RESUMEN
This research investigates the intricate dynamics of DNA methylation in the hours following CD8+ T cell activation, during a critical yet understudied temporal window. DNA methylation is an epigenetic modification central to regulation of gene expression and directing immune responses. Our investigation spanned 96-h post-activation and unveils a nuanced tapestry of global and site-specific methylation changes. We identified 15,626 significant differentially methylated CpGs spread across the genome, with the most significant changes occurring within the genes ADAM10, ICA1, and LAPTM5. While many changes had modest effect sizes, approximately 120 CpGs exhibited a log2FC above 1.5, with cell activation and proliferation pathways the most affected. Relatively few of the differentially methylated CpGs occurred along adjacent gene regions. The exceptions were seven differentially methylated gene regions, with the Human T cell Receptor Alpha Joining Genes demonstrating consistent methylation change over a 3kb window. We also investigated whether an inflammatory environment could alter DNA methylation during activation, with proliferating cells exposed to the oxidant glycine chloramine. No substantial differential methylation was observed in this context. The temporal perspective of early activation adds depth to the evolving field of epigenetic immunology, offering insights with implications for therapeutic innovation and expanding our understanding of epigenetic modulation in immune function.
Asunto(s)
Linfocitos T CD8-positivos , Proliferación Celular , Metilación de ADN , Activación de Linfocitos , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Humanos , Islas de CpG , Epigénesis Genética , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Proteínas de la Membrana/genéticaRESUMEN
Bacillus Calmette-Guérin (BCG) treatment for non-muscle invasive bladder cancer (NMIBC) is an established immunotherapeutic, however, a significant portion of patients do not respond to treatment. Despite extensive research into the therapeutic mechanism of BCG, gaps remain in our understanding. This review specifically focuses on the epigenomic contributions in the immune microenvironment, in the context of BCG treatment for NMIBC. We also summarise the current understanding of NMIBC epigenetic characteristics, and discuss how future targeted strategies for BCG therapy should incorporate epigenomic biomarkers in conjunction with genomic biomarkers.
RESUMEN
G-quadruplexes (G4s) within the human genome have undergone extensive molecular investigation, with a strong focus on telomeres, gene promoters and repetitive regulatory sequences. G4s play central roles in regulating essential biological processes, including telomere maintenance, replication, transcription and translation. Targeting these molecular processes with G4-binding ligands holds substantial therapeutic potential in anticancer treatments and has also shown promise in treating neurological, skeletal and muscular disorders. The presence of G4s in bacterial and viral genomes also suggests that G4-binding ligands could be a critical tool in fighting infections. This review provides an overview of the progress and applications of G4-binding ligands, their proposed mechanisms of action, challenges faced and prospects for their utilization in anticancer treatments, neurological disorders and antiviral activities.
Asunto(s)
G-Cuádruplex , Neoplasias , Enfermedades del Sistema Nervioso , Virosis , Humanos , Virosis/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Ligandos , TelómeroRESUMEN
BACKGROUND: Cancer cell lines are invaluable model systems for biomedical research because they provide an almost unlimited supply of biological materials. However, there is considerable skepticism regarding the reproducibility of data derived from these in vitro models. RECENT FINDINGS: Chromosomal instability (CIN) is one of the primary issues associated with cell lines, which can cause genetic heterogeneity and unstable cell properties within a cell population. Many of these problems can be avoided with some precautions. Here we review the underlying causes of CIN, including merotelic attachment, telomere dysfunction, DNA damage response defects, mitotic checkpoint defects and cell cycle disturbances. CONCLUSION: In this review we summarize studies highlighting the consequences of CIN in various cell lines and provide suggestions on monitoring and controlling CIN during cell culture.
Asunto(s)
Mitosis , Neoplasias , Humanos , Mitosis/genética , Reproducibilidad de los Resultados , Inestabilidad Cromosómica , Línea Celular , Neoplasias/genéticaRESUMEN
Oxidative stress is a common feature of inflammation-driven cancers, and it promotes genomic instability and aggressive tumour phenotypes. It is known that oxidative stress transiently modulates gene expression through the oxidation of transcription factors and associated regulatory proteins. Neutrophils are our most abundant white blood cells and accumulate at sites of infection and inflammation. Activated neutrophils produce hypochlorous acid and chloramines, which can disrupt DNA methylation by oxidizing methionine. The goal of the current study was to determine whether chloramine exposure results in sequence-specific modifications in DNA methylation that enable long-term alterations in transcriptional output. Proliferating Jurkat T-lymphoma cells were exposed to sublethal doses of glycine chloramine and differential methylation patterns were compared using Illumina EPIC 850 K bead chip arrays. There was a substantial genome-wide decrease in methylation 4 h after exposure that correlated with altered RNA expression for 24 and 48 h, indicating sustained impacts on exposed cells. A large proportion of the most significant differentially methylated CpG sites were situated towards chromosomal ends, suggesting that these regions are most susceptible to inhibition of maintenance DNA methylation. This may contribute to epigenetic instability of chromosomal ends in rapidly dividing cells, with potential implications for the regulation of telomere length and cellular longevity.
