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Chronic pain exerts a significant impact on the quality of life, giving rise to both physical and psycho-social vulnerabilities. It not only leads to direct costs associated with treatments, but also results in indirect costs due to the reduced productivity of affected individuals. Chronic conditions can be improved by reducing modifiable risk factors. Various educational programs, including the Chronic Disease Self-Management Programme (CDSMP), have demonstrated the advantages of enhancing patient empowerment and health literacy. Nevertheless, their efficacy in addressing pain symptoms has received limited attention, especially concerning vulnerable populations. This research aims to assess the effectiveness of the CDSMP in alleviating pain among socio-economically vulnerable participants with chronic conditions. By accounting for a wide range of variables, and using data from the EFFICHRONIC project (EU health programme), we investigated the changes in pain levels after the intervention, among 1070 participants from five European countries. Our analyses revealed a significant reduction in pain following the intervention. This finding supports the notion that training programs can effectively ameliorate pain and alleviate its impact on the quality of life, particularly in vulnerable populations. Younger participants, as well as those with higher education levels and individuals experiencing higher levels of pain at baseline, were more likely to experience a reduction in their pain levels. These findings underscore the importance of recognising the social determinants of health. The study was registered at ClinicalTrials.gov (ISRCTN70517103)., (© 2024 The Authors. The International Journal of Health Planning and Management published by John Wiley & Sons Ltd.)

Different modes of gene regulation, such as histone modification, transcription factor binding, DNA methylation, and microRNA (miRNA) expression, are critical for the spatiotemporal expression of genes in developing orofacial tissues. Aberrant regulation in any of these modes may contribute to orofacial defects. Noncoding RNAs (ncRNAs), such as long ncRNAs (lncRNAs) and circular RNAs (circRNAs), have been shown to alter miRNA expression, and are thus emerging as novel contributors to gene regulation. Some of these appear to function as 'miRNA sponges', thereby diminishing the availability of these miRNAs to inhibit the expression of target genes. Such ncRNAs are also termed competitive endogenous RNAs (ceRNAs). Here, we examine emerging data that shed light on how lncRNAs and circRNAs may alter miRNA regulation, thus affecting orofacial development and potentially contributing to orofacial clefting., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Objective: Normal development of the embryonic orofacial region requires precise spatiotemporal coordination between numerous genes. MicroRNAs represent small, single-stranded, non-coding molecules that regulate gene expression. This study examines the role of microRNA-22 (miR-22) in murine orofacial ontogeny., Methods: Spatiotemporal and differential expression of miR-22 (mmu-miR-22-3p) within the developing secondary palate was determined by in situ hybridization and quantitative real-time PCR, respectively. Bioinformatic approaches were used to predict potential mRNA targets of miR-22 and analyze their association with cellular functions indispensable for normal orofacial ontogeny. An in vitro palate organ culture system was used to assess the role of miR-22 in secondary palate development., Results: There was a progressive increase in miR-22 expression from GD12.5 to GD14.5 in palatal processes. On GD12.5 and GD13.5, miR-22 was expressed in the future oral, nasal, and medial edge epithelia. On GD14.5, miR-22 expression was observed in the residual midline epithelial seam (MES), the nasal epithelium and the mesenchyme, but not in the oral epithelium. Inhibition of miR-22 activity in palate organ cultures resulted in failure of MES removal. Bioinformatic analyses revealed potential mRNA targets of miR-22 that may play significant roles in regulating apoptosis, migration, and/or convergence/extrusion, developmental processes that modulate MES removal during palatogenesis., Conclusions: Results from the current study suggest a key role for miR-22 in the removal of the MES during palatogenesis and that miR-22 may represent a potential contributor to the etiology of cleft palate.

Background: Neural tube (NT) morphogenesis is reliant on the proper temporospatial expression of numerous genes and synchronized crosstalk between diverse signaling cascades and gene regulatory networks governing key cellular processes. MicroRNAs (miRNAs), a group of small non-coding regulatory RNAs, execute defining roles in directing key canonical pathways during embryogenesis., Objective: In order to comprehend the mechanistic underpinnings of miRNA regulation of NT morphogenesis, we have identified in the current study various miRNAs and their target mRNAs associated with BMP signaling during critical stages of neurulation., Methods: We previously demonstrated the expression of several miRNAs during the critical stages of neurulation (gestational days (GD) 8.5, 9.0, and 9.5) employing high-sensitivity, high-coverage microarrays. In the present study, bioinformatic analyses were used to identify miRNAs differentially expressed (DE) in the embryonic NT that target messenger RNAs (mRNAs) associated with the bone morphogenetic protein (BMP) signaling pathway. RNAs extracted from the developing NT were hybridized to both miRNA and mRNA arrays to evaluate miRNA-mRNA interactions., Results: Bioinformatic analysis identified several DE miRNAs that targeted mRNAs encoding members of (and proteins associated with) the BMP signaling pathway - a signaling cascade central to normal NT development., Conclusion: Identification of the miRNAs and their mRNA targets associated with BMP signaling facilitates a better understanding of the crucial epigenetic mechanisms underlying normal NT development as well as the pathogenesis of NT defects. The current study supports the notion that miRNAs function as key regulators of neural tube morphogenesis via modulation of the BMP signaling cascade. Altered expression of these miRNAs during neurulation may therefore result in NT defects., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Introduction: The Chronic Disease Self-Management Programme (CDSMP) has resulted in improved health outcomes for patients. However, research has focused mainly on those with chronic conditions and has not extensively explored prevention programmes targeting individuals with specific vulnerability profiles., Aim: This study aimed to understand the effects of the CDSMP on the lived experience of vulnerable patients included in the EFFICHRONIC project in France, based on their needs and expectations before and after participation., Methods: We conducted a qualitative phenomenological semio-pragmatic study based on 37 in-depth interviews with 20 patients (20 before/17 after CDSMP)., Results: By transforming existential dimensions (identity, relationship with others and bodily experience), chronic illness generates new needs in the vulnerable person. By resonating with the expectations and needs of participants, the CDSMP induces motivation and a sense of belonging to a community of peers. It has enabled the participants to become actors of their own health until empowerment. Although some limitations are reported, the programme has awakened a desire in the participants to take better care of their health and to develop personal skills with, for some, a desire to become involved in health education., Conclusion: Our phenomenological approach highlighted the resonance between the programme (its design and implementation) and the lived experience of patients, as an effective element of empowerment. This necessitates training the facilitators to elicit the lived experience of patients. Furthermore, as a patient-centred approach is required, the facilitators need to learn how to adapt the design of the programme to the singularity of the patient., Patient or Public Contribution: Patients provided the data that were collected through in-depth interviews, and their experiences before and after the programme were analysed., (© 2022 The Authors. Health Expectations published by John Wiley & Sons Ltd.)

