the general population. The flaws in the proportionality argument and several other misconceptions are discussed.
Assuntos
Abuso Sexual na Infância , Clero/psicologia , Heterossexualidade/psicologia , Homossexualidade Masculina/psicologia , Pedofilia , Catolicismo/psicologia , Criança , Abuso Sexual na Infância/psicologia , Abuso Sexual na Infância/estatística & dados numéricos , Feminino , Humanos , Masculino , Meios de Comunicação de Massa , Pedofilia/psicologia , Terminologia como AssuntoRESUMO
Emerging single-cell epigenomic methods are being developed with the exciting potential to transform our knowledge of gene regulation. Here we review available techniques and future possibilities, arguing that the full potential of single-cell epigenetic studies will be realized through parallel profiling of genomic, transcriptional, and epigenetic information.
Assuntos
Epigenômica/métodos , Análise de Célula Única/métodos , Animais , Metilação de DNA , Regulação da Expressão Gênica , HumanosRESUMO
Most previous studies on pain in endodontics have focused on pain that occurs after root canal therapy. Very few studies have compared pain during the root canal procedure with pain occurring during other dental procedures. In the present study, 250 patients were queried following dental procedures regarding their pain levels prior to treatment and their pain levels during the treatment procedure. Of the total number of patients, 150 had a pulpectomy, 50 patients had a single extraction, and 50 patients had a single restoration. These patients reported significantly more pain during extractions than during root canal therapy. Ninety-two percent of patients undergoing root canal therapy reported that pain during the procedure was less than or much less than anticipated. Eighty-three percent of the patients undergoing root canal therapy experienced less pain during the treatment procedure than they experienced prior to the treatment.
Assuntos
Restauração Dentária Permanente/efeitos adversos , Dor Facial/etiologia , Pulpectomia/efeitos adversos , Tratamento do Canal Radicular/psicologia , Extração Dentária/efeitos adversos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ansiedade ao Tratamento Odontológico/psicologia , Restauração Dentária Permanente/psicologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição da Dor , Pulpectomia/psicologia , Extração Dentária/psicologiaRESUMO
OBJECTIVE: We sought to evaluate the detectability of second mesiobuccal (MB2) canals in the MB root of maxillary first molar teeth by comparing (1) parallax with pairs of conventional direct-exposure film intraoral radiographs (both D-speed and F-speed), (2) parallax with pairs of charge-coupled device-based digital images acquired through the use of Trophy RVG-ui, and (3) charge-coupled device-based images acquired through the use of a Trophy RVG-ui sensor and tomosynthetically reconstructed by TACT (tuned-aperture computed tomography) Workbench Software. STUDY DESIGN: Maxillary first molars were mounted in simulated bone. Pairs of images were generated by conventional D-speed and F-speed radiography and digital radiography with a charge-coupled device-based sensor, the RVG-ui. Sequences of images were also acquired for TACT reconstruction by using the digital sensor. Observers viewed sets of images to determine the number of canals present within the MB root of each tooth. Roots were horizontally cross-sectioned and viewed under an operating microscope to determine the actual number of canals present. RESULTS: The frequency of detection of MB2 canals was remarkably similar across techniques: 39.2% to 39.6% with parallax for both types of film and for RVG-ui images, and 37.9% with TACT. No statistically significant difference was found in the detectability of MB2 canals between the modalities tested. TACT had higher correlation coefficients than the other 3 modalities with respect to intrarater and interrater reliabilities. CONCLUSIONS: (1) There was a less than 40% chance of locating MB2 canals in the MB root of maxillary first molar teeth by using parallax with pairs of digital or analog radiographs. (2) TACT did not significantly affect the rate of detection of MB2 canals.