Asunto(s)
Metilación de ADN , Factores de Transcripción , Metilación de ADN/genética , Oxidación-Reducción , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo/genética , Islas de CpG/genética , Epigénesis GenéticaRESUMEN
Cells around epithelial wounds must first become aware of the wound's presence in order to initiate the wound-healing process. An initial response to an epithelial wound is an increase in cytosolic calcium followed by complex calcium-signaling events. While these calcium signals are driven by both physical and chemical wound responses, cells around the wound will all be equipped with the same cellular components to produce and interact with the calcium signals. Here we have developed a mathematical model in the context of laser ablation of the Drosophila pupal notum that integrates tissue-level damage models with a cellular calcium-signaling toolkit. The model replicates experiments in the contexts of control wounds as well as knockdowns of specific cellular components, but it also provides new insights that are not easily accessible experimentally. The model suggests that cell-cell variability is necessary to produce calcium-signaling events observed in experiments; it quantifies calcium concentrations during wound-induced signaling events, and it shows that intercellular transfer of the molecule IP3 is required to coordinate calcium signals across distal cells around the wound. The mathematical model developed here serves as a framework for quantitative studies in both wound signaling and calcium signaling in the Drosophila system.
Asunto(s)
Calcio , Drosophila , Animales , Calcio/metabolismo , Drosophila/metabolismo , Rayos Láser , Señalización del Calcio , Modelos TeóricosRESUMEN
It is well known that certain non B-DNA structures, including G-quadruplexes, are key elements that can regulate gene expression. Here, we explore the theory that DNA modifications, such as methylation of cytosine, could act as a dynamic switch by promoting or alleviating the structural formation of G-quadruplex structures in DNA or RNA. The interaction between epigenetic DNA modifications, G4 formation, and the 3D architecture of the genome is a complex and developing area of research. Although there is growing evidence for such interactions, a great deal still remains to be discovered. In vivo, the potential effect that cytosine methylation may have on the formation of DNA structures has remained largely unresearched, despite this being a potential mechanism through which epigenetic factors could regulate gene activity. Such interactions could represent novel mechanisms for important biological functions, including altering nucleosome positioning or regulation of gene expression. Furthermore, promotion of strand-specific G-quadruplex formation in differentially methylated genes could have a dynamic role in directing X-inactivation or the control of imprinting, and would be a worthwhile focus for future research.
Asunto(s)
Citosina/metabolismo , ADN/química , Animales , Metilación de ADN , Epigénesis Genética , G-Cuádruplex , Regulación de la Expresión Génica , HumanosRESUMEN
The presence of a wound triggers surrounding cells to initiate repair mechanisms, but it is not clear how cells initially detect wounds. In epithelial cells, the earliest known wound response, occurring within seconds, is a dramatic increase in cytosolic calcium. Here, we show that wounds in the Drosophila notum trigger cytoplasmic calcium increase by activating extracellular cytokines, Growth-blocking peptides (Gbps), which initiate signaling in surrounding epithelial cells through the G-protein-coupled receptor Methuselah-like 10 (Mthl10). Latent Gbps are present in unwounded tissue and are activated by proteolytic cleavage. Using wing discs, we show that multiple protease families can activate Gbps, suggesting that they act as a generalized protease-detector system. We present experimental and computational evidence that proteases released during wound-induced cell damage and lysis serve as the instructive signal: these proteases liberate Gbp ligands, which bind to Mthl10 receptors on surrounding epithelial cells, and activate downstream release of calcium.