Environmental and genetic factors contribute significantly to the etiology of orofacial clefting, which is one of the most common of human congenital craniofacial malformations. Current biological thought now recognizes that epigenetics represents a fundamental contributing process in embryogenesis. Indeed, many of the mechanisms whereby environmental insults affect key pathways crucial for proper embryonic growth and development are increasingly thought to be mediated via the epigenome. Epigenetic regulators, such as microRNAs (miRNAs), play vital roles in the ontogeny of the orofacial region. Evidence for this comes from conditional knockouts of Dicer or DGCR8, genes encoding key enzymes in the miRNA biosynthetic machinery, in neural crest cells. Such knockouts result in a range of craniofacial/orofacial anomalies, including cleft palate and cleft lip. Epigenetic pathways may thus represent key vehicles in the regulation, and misregulation, of gene expression during normal and abnormal orofacial embryogenesis. Significant strides have been made in the last decade in identifying miRNAs and their target genes involved in lip and palate morphogenesis. Such morphogenetic processes include apoptosis, cell proliferation, cell differentiation, and epithelial-mesenchymal transition (EMT). While some of the miRNA-target gene interactions have been functionally validated, many exhibit causal relationships that await functional confirmation. A plethora of genes associated with cleft palate/cleft lip have now been identified that provides a veritable treasure trove of information that could be harnessed to identify novel miRNA candidates for further analysis. In this review, we summarize studies identifying miRNAs involved in various aspects of lip and palate morphogenesis and whose aberrant expression may result in orofacial clefts., (Copyright © 2022 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

It is estimated that 2-4% of live births will have a birth defect (BD). The availability of biomarkers for the prenatal detection of BDs will facilitate early risk assessment, prompt medical intervention and ameliorating disease severity. miRNA expression levels are often found to be altered in many diseases. There is, thus, a growing interest in determining whether miRNAs, particularly extracellular miRNAs, can predict, diagnose, or monitor BDs. These miRNAs, typically encapsulated in exosomes, are released by cells (including those of the fetus and placenta) into the extracellular milieu, such as blood, urine, saliva and cerebrospinal fluid, thereby enabling interaction with target cells. Exosomal miRNAs are stable, protected from degradation, and retain functionality. The observation that placental and fetal miRNAs can be detected in maternal serum, provides a strong rationale for adopting miRNAs as noninvasive prenatal biomarkers for BDs. In this mini-review, we examine the current state of research involving the use of miRNAs as prognostic and diagnostic biomarkers for BD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Folic acid is a nutrient essential for embryonic development. Folate deficiency can cause embryonic lethality or neural tube defects and orofacial anomalies. Folate receptor 1 (Folr1) is a folate binding protein that facilitates the cellular uptake of dietary folate. To better understand the biological processes affected by folate deficiency, gene expression profiles of gestational day 9.5 (gd9.5) Folr1 -/- embryos were compared to those of gd9.5 Folr1 +/+ embryos. The expression of 837 genes/ESTs was found to be differentially altered in Folr1 -/- embryos, relative to those observed in wild-type embryos. The 837 differentially expressed genes were subjected to Ingenuity Pathway Analysis. Among the major biological functions affected in Folr1 -/- mice were those related to 'digestive system development/function', 'cardiovascular system development/function', 'tissue development', 'cellular development', and 'cell growth and differentiation', while the major canonical pathways affected were those associated with blood coagulation, embryonic stem cell transcription and cardiomyocyte differentiation (via BMP receptors). Cellular proliferation, apoptosis and migration were all significantly affected in the Folr1 -/- embryos. Cranial neural crest cells (NCCs) and neural tube explants, grown under folate-deficient conditions, exhibited marked reduction in directed migration that can be attributed, in part, to an altered cytoskeleton caused by perturbations in F-actin formation and/or assembly. The present study revealed that several developmentally relevant biological processes were compromised in Folr1 -/- embryos., (Copyright © 2020 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) provide context-dependent transcriptional regulation of genes comprising signalling networks throughout the developing organism including morphogenesis of the embryonic neural tube (NT). Using a high-sensitivity, high-coverage microarray analysis platform, miRNA expression in the murine embryonic NT during the critical stages of its formation was examined. Analysis of a number of differentially expressed (DE) miRNAs enabled identification of several gene targets associated with cellular processes essential for normal NT development. Using computational pathway analysis, interactive biologic networks and functional relationships connecting DE miRNAs with their targeted messenger RNAs (mRNAs) were identified. Potential mRNA targets and a key signal transduction pathway governing critical cellular processes indispensable for normal mammalian neurulation were also identified. RNA preparations were also used to hybridize both miRNA arrays and mRNA arrays allowing miRNA-mRNA target analysis using data of DE miRNAs and DE mRNAs - co-expressed in the same developing NT tissue samples. Identification of these miRNA targets provides key insight into the epigenetic regulation of NT development as well as into potential mechanistic underpinning of NT defects. SIGNIFICANCE OF THE STUDY: This study underscores the premise that microRNAs are potential coordinators of normal neural tube (NT) formation, via regulation of the crucial, planar cell polarity pathway. Any alteration in their expression during neurulation would result in abnormal NT development., (© 2020 John Wiley & Sons Ltd.)

