Detection of long non-coding RNAs in human breastmilk extracellular vesicles: Implications for early child development

<p>Breastmilk has many documented beneficial effects on the developing human infant, but the components of breastmilk that influence these developmental pathways have not been fully elucidated. Increasing evidence suggests that non-coding RNAs encapsulated in extracellular vesicles (EVs) represent an important mechanism of communication between the mother and child. Long non-coding RNAs (lncRNAs) are of particular interest given their key role in gene expression and development. However, it is not known whether breastmilk EVs contain lncRNAs. We used qRT-PCR to determine whether EVs isolated from human breastmilk contain lncRNAs previously reported to be important for developmental processes. We detected 55 of the 87 screened lncRNAs in EVs from the 30 analyzed breastmilk samples, and CRNDE, DANCR, GAS5, SRA1 and ZFAS1 were detected in >90% of the samples. GAS5, SNHG8 and ZFAS1 levels were highly correlated (Spearman's rho > 0.9; <i>P</i> < 0.0001), which may indicate that the loading of these lncRNAs into breastmilk EVs is regulated by the same pathways. The detected lncRNAs are important epigenetic regulators involved in processes such as immune cell regulation and metabolism. They may target a repertoire of recipient cells in offspring and could be essential for child development and health. Further experimental and epidemiological studies are warranted to determine the impact of breastmilk EV-encapsulated lnRNAs in mother to child signaling.</p

Changes in DNA Methylation in Mouse Lungs after a Single Intra-Tracheal Administration of Nanomaterials

<div><p>Aims</p><p>This study aimed to investigate the effects of nanomaterial (NM) exposure on DNA methylation.</p><p>Methods and Results</p><p>Intra-tracheal administration of NM: gold nanoparticles (AuNPs) of 5-, 60- and 250-nm diameter; single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) at high dose of 2.5 mg/kg and low dose of 0.25 mg/kg for 48 h to BALB/c mice. Study showed deregulations in immune pathways in NM-induced toxicity <i>in vivo</i>. NM administration had the following DNA methylation effects: AuNP 60 nm induced CpG hypermethylation in <i>Atm</i>, <i>Cdk</i> and <i>Gsr</i> genes and hypomethylation in <i>Gpx</i>; <i>Gsr</i> and <i>Trp53</i> showed changes in methylation between low- and high-dose AuNP, 60 and 250 nm respectively, and AuNP had size effects on methylation for <i>Trp53</i>.</p><p>Conclusion</p><p>Epigenetics may be implicated in NM-induced disease pathways.</p></div

Bronchoalveolar lavage (BAL) fluid analysis in mice exposed to nanomaterial.

<p>a): total cell count; b): differential cell count.; c): uptake/association by/with BAL macrophages. For clarity of presentation in panel b, significant groups are not annotated. In panel b, macrophages count was significant in following exposure groups: AuNPs 5nm 50μg and AuNPs 60nm 50μg compared to the vehicle; AuNP 5nm 50μg compared to AuNP 250nm 5μg; and AuNPs 60 nm 50μg compared to the AuNP 250nm 5μg dose categories. Neutrophils count was significant in following exposure groups: SWCNT 50μg and MWCNTs 50μg compared to vehicle. Lymphocytes count was significant in following exposure groups: SWCNT 50μg and MWCNTs 50μg compared to vehicle. In panel c, 100 macrophages were randomly counted for the microscopic presence or absence of NM aggregated inside the cytoplasm at 1000X magnification. Representative images of macrophage d): vehicle; e): AuNPs 5nm; f): AuNPs 60nm; g): AuNPs 250 nm; h): SWCNTs; i): MWCNTs. In panel a and c; the box plot describes the median (line across the box), inter-quartile range and maximum and minimum values (whiskers). Outliers are shown as colored circles outside the ends of whiskers. Data in panel b is represented as median ±SD. Asterisk sign (*) shows significance levels at <i>p</i> = 0.05 (dunn’s statistics). Gold nanoparticles: AuNPs; single-walled- and multi-walled carbon nanotubes: SWCNTs and MWCNTs.</p

Global DNA methylation (5mC) and hydroxymethylation (5hmC) in lungs.

<p>a): no significant effects (Wilcoxon test) of gold nanoparticle (AuNPs) and single-walled and multi-walled carbon nanotubes (SWCNTs and MWCNTs) were observed on 5mC (<i>p</i> = 0.667 and 0.284 respectively). b): also no significant effect of AuNPs exposure on lung 5hmC were observed (<i>p</i> = 0.107). However, CNTs exposure showed significant effect on 5hmC (<i>p</i> = 0.024) levels by Wilcoxon statistics, while no group remained significant after multiple comparisons (Dunn all pairs post-hoc). In panel a and b; box plot describes the median (line across the box), inter-quartile range and maximum and minimum values (whiskers). Outliers are shown as colored circles outside the ends of whiskers.</p

Effect of shapes of single and multiwalled carbon nanotubes (SWCNTs, MWCNTs) upon exposure on <i>Atm</i> gene methylation.

<p>Bars connect exposure groups with significant methylation difference. In panels, box plot describes the median (line across the box), inter-quartile ranges and maximum and minimum values (whiskers). Outliers are shown as colored circles outside the ends of whiskers. Asterisk sign (*) shows significance levels at <i>p</i> = 0.5 (dunn’s statistics). <i>Atm</i>: ataxia telangiectasia mutated; <i>Trp53</i>: tumor protein P53.</p

Effect of nanoparticles (NPs) dose and size on gene promoter methylation upon exposure.

<p>Bars connect exposure groups with significant methylation difference, a-d): effects of gold NPs (AuNPs) exposure dose on promoter methylation levels of <i>Gsr</i> (a, and b), <i>Trp53</i> (c) in lungs, and <i>Pparg</i> (d) genes in blood. AuNPs size effect on CpG methylation of <i>Trp53</i> gene was observed between 60 nm and 250 nm AuNPs. In panels, box plot describes the median (line across the box), inter-quartile range and maximum and minimum values (whiskers). Outliers are