Asunto(s)
Epitelio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Cicatrización de Heridas/fisiología , Animales , Calcio/metabolismo , Señalización del Calcio , Citosol/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Epiteliales/metabolismo , Epitelio/fisiología , Péptidos/metabolismo , Proteolisis , Heridas y Lesiones/metabolismoRESUMEN
BACKGROUND: Environmental factors, such as oxidative stress, have the potential to modify the epigenetic landscape of cells. We have previously shown that DNA methyltransferase (DNMT) activity can be inhibited by sublethal doses of hydrogen peroxide (H2O2). However, site-specific changes in DNA methylation and the reversibility of any changes have not been explored. Using bead chip array technology, differential methylation was assessed in Jurkat T-lymphoma cells following exposure to H2O2. RESULTS: Sublethal H2O2 exposure was associated with an initial genome-wide decrease in DNA methylation in replicating cells, which was largely corrected 72 h later. However, some alterations were conserved through subsequent cycles of cell division. Significant changes to the variability of DNA methylation were also observed both globally and at the site-specific level. CONCLUSIONS: This research indicates that increased exposure to H2O2 can result in long-term alterations to DNA methylation patterns, providing a mechanism for environmental factors to have prolonged impact on gene expression.
Asunto(s)
Metilación de ADN , Peróxido de Hidrógeno , Genoma , Peróxido de Hidrógeno/toxicidad , Estrés OxidativoRESUMEN
Dendritic cells are potently activated by the synergistic action of CD40 stimulation in conjunction with signaling through toll like receptors, subsequently priming T cells. Cancer vaccines targeting the activation of dendritic cells in this manner show promise in murine models and are being developed for human patients. While the efficacy of vaccines based on CD40 and toll like receptor stimulation has been established, further investigation is needed to understand the mechanism of tumor control and how vaccination alters tumor infiltrating immune cells. In this study we vaccinated mice bearing established murine melanoma tumors with agonistic anti-CD40, polyI:C, and tumor antigen. Vaccination led to increased intratumoral T cell numbers and delayed tumor growth, yet did not require trafficking of T cells from the periphery. Pre-existing intratumoral T cells exhibited an acute burst in proliferation but became less functional in response to vaccination. However, the increased intratumoral T cell numbers yielded increased numbers of effector T cells per tumor. Together, our data indicate that the existing T cell response and intratumoral dendritic cells are critical for vaccination efficacy. It also suggests that circulating T cells responding to vaccination may not be an appropriate biomarker for vaccine efficacy.
Asunto(s)
Antígenos CD40/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma Experimental/inmunología , Receptor Toll-Like 3/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Vacunación/métodosRESUMEN
Background: Focused ultrasound (FUS) activation of microbubbles (MBs) for blood-brain (BBB) and blood-tumor barrier (BTB) opening permits targeted therapeutic delivery. While the effects of FUS+MBs mediated BBB opening have been investigated for normal brain tissue, no such studies exist for intracranial tumors. As this technology advances into clinical immunotherapy trials, it will be crucial to understand how FUS+MBs modulates the tumor immune microenvironment. Methods and Results: Bulk RNA sequencing revealed that FUS+MBs BTB/BBB opening (1 MHz, 0.5 MPa peak-negative pressure) of intracranial B16F1cOVA tumors increases the expression of genes related to proinflammatory cytokine and chemokine signaling, pattern recognition receptor signaling, and antigen processing and presentation. Flow cytometry revealed increased maturation (i.e. CD86) of dendritic cells (DCs) in the meninges and altered antigen loading of DCs in both the tumor and meninges. For DCs in tumor draining lymph nodes, FUS+MBs had no effect on maturation and elicited only a trend towards increased presentation of tumor-derived peptide by MHC. Neither tumor endothelial cell adhesion molecule expression nor homing of activated T cells was affected by FUS+MBs. Conclusion: FUS+MBs-mediated BTB/BBB opening elicits signatures of inflammation; however, the response is mild, transient, and unlikely to elicit a systemic response independent of administration of immune adjuvants.