The adverse developmental effects of exposure to Cigarette Smoke (CS) during pregnancy are documented in this paper. These include low birth weight, congenital anomalies, preterm birth, fetal mortality and morbidity. The current biological thought now recognizes that epigenetics represents a fundamental contributing process in embryogenesis, and that the environment can have a profound effect on shaping the epigenome. It has become increasingly recognized that genes encoding microRNAs (miRNAs) might be potential loci for congenital disabilities. One means by which CS can cause developmental anomalies may be through epigenetic mechanisms involving altered miRNA expression. While several studies have focused on genes affected by CS during embryonic/ fetal development, there is a paucity of knowledge on the involvement of miRNAs in this process. This brief review summarizes the current state of knowledge in this area., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Maternal smoking during pregnancy represents a major public health concern increasing the risk for low birth weight, congenital anomalies, preterm birth, fetal mortality, and morbidity. In an effort to diminish adverse developmental effects of exposure to cigarette smoking, pregnant women, and women of reproductive age, are increasingly turning to electronic nicotine delivery systems (ENDS), such as e-cigarettes, as an alternative. Given that health risks associated with ENDS use during pregnancy are largely unknown, there is an acute need to determine risks vs. benefits of e-cigarette use by pregnant women. While the most recent Surgeon General's Report on the "Health Consequences of Smoking" states that "the evidence is sufficient to infer that nicotine adversely affects maternal and fetal health during pregnancy, contributing to multiple adverse outcomes," it remains unclear whether use of ENDS represents a "safer alternative" to tobacco smoking during pregnancy. This is due, in part, to the lack of sufficient and conclusive evidence concerning whether or not maternal e-cigarette use adversely affects embryonic/fetal development. While several recent developmental studies have challenged the safety of nicotine inhalation via ENDS, the true risks of smoking e-cigarettes during the first trimester of pregnancy-the period of organogenesis-are largely unknown. Moreover, evidence is emerging that even nicotine-free e-cigarette aerosols may harm the developing conceptus, suggesting that components of e-cigarette liquid, including flavorings, may be developmentally toxicity. Focused human epidemiological analyses, and carefully designed animal studies are critically needed to address the question of the safety of ENDS use during pregnancy., (© 2019 Wiley Periodicals, Inc.)

Prenatal exposure to arsenic, a naturally occurring toxic element, causes neural tube defects (NTDs) and, in animal models, orofacial anomalies. Since aberrant development or migration of cranial neural crest cells (CNCCs) can also cause similar anomalies within developing embryos, we examined the effects of in utero exposure to sodium arsenate on gene expression patterns in pure populations of CNCCs, isolated by fluorescence activated cell sorting (FACS), from Cre/LoxP reporter mice. Changes in gene expression were analyzed using Affymetrix GeneChip ® microarrays and expression of selected genes was verified by TaqMan quantitative real-time PCR. We report, for the first time, arsenate-induced alterations in the expression of a number of novel candidate genes and canonical cascades that may contribute to the pathogenesis of orofacial defects. Ingenuity Pathway and NIH-DAVID analyses revealed cellular response pathways, biological themes, and potential upstream regulators, that may underlie altered fetal programming of arsenate exposed CNCCs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background/rationale : The Chronic Disease Self-Management Programme (CDSMP) intervention is an evidence-based program that aims to encourage citizens with a chronic condition, as well as their caregivers, to better manage and maintain their own health. CDSMP intervention is expected to achieve greater health gains in citizens with a low socioeconomic position (SEP), because citizens with a low SEP have fewer opportunities to adhere to a healthy lifestyle, more adverse chronic conditions and a poorer overall health compared to citizens with a higher SEP. In the EFFICHRONIC project, CDSMP intervention is offered specifically to adults with a chronic condition and a low SEP, as well as to their caregivers (target population). Study objective : The objective of our study is to evaluate the benefits of offering CDSMP intervention to the target population. Methods : A total of 2500 participants (500 in each study site) are recruited to receive the CDSMP intervention. The evaluation study has a pre-post design. Data will be collected from participants before the start of the intervention (baseline) and six months later (follow up). Benefits of the intervention include self-management in healthy lifestyle, depression, sleep and fatigue, medication adherence and health-related quality of life, health literacy, communication with healthcare professionals, prevalence of perceived medical errors and satisfaction with the intervention. The study further includes a preliminary cost-effectiveness analysis with a time horizon of six months. Conclusion : The EFFICHRONIC project will measure the effects of the CDSMP intervention on the target population and the societal cost savings in five European settings.

Background: Development of the mammalian palate is dependent on precise, spatiotemporal expression of a panoply of genes. MicroRNAs (miRNAs), the largest family of noncoding RNAs, function as crucial modulators of cell and tissue differentiation, regulating expression of key downstream genes., Observations: Our laboratory has previously identified several developmentally regulated miRNAs, including miR-206, during critical stages of palatal morphogenesis. The current study reports spatiotemporal distribution of miR-206 during development of the murine secondary palate (gestational days 12.5-14.5)., Result and Conclusion: Potential cellular functions and downstream gene targets of miR-206 were investigated using functional assays and expression profiling, respectively. Functional analyses highlighted potential roles of miR-206 in governing TGFß- and Wnt signaling in mesenchymal cells of the developing secondary palate. In addition, altered expression of miR-206 within developing palatal tissue of TGFß3-/- fetuses reinforced the premise that crosstalk between this miRNA and TGFß3 is crucial for secondary palate development., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

5-Aza-2'-deoxycytidine (AzaD), also known as Decitabine, is a deoxycytidine analog that is typically used to activate methylated and silenced genes by promoter demethylation. However, a survey of the scientific literature indicates that promoter demethylation may not be the only (or, indeed, the major) mechanism by which AzaD affects gene expression. Regulation of gene expression by AzaD can occur in several ways, including some that are independent of DNA demethylation. Results from several studies indicate that the effect of AzaD on gene expression is highly context-dependent and can differ for the same gene under different environmental settings. This may, in part, be due to the nature of the silencing mechanism(s) involved - DNA methylation, repressive histone modifications, or a combination of both. The varied effects of AzaD on such context-dependent regulation of gene expression may underlie some of the diverse responses exhibited by patients undergoing AzaD therapy. In this review, we describe the salient properties of AzaD with particular emphasis on its diverse effects on gene expression, aspects that have barely been discussed in most reviews of this interesting drug.

In this study, we identify gene targets and cellular events mediating the teratogenic action(s) of 5-Aza-2'-deoxycytidine (AzaD), an inhibitor of DNA methylation, on secondary palate development. Exposure of pregnant mice (on gestation day (GD) 9.5) to AzaD for 12h resulted in the complete penetrance of cleft palate (CP) in fetuses. Analysis of cells of the embryonic first branchial arch (1-BA), in fetuses exposed to AzaD, revealed: 1) significant alteration in expression of genes encoding several morphogenetic factors, cell cycle inhibitors and regulators of apoptosis; 2) a decrease in cell proliferation; and, 3) an increase in apoptosis. Pyrosequencing of selected genes, displaying pronounced differential expression in AzaD-exposed 1-BAs, failed to reveal significant alterations in CpG methylation levels in their putative promoters or gene bodies. CpG methylation analysis suggested that the effects of AzaD on gene expression were likely indirect., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Defects in development of the secondary palate, which arise from the embryonic first branchial arch (1-BA), can cause cleft palate (CP). Administration of 5-Aza-2'-deoxycytidine (AzaD), a demethylating agent, to pregnant mice on gestational day 9.5 resulted in complete penetrance of CP in fetuses. Several genes critical for normal palatogenesis were found to be upregulated in 1-BA, 12h after AzaD exposure. MethylCap-Seq (MCS) analysis identified several differentially methylated regions (DMRs) in DNA extracted from AzaD-exposed 1-BAs. Hypomethylated DMRs did not correlate with the upregulation of genes in AzaD-exposed 1-BAs. However, most DMRs were associated with endogenous retroviral elements. Expression analyses suggested that interferon signaling was activated in AzaD-exposed 1-BAs. Our data, thus, suggest that a 12-h in utero AzaD exposure demethylates and activates endogenous retroviral elements in the 1-BA, thereby triggering an interferon-mediated response. This may result in the dysregulation of key signaling pathways during palatogenesis, causing CP., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Exposure to cigarette smoke during development is linked to neurodevelopmental delays and cognitive impairment including impulsivity, attention deficit disorder, and lower IQ. Utilizing a murine experimental model of "active" inhalation exposure to cigarette smoke spanning the entirety of gestation and through human third trimester equivalent hippocampal development [gestation day 1 (GD1) through postnatal day 21 (PD21)], we examined hippocampus proteome and metabolome alterations present at a time during which developmental cigarette smoke exposure (CSE)-induced behavioral and cognitive impairments are evident in adult animals from this model system. At six month of age, carbohydrate metabolism and lipid content in the hippocampus of adult offspring remained impacted by prior exposure to cigarette smoke during the critical period of hippocampal ontogenesis indicating limited glycolysis. These findings indicate developmental CSE-induced systemic glucose availability may limit both organism growth and developmental trajectory, including the capacity for learning and memory., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cigarette smoke exposure (CSE) during gestation and early development suppresses the growth trajectory in offspring. In prior studies utilizing a mouse model of 'active' developmental CSE (GD1-PD21), low birth weight induced by CSE persisted throughout the neonatal period and was present at the cessation of exposure at weaning with proportionally smaller kidney mass that was accompanied by impairment of carbohydrate metabolism. In the present study, littermates of those characterized in the prior study were maintained until 6 months of age at which time the impact of developmental CSE on the abundance of proteins associated with cellular metabolism in the kidney was examined. Kidney protein abundances were examined by 2D-SDS-PAGE based proteome profiling with statistical analysis by Partial Least Squares-Discriminant Analysis. Key findings of this study include a persistence of impact of developmental CSE past the original exposure period on the nucleic acid and carbohydrate metabolism networks and oxidant scavenging pathways., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Utilizing a mouse model of 'active' developmental cigarette smoke exposure (CSE) [gestational day (GD) 1 through postnatal day (PD) 21] characterized by offspring low birth weight, the impact of developmental CSE on liver proteome profiles of adult offspring at 6 months of age was determined. Liver tissue was collected from Sham- and CSE-offspring for 2D-SDS-PAGE based proteome analysis with Partial Least Squares-Discriminant Analysis (PLS-DA). A similar study conducted at the cessation of exposure to cigarette smoke documented decreased gluconeogenesis coupled to oxidative stress in weanling offspring. In the current study, exposure throughout development to cigarette smoke resulted in impaired hepatic carbohydrate metabolism, decreased serum glucose levels, and increased gluconeogenic regulatory enzyme abundances during the fed-state coupled to decreased expression of SIRT1 as well as increased PEPCK and PGC1α expression. Together these findings indicate inappropriately timed gluconeogenesis that may reflect impaired insulin signaling in mature offspring exposed to 'active' developmental CSE., (Copyright © 2016 Elsevier Inc. All rights reserved.)

p300 is a multifunctional transcriptional coactivator that interacts with numerous transcription factors and exhibits protein/histone acetyltransferase activity. Loss of p300 function in humans and in mice leads to craniofacial defects. In this study, we demonstrated that inhibition of p300 histone acetyltransferase activity with the compound, C646, altered the expression of several genes, including Cdh1 (E-cadherin) in mouse maxillary mesenchyme cells, which are the cells that give rise to the secondary palate. The increased expression of plasma membrane-bound E-cadherin was associated with reduced cytosolic β-catenin, that led to attenuated signaling through the canonical Wnt pathway. Furthermore, C646 reduced both cell proliferation and the migratory ability of these cells. These results suggest that p300 histone acetyltransferase activity is critical for Wnt-dependent palate mesenchymal cell proliferation and migration, both processes that play a significant role in morphogenesis of the palate., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Orofacial clefts, the most prevalent of developmental anomalies, occur with a frequency of 1 in 700 live births. Maternal cigarette smoking during pregnancy represents a risk factor for having a child with a cleft lip and/or cleft palate. Using primary cultures of first branchial arch-derived cells (1-BA cells), which contribute to the formation of the lip and palate, the present study addressed the hypothesis that components of cigarette smoke alter global DNA methylation, and/or expression of DNA methyltransferases (Dnmts) and various methyl CpG-binding proteins. Primary cultures of 1-BA cells, exposed to 80μg/mL cigarette smoke extract (CSE) for 24h, exhibited a >13% decline in global DNA methylation and triggered proteasomal-mediated degradation of Dnmts (DNMT-1 and -3a), methyl CpG binding protein 2 (MeCP2) and methyl-CpG binding domain protein 3 (MBD-3). Pretreatment of 1-BA cells with the proteasomal inhibitor MG-132 completely reversed such degradation. Collectively, these data allow the suggestion of a potential epigenetic mechanism underlying maternal cigarette smoke exposure-induced orofacial clefting., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Clefting of the secondary palate is the most common birth defect in humans. Midline fusion of the bilateral palatal processes is thought to involve apoptosis, epithelial to mesenchymal transition, and cell migration of the medial edge epithelium (MEE), the specialized cells of the palate that mediate fusion of the palatal processes during fetal development. Data presented in this manuscript are the result of analyses designed to identify microRNAs that are expressed and regulated by TGFβ3 in developing palatal MEE. The expression of 7 microRNAs was downregulated and 1 upregulated in isolated MEE from wildtype murine fetuses on gestational day (GD) 13.5 to GD14.5 (prior to and during epithelial fusion of the palatal processes, respectively). Among this group were miRNAs linked to apoptosis (miR-378) and epithelial to mesenchymal transformation (miR-200b, miR-205, and miR-93). Tgfβ3(-/-) fetuses, which present with a complete and isolated cleft of the secondary palate, exhibited marked dysregulation of distinct miRNAs both in the palatal MEE and mesenchyme when compared to comparable wild-type tissue. These included, among others, miRNAs known to affect apoptosis (miR-206 and miR-186). Dysregulation of miRNAs in the mesenchyme underlying the palatal MEE of Tgfβ3(-/-) fetuses is also discussed in relation to epithelial-mesenchymal transformation of the MEE. These results are the first systematic analysis of the expression of microRNAs in isolated fetal palatal epithelium and mesenchyme. Moreover, analysis of the Tgfβ3 knockout mouse model has enabled identification of miRNAs with altered expression that may contribute to the cleft palate phenotype.

Background: Transforming growth factor-β3 (TGF-β3) plays a central role in mediating secondary palate fusion along the facial midline. However, the mechanisms by which TGF-β3 functions during secondary palate fusion are still poorly understood., Results: We found that mouse cytokeratin 6α and 17 mRNAs were expressed exclusively in the palate medial edge epithelium on embryonic day 14.5, and this expression was completely abolished in Tgf-β3 mutant embryos. In contrast, we found that Jagged2 was initially expressed throughout the palate epithelium, but was specifically down-regulated in the medial edge epithelium during palatal fusion. Jagged2 down-regulation was regulated by TGF-β3, since Jagged2 was persistently expressed in palatal medial edge epithelium in Tgf-β3 null mutant embryos. Moreover, addition of DAPT, a specific inhibitor of Notch signaling, partially rescued the fusion defects in Tgf-β3 null mutant palatal shelves., Conclusions: Based on these results, together with the previous study indicating that the loss of Jagged2 function promotes embryonic oral epithelial fusion, we concluded that TGF-β3 mediates palate fusion in part by down-regulating Jagged2 expression in palatal medial edge epithelium. In addition, cytokeratin 6α and 17 are two TGF-β3 downstream target genes in palate medial edge epithelium differentiation., (© 2014 Wiley Periodicals, Inc.)

Exposure to cigarette smoke during development is linked to neurodevelopmental delays and cognitive impairment including impulsivity, attention deficit disorder, and lower IQ. However, brain region specific biomolecular alterations induced by developmental cigarette smoke exposure (CSE) remain largely unexplored. In the current molecular phenotyping study, a mouse model of 'active' developmental CSE (serum cotinine > 50 ng/mL) spanning pre-implantation through third trimester-equivalent brain development (gestational day (GD) 1 through postnatal day (PD) 21) was utilized. Hippocampus tissue collected at the time of cessation of exposure was processed for gel-based proteomic and non-targeted metabolomic profiling with partial least squares-discriminant analysis (PLS-DA) for selection of features of interest. Ingenuity pathway analysis was utilized to identify candidate molecular and metabolic pathways impacted within the hippocampus. CSE impacted glycolysis, oxidative phosphorylation, fatty acid metabolism, and neurodevelopment pathways within the developing hippocampus., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Environmental factors contribute to the etiology of cleft palate (CP). Environmental factors can also affect gene expression via alterations in DNA methylation suggesting a possible mechanism for the induction of CP. Identification of genes methylated during development of the secondary palate provides the basis for examination of the means by which environmental factors may adversely influence palatal ontogeny. We previously characterized the methylome of the developing murine secondary palate focusing primarily on protein- encoding genes. We now extend this study to include methylated microRNA (miRNA) genes. A total of 42 miRNA genes were found to be stably methylated in developing murine palatal tissue. Twenty eight of these were localized within host genes. Gene methylation was confirmed by pyrosequencing of selected miRNA genes. Integration of methylated miRNA gene and expression datasets identified 62 miRNAs, 69% of which were non-expressed. For a majority of genes (83%), upstream CpG islands (CGIs) were highly methylated suggesting down-regulation of CGI-associated promoters. DAVID and IPA analyses indicated that both expressed and non-expressed miRNAs target identical signaling pathways and biological processes associated with palatogenesis. Furthermore, these analyses also identified novel signaling pathways whose roles in palatogenesis remain to be elucidated. In summary, we identify methylated miRNA genes in the developing murine secondary palate, correlate miRNA gene methylation with expression of their cognate miRNA transcripts, and identify pathways and biological processes potentially mediated by these miRNAs.

Increased accumulation of p53 tumor suppressor protein is an early response to low-level stressors. To investigate the fate of mitochondrial-sequestered p53, mouse embryonic fibroblast cells (MEFs) on a p53-deficient genetic background were transfected with p53-EGFP fusion protein led by a sense (m53-EGFP) or antisense (c53-EGFP) mitochondrial import signal. Rotenone exposure (100nM, 1h) triggered the translocation of m53-EGFP from the mitochondrion to the nucleus, thus shifting the transfected cells from a mitochondrial p53 to a nuclear p53 state. Antibodies for p53 serine phosphorylation or lysine acetylation indicated a different post-translational status of recombinant p53 in the nucleus and mitochondrion, respectively. These data suggest that cycling of p53 through the mitochondria may establish a direct pathway for p53 signaling from the mitochondria to the nucleus during mitochondrial dysfunction. PK11195, a pharmacological ligand of mitochondrial TSPO (formerly known as the peripheral-type benzodiazepine receptor), partially suppressed the release of mitochondria-sequestered p53. These findings support the notion that p53 function mediates a direct signaling pathway from the mitochondria to nucleus during mitochondrial dysfunction., (© 2013. Published by Elsevier Inc. All rights reserved.)

In this study, we demonstrate that sphere formation triggers immortalization and stable reprogramming of mouse fibroblasts. Cell contact signaling in spheres causes downregulation of the epithelial-to-mesenchymal transition transcription factor Zeb1 leading to rapid mesenchymal-to-epithelial transition. Hypoxia within spheres together with loss of Zeb1 repression synergize to cause superinduction of Hif1a, which in turn leads to induction of the DNA demethylase Aid/Aicda, demethylation of the Oct4 promoter/enhancer and multipotency. Oct4 and Nanog expression diminish when cells are removed from the hypoxic environment of spheres and placed in monolayer culture, but the cells retain multipotential capacity, demonstrating stable reprogramming and a gene expression pattern resembling adult stem cells. Oct4 has been shown to induce Dnmt1 in mesenchymal stem cells, and we link Oct4 and Dnmt1 to silencing of cell cycle inhibitory cyclin dependent kinase inhibitors and Arf, and immortalization of the reprogrammed fibroblasts. Sphere formation then represents a novel and rapid protocol for immortalization and stable reprogramming of fibroblasts to multipotency that does not require exogenous expression of a stem cell factor or a lineage-specifying transcription factor., (Copyright © 2013 AlphaMed Press.)

Fetal alcohol syndrome (FAS), presenting with a constellation of neuro-/psychological, craniofacial and cardiac abnormalities, occurs frequently in offspring of women who consume alcohol during pregnancy, with a prevalence of 1-3 per 1000 livebirths. The present study was designed to test the hypothesis that alcohol alters global DNA methylation, and modulates expression of the DNA methyltransferases (DNMTs) and various methyl CpG-binding proteins. Murine embryonic fibroblasts (MEFs), utilized as an in vitro embryonic model system, demonstrated ∼5% reduction in global DNA methylation following exposure to 200mM ethanol. In addition, ethanol induced degradation of DNA methyltransferases (DNMT-1, DNMT-3a, and DNMT-3b), as well as the methyl CpG-binding proteins (MeCP-2, MBD-2 and MBD-3), in MEF cells by the proteasomal pathway. Such degradation could be completely rescued by pretreatment of MEF cells with the proteasomal inhibitor, MG-132. These data support a potential epigenetic molecular mechanism underlying the pathogenesis of FAS during mammalian development., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Environmental factors contribute to the etiology of cleft palate (CP). Identification of genes that are methylated during development of the secondary palate will contribute to a better understanding of the gene-environment link contributing to CP., Methods: Genomic DNA fragments from secondary palate tissue from gestational days (GDs) 12 to 14 were subjected to Selective Enrichment of Methylated DNA (SEMD) and used to probe NimbleGen 2.1M mouse promoter arrays. Input (control) and SEMD samples were labeled with Cy3 and Cy5, respectively, and used for array hybridization (three arrays per GD). Data were analyzed using the Bioconductor package Ringo. Gene methylation was verified by pyrosequencing analysis and expression by quantitative real-time PCR., Results: A total of 5577 methylated genes were identified during palate development: (1) 74% of genes were methylated on all three GDs; (2) CpG islands accounted for only 30% of methylated regions of interest (MRIs); (3) location of MRIs was more often observed in gene bodies (73%) than in promoters; (4) evaluation of MRIs on GDs 12-14 revealed no significant differentially methylated regions; (5) DAVID analysis of MRIs revealed that the cadherin and Wnt signaling pathways, as well as pathways involved in proteoglycan synthesis, were significantly enriched for methylated genes., Conclusions: Our prior studies identified differentially expressed mRNAs and microRNAs in the developing palate. The current study complements these studies by identifying genes whose expression may be altered as a result of DNA methylation., (Copyright © 2013 Wiley Periodicals, Inc.)

Loss of Prdm16 expression in the mouse leads to a complete cleft of the secondary palate. We have now determined changes in gene expression in the secondary palates of Prdm16(-/-) fetuses in an attempt to reveal the mechanism(s) leading to the failure of palate closure in these mice. Defined pathway-based polymerase chain reaction arrays were used to analyze the expression of genes associated with the extracellular matrix and the transforming growth factor-β and bone morphogenetic protein signaling networks, perturbations of which can lead to palatal clefting. Loss of Prdm16 expression in the secondary palate leads to alterations in numerous genes within these groups, many of which have been linked to chondrogenesis and osteogenesis. The expression of several genes linked to bone development was significantly changed in the developing secondary palate. Analysis of gene expression in the mandibles of Prdm16(-/-) fetuses revealed similar alterations in the same gene set. These data suggest that one function of Prdm16 is the regulation of genes that play a role in the differentiation of mesenchymal cells into chondro-/osteocytes.

Background: MicroRNAs (miRNAs) constitute the largest family of noncoding RNAs involved in gene silencing and represent critical regulators of cell and tissue differentiation. Microarray expression profiling of miRNAs is an effective means of acquiring genome-level information of miRNA activation and inhibition, as well as the potential regulatory role that these genes play within a biological system. As with mRNA expression profiling arrays, miRNA microarrays come in a variety of platforms from numerous manufacturers, and there are a multitude of techniques available for reducing and analyzing these data., Results: In this paper, we present an analysis of a typical two-color miRNA microarray experiment using publicly available packages from R and Bioconductor, the open-source software project for the analysis of genomic data. Covered topics include visualization, normalization, quality checking, differential expression, cluster analysis, miRNA target identification, and gene set enrichment analysis. Many of these tools carry-over from the analysis of mRNA microarrays, but with some notable differences that require special attention. The paper is presented as a "compendium" which, along with the accompanying R package MmPalateMiRNA, contains all of the experimental data and source code to reproduce the analyses contained in the paper., Conclusions: The compendium presented in this paper will provide investigators with an access point for applying the methods available in R and Bioconductor for analysis of their own miRNA array data.

Although maternal cigarette smoking during pregnancy is a well-documented risk factor for a variety of adverse pregnancy outcomes, how prenatal cigarette smoke exposure affects postnatal neurobehavioral/cognitive development remains poorly defined. In order to investigate the cause of an altered behavioral phenotype, mice developmentally exposed to a paradigm of 'active' maternal cigarette smoke is needed. Accordingly, cigarette smoke exposed (CSE) and air-exposed C57BL/6J mice were treated for 6h per day in paired inhalation chambers throughout gestation and lactation and were tested for neurobehavioral effects while controlling for litter effects. CSE mice exhibited less than normal anxiety in the elevated zero maze, transient hypoactivity during a 1h locomotor activity test, had longer latencies on the last day of cued Morris water maze testing, impaired hidden platform learning in the Morris water maze during acquisition, reversal, and shift trials, and impaired retention for platform location on probe trials after reversal but not after acquisition or shift. CSE mice also showed a sexually dimorphic response in central zone locomotion to a methamphetamine challenge (males under-responded and females over-responded), and showed reduced anxiety in the light-dark test by spending more time on the light side. No differences on tests of marble burying, acoustic startle response with prepulse inhibition, Cincinnati water maze, matching-to-sample Morris water maze, conditioned fear, forced swim, or MK-801-induced locomotor activation were found. Collectively, the data indicate that developmental cigarette smoke exposure induces subnormal anxiety in a novel environment, impairs spatial learning and reference memory while sparing other behaviors (route-based learning, fear conditioning, and forced swim immobility). The findings add support to mounting evidence that developmental cigarette smoke exposure has long-term adverse effects on brain function., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Exposure to an imbalance of nutrients prior to conception and during critical developmental periods can have lasting consequences on physiological processes resulting in chronic diseases later in life. Developmental programming has been shown to involve structural and functional changes in important tissues. The aim of the present study was to investigate whether early life diet has a programming effect on the mammary gland. Wild-type mice were exposed from 2 weeks prior to conception to 6 weeks of age to a regular low-fat diet, or to high-fat diets based on either corn oil or flaxseed oil. At 6 weeks of age, all mice were shifted to the regular low-fat diet until termination at 10 weeks of age. Early life exposure to a high-fat diet, either high in n-6 (corn oil) or in n-3 (flaxseed oil) polyunsaturated fatty acids, did not affect birth weight, but resulted in an increased body weight at 10 weeks of age. Transcriptome analyses of the fourth abdominal mammary gland revealed differentially expressed genes between the different treatment groups. Exposure to high-fat diet based on flaxseed oil, but not on corn oil, resulted in regulation of pathways involved in energy metabolism, immune response and inflammation. Our findings suggest that diet during early life indeed has a lasting effect on the mammary gland and significantly influences postnatal body weight gain, metabolic status, and signaling networks in the mammary gland of female offspring.

Cigarette smoke is composed of over 4000 chemicals many of which are strong oxidizing agents and chemical carcinogens. Chronic cigarette smoke exposure (CSE) induces mild alterations in liver histology indicative of toxicity though the molecular pathways underlying these alterations remain to be explored. Utilizing a mouse model of 'active' developmental CSE (gestational day (GD) 1 through postnatal day (PD) 21; cotinine >50ng/mL) characterized by low birth weight offspring, the impact of developmental CSE on liver protein abundances was determined. On PD21, liver tissue was collected from pups for 2D SDS-PAGE based proteome analysis with statistical analysis by Partial Least Squares-Discriminant Analysis (PLS-DA). Protein spots of interest were identified by ESI-MS/MS with impacted molecular pathways identified by Ingenuity Pathway Analysis. Developmental CSE decreased the abundance of proteins associated with the small molecule biochemistry (includes glucose metabolism), lipid metabolism, amino acid metabolism, and inflammatory response pathways. Decreased gluconeogenic enzyme activity and lysophosphatidylcholine availability following developmental CSE were found and supports the impact of CSE on these pathways. Proteins with increased abundance belonged to the cell death and drug metabolism networks. Liver antioxidant enzyme abundances [glutathione-S-transferase (GST) and peroxiredoxins] were also altered by CSE, but GST enzymatic activity was unchanged. In summary, cigarette smoke exposure spanning pre- and post-natal development resulted in persistent decreased offspring weights, decreased abundances of liver metabolic proteins, decreased gluconeogenic activity, and altered lipid metabolism. The companion paper details the kidney proteome alterations in the same offspring., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The Brenner hypothesis states that a congenital reduction in nephron number predisposes to adult-onset hypertension and renal failure. The reduction in nephron number induced by proportionally smaller kidney mass may predispose offspring to glomerular hyperfiltration with maturity onset obesity. Developmental cigarette smoke exposure (CSE) results in intrauterine growth retardation with a predisposition to obesity and cardiovascular disease at maturity. Utilizing a mouse model of 'active' developmental CSE (gestational day [GD] 1-postnatal day [PD] 21; cotinine>50 ng/mL) characterized by persistently smaller offspring with proportionally decreased kidney mass, the present study examined the impact of developmental CSE on the abundance of proteins associated with cellular metabolism in the kidney. Following cessation of CSE on PD21, kidney tissue was collected from CSE and Sham exposed pups for 2D-SDS-PAGE based proteome profiling with statistical analysis by partial least squares-discriminant analysis (PLS-DA) with affected molecular pathways identified by ingenuity pathway analysis. Proteins whose expression in the kidney were affected by developmental CSE belonged to the inflammatory disease, cell to cell signaling/interaction, lipid metabolism, small molecule biochemistry, cell cycle, respiratory disease, nucleic acid and carbohydrate metabolism networks. The present findings indicate that developmental CSE alters the kidney proteome. The companion paper details the liver proteome alterations in the same offspring., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The transcription factor PRDM16 regulates differentiation of brown adipocyte tissue in mice. Recently, however, it has been demonstrated that genetic knockout of Prdm16 in mice leads to a complete cleft of the secondary palate in offspring. To identify genes whose promoters bind PRDM16 in mouse embryonic palate/maxillary mesenchymal cells, we have conducted a chromatin immunoprecipitation-promoter microarray analysis (ChIP-Chip). One hundred and twenty-two gene promoters were identified as capable of binding PRDM16. These could be functionally grouped to include those on genes linked to muscle development, chondrogenesis and osteogenesis, in addition to many transcription factors. These results suggest that PRDM16 may play a role in differentiation of mesenchymal cells in the embryonic secondary palate that contribute to the anterior, bony palate and posterior, muscular palate.

Background: The presence of strain-specific modifier genes is known to modulate the phenotype and pathophysiology of mice harboring genetically engineered mutations. Thus, identification of genetic modifier genes is requisite to understanding control of phenotypic expression. c-Ski is a transcriptional regulator. Ski(-/-) mice on a C57BL6J (B6) background exhibit facial clefting, while Ski(-/-) mice on a 129P3 (129) background present with exencephaly., Methods: In the present study, oligonucleotide-based gene expression profiling was used to identify potential strain-specific modifier gene candidates present in wild type mice of B6 and 129 genetic backgrounds. Changes in gene expression were verified by TaqMan quantitative real-time PCR., Results: Steady-state levels of 89 genes demonstrated a significantly higher level of expression, and those of 68 genes demonstrated a significantly lower level of expression in the developing neural tubes from embryonic day (E) 8.5, B6 embryos when compared to expression levels in neural tubes derived from E 8.5, 129 embryos., Conclusions: Based on the results from the current comparative microarray study, and taking into consideration a number of relevant published reports, several potential strain-specific gene candidates, likely to modify the craniofacial phenotypes in various knockout mouse models have been identified., (Copyright © 2012 Wiley Periodicals, Inc.)

Orofacial clefts occur with a frequency of 1 to 2 per 1000 live births. Cleft palate, which accounts for 30% of orofacial clefts, is caused by the failure of the secondary palatal processes--medially directed, oral projections of the paired embryonic maxillary processes--to fuse. Both gene mutations and environmental effects contribute to the complex etiology of this disorder. Although much progress has been made in identifying genes whose mutations are associated with cleft palate, little is known about the mechanisms by which the environment adversely influences gene expression during secondary palate development. An increasing body of evidence, however, implicates epigenetic processes as playing a role in adversely influencing orofacial development. Epigenetics refers to inherited changes in phenotype or gene expression caused by processes other than changes in the underlying DNA sequence. Such processes include, but are not limited to, DNA methylation, microRNA effects, and histone modifications that alter chromatin conformation. In this review, we describe our current understanding of the possible role epigenetics may play during development of the secondary palate. Specifically, we present the salient features of the embryonic palatal methylome and profile the expression of numerous microRNAs that regulate protein-encoding genes crucial to normal orofacial ontogeny.