Asunto(s)
Neoplasias Encefálicas/terapia , Células Dendríticas/metabolismo , Melanoma Experimental/terapia , Ovalbúmina/inmunología , Terapia por Ultrasonido/métodos , Animales , Barrera Hematoencefálica , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/inmunología , Regulación Neoplásica de la Expresión Génica , Masculino , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Ratones , Microburbujas , Análisis de Secuencia de ARN , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Sequences encoding Olduvai protein domains (formerly DUF1220) show the greatest human lineage-specific increase in copy number of any coding region in the genome and have been associated, in a dosage-dependent manner, with brain size, cognitive aptitude, autism, and schizophrenia. Tandem intragenic duplications of a three-domain block, termed the Olduvai triplet, in four NBPF genes in the chromosomal 1q21.1-0.2 region, are primarily responsible for the striking human-specific copy number increase. Interestingly, most of the Olduvai triplets are adjacent to, and transcriptionally coregulated with, three human-specific NOTCH2NL genes that have been shown to promote cortical neurogenesis. Until now, the underlying genomic events that drove the Olduvai hyperamplification in humans have remained unexplained. Here, we show that the presence or absence of an alternative first exon of the Olduvai triplet perfectly discriminates between amplified (58/58) and unamplified (0/12) triplets. We provide sequence and breakpoint analyses that suggest the alternative exon was produced by an nonallelic homologous recombination-based mechanism involving the duplicative transposition of an existing Olduvai exon found in the CON3 domain, which typically occurs at the C-terminal end of NBPF genes. We also provide suggestive in vitro evidence that the alternative exon may promote instability through a putative G-quadraplex (pG4)-based mechanism. Lastly, we use single-molecule optical mapping to characterize the intragenic structural variation observed in NBPF genes in 154 unrelated individuals and 52 related individuals from 16 families and show that the presence of pG4-containing Olduvai triplets is strongly correlated with high levels of Olduvai copy number variation. These results suggest that the same driver of genomic instability that allowed the evolutionarily recent, rapid, and extreme human-specific Olduvai expansion remains highly active in the human genome.
Asunto(s)
Proteínas Portadoras/genética , Genoma Humano , Expansión de Repetición de Trinucleótido , Animales , Secuencia de Bases , Variaciones en el Número de Copia de ADN , Evolución Molecular , G-Cuádruplex , Amplificación de Genes , Dosificación de Gen , Inestabilidad Genómica , Recombinación Homóloga , Humanos , Primates , Dominios Proteicos , Homología de SecuenciaRESUMEN
It has been widely hypothesized that both diet and the microbiome play a role in the regulation of attention-deficit/hyperactivity disorder (ADHD) behaviour. However, there has been very limited scientific investigation into the potential biological connection. We performed a 10-week pilot study investigating the effects of a broad spectrum micronutrient administration on faecal microbiome content, using 16S rRNA gene sequencing. The study consisted of 17 children (seven in the placebo and ten in the treatment group) between the ages of seven and 12 years, who were diagnosed with ADHD. We found that micronutrient treatment did not drive large-scale changes in composition or structure of the microbiome. However, observed OTUs significantly increased in the treatment group, and showed no mean change in the placebo group. The differential abundance and relative frequency of Actinobacteria significantly decreased post- micronutrient treatment, and this was largely attributed to species from the genus Bifidobacterium. This was compensated by an increase in the relative frequency of species from the genus Collinsella. Further research is required to establish the role that Bifidobacterium contribute towards neuropsychiatric disorders; however, these findings suggest that micronutrient administration could be used as a safe, therapeutic method to modulate Bifidobacterium abundance, which could have potential implications for modulating and regulating ADHD behaviour. Our pilot study provides an initial observation into this area of research, and highlights an interesting avenue for further investigation in a larger cohort. Furthermore, these novel results provide a basis for future research on the biological connection between ADHD, diet and the microbiome.
Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad/microbiología , Trastorno por Déficit de Atención con Hiperactividad/psicología , Microbioma Gastrointestinal , Micronutrientes/uso terapéutico , Actinobacteria , Trastorno por Déficit de Atención con Hiperactividad/dietoterapia , Niño , Suplementos Dietéticos , Método Doble Ciego , Heces/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/genética , Humanos , FilogeniaRESUMEN
BACKGROUND: Intent-to-treat analyses from a randomized controlled trial showed significant between-group differences favouring micronutrient treatment on the Clinical Global Impression-Improvement, but no group differences on clinician, parent and teacher ratings of overall ADHD symptoms. There was an advantage of micronutrients over placebo in improving overall function, emotional regulation, aggression, and reducing impairment as well as improving inattention based on clinician but not parent observation. No group differences were observed on hyperactive-impulsive symptoms. We investigated predictors of response defined by pre-treatment variables. METHOD: We conducted analyses of data from a clinical trial of children (7-12â¯years) with ADHD, whereby participants were randomized to receive micronutrients or placebo for 10â¯weeks followed by a 10â¯week open-label (OL) phase. We included only children who had been exposed to micronutrients for a full 10â¯week period and demonstrated satisfactory adherence, either in RCT phase (nâ¯=â¯40) or OL phase (those who received placebo during RCT phase; nâ¯=â¯31). Seven outcomes were examined: change in ADHD symptoms (clinician/parent), ADHD responder, overall responder, change in mood, change in functioning, and change in aggression. Demographic, developmental variables, current clinical and physical characteristics, MTHFR genotype at two common variants, and pre-treatment serum/plasma levels (vitamin D, B12, folate, zinc, copper, iron, ferritin, potassium, calcium, magnesium, and homocysteine) were all considered as putative predictors. RESULTS: Substantial nutrient deficiencies pre-treatment were observed only for vitamin D (13%) and copper (15%), otherwise most children entered the trial with nutrient levels falling within expected ranges. Regression analyses showed varying predictors across outcomes with no one predictor being consistently identified across different variables. Lower pre-treatment folate and B12 levels, being female, greater severity of symptoms and co-occurring disorders pre-treatment, more pregnancy complications and fewer birth problems were identified as possible predictors of greater improvement for some but not all outcome measures although predictive values were weak. Lower IQ and higher BMI predicted greater improvement in aggression. CONCLUSIONS: This study replicates Rucklidge et al. (2014b) showing the limited value of using serum nutrient levels to predict treatment response although we cannot rule out that other non-assayed nutrient levels may be more valuable. Additionally, no specific demographic or clinical characteristics, including MTHFR genetic status, were identified that would preclude children with ADHD from trying this treatment approach.
Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad/terapia , Suplementos Dietéticos , Micronutrientes/uso terapéutico , Vitaminas/uso terapéutico , Trastorno por Déficit de Atención con Hiperactividad/sangre , Trastorno por Déficit de Atención con Hiperactividad/diagnóstico , Trastorno por Déficit de Atención con Hiperactividad/genética , Biomarcadores/sangre , Índice de Masa Corporal , Niño , Femenino , Humanos , Inteligencia , Masculino , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Trastornos Nutricionales/sangre , Trastornos Nutricionales/diagnóstico , Trastornos Nutricionales/genética , Trastornos Nutricionales/terapia , Polimorfismo de Nucleótido Simple , Pronóstico , Índice de Severidad de la EnfermedadRESUMEN
Exposure times and dosage required for dietary components to modify DNA methylation patterns are largely unknown. AIM: This exploratory research represents the first genome-wide analysis of DNA methylation changes during a randomized-controlled-trial (RCT) for dietary supplementation with broad spectrum vitamins, minerals and amino acids in humans. METHODS: Genome-wide changes in methylation from paired, peripheral blood samples were assessed using the Infinium Methylation EPIC 850 K array. RESULTS: Methylation increased at 84% of the most significant differentially methylated CpGs; however, none showed significance after adjustment for genome-wide testing. CONCLUSION: Micronutrient supplementation is unlikely to have a substantial biological effect on DNA methylation over 10 weeks; however, the trend toward hypermethylation that we observed is likely to become more marked with longer exposure periods.
Asunto(s)
Aminoácidos/farmacología , Trastorno por Déficit de Atención con Hiperactividad/dietoterapia , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Suplementos Dietéticos , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Minerales/farmacología , Vitaminas/farmacología , Aminoácidos/administración & dosificación , Niño , Islas de CpG , Epigénesis Genética/efectos de los fármacos , Femenino , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Masculino , Minerales/administración & dosificación , Vitaminas/administración & dosificaciónRESUMEN
Many aspects of human development and disease are influenced by the interaction between genetic and environmental factors. Understanding how our genes respond to the environment is central to managing health and disease, and is one of the major contemporary challenges in human genetics. Various epigenetic processes affect chromosome structure and accessibility of deoxyribonucleic acid (DNA) to the enzymatic machinery that leads to expression of genes. One important epigenetic mechanism that appears to underlie the interaction between environmental factors, including diet, and our genome, is chemical modification of the DNA. The best understood of these modifications is methylation of cytosine residues in DNA. It is now recognized that the pattern of methylated cytosines throughout our genomes (the 'methylome') can change during development and in response to environmental cues, often with profound effects on gene expression. Many dietary constituents may indirectly influence genomic pathways that methylate DNA, and there is evidence for biochemical links between nutritional quality and mental health. Deficiency of both macro- and micronutrients has been associated with increased behavioural problems, and nutritional supplementation has proven efficacious in treatment of certain neuropsychiatric disorders. In this review we examine evidence from the fields of nutrition, developmental biology, and mental health that supports dietary impacts on epigenetic processes, particularly DNA methylation. We then consider whether such processes could underlie the demonstrated efficacy of dietary supplementation in treatment of mental disorders, and whether targeted manipulation of DNA methylation patterns using controlled dietary supplementation may be of wider clinical value.
Asunto(s)
Epigénesis Genética , Salud Mental , Estado Nutricional , Animales , Metilación de ADN/efectos de los fármacos , Dieta , Suplementos Dietéticos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Trastornos Mentales/diagnóstico , Trastornos Mentales/dietoterapia , Trastornos Mentales/genética , Micronutrientes/administración & dosificación , Micronutrientes/deficiencia , Trastornos del Neurodesarrollo/diagnóstico , Trastornos del Neurodesarrollo/dietoterapia , Trastornos del Neurodesarrollo/genéticaRESUMEN
Epithelial wound healing is an evolutionarily conserved process that requires coordination across a field of cells. Studies in many organisms have shown that cytosolic calcium levels rise within a field of cells around the wound and spread to neighboring cells, within seconds of wounding. Although calcium is a known potent second messenger and master regulator of wound-healing programs, it is unknown what initiates the rise of cytosolic calcium across the wound field. Here we use laser ablation, a commonly used technique for the precision removal of cells or subcellular components, as a tool to investigate mechanisms of calcium entry upon wounding. Despite its precise ablation capabilities, we find that this technique damages cells outside the primary wound via a laser-induced cavitation bubble, which forms and collapses within microseconds of ablation. This cavitation bubble damages the plasma membranes of cells it contacts, tens of microns away from the wound, allowing direct calcium entry from extracellular fluid into damaged cells. Approximately 45 s after this rapid influx of calcium, we observe a second influx of calcium that spreads to neighboring cells beyond the footprint of cavitation. The occurrence of this second, delayed calcium expansion event is predicted by wound size, indicating that a separate mechanism of calcium entry exists, corresponding to cell loss at the primary wound. Our research demonstrates that the damage profile of laser ablation is more similar to a crush injury than the precision removal of individual cells. The generation of membrane microtears upon ablation is consistent with studies in the field of optoporation, which investigate ablation-induced cellular permeability. We conclude that multiple types of damage, including microtears and cell loss, result in multiple mechanisms of calcium influx around epithelial wounds.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Cicatrización de Heridas/fisiología , Animales , Animales Modificados Genéticamente , Membrana Celular/fisiología , Citosol/metabolismo , Drosophila , Células Epiteliales/patología , Células Epiteliales/fisiología , Rayos Láser , Microscopía Confocal , Imagen de Colorante Sensible al Voltaje , Alas de AnimalesRESUMEN
The promoter of the human imprinted gene MEST is differentially methylated with respect to the parent of origin and contains several non B-DNA motifs that are capable of forming G-quadruplexes. These factors can contribute to a consistent allelic dropout (ADO) of the maternally methylated DNA during polymerase chain reaction (PCR) analysis of such gene regions. Here, we directly investigate the cause of allelic dropout by applying fluorescent techniques to visualize polymerase amplification and arrest during PCR of differentially methylated DNA templates. We demonstrate that polymerase arrest corresponds to previously characterized G-quadruplex-forming motifs at the MEST promoter region and occurs at equivalent sites on both methylated and nonmethylated DNA templates. However, during PCR, polymerase arrest can be observed on the methylated template for several cycles longer than on the nonmethylated template, and this results in an amplification lag and a lower yield of full length amplicons. We demonstrate that this delay in amplification is sufficient to cause complete ADO during PCR, providing a mechanistic basis for the previously observed genotyping error at this locus.
Asunto(s)
Metilación de ADN , G-Cuádruplex , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas , HumanosRESUMEN
Loss of one allele during polymerase chain reaction (PCR) amplification of DNA, known as allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation and G-quadruplex formation was previously described for a differentially methylated region of the human imprinted gene, MEST We now demonstrate that this parent-of-origin specific allelic dropout can potentially occur at several other genomic regions that display genomic imprinting and have propensity for G-quadruplex formation, including AIM1, BLCAP, DNMT1, PLAGL1, KCNQ1, and GRB10 These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the genome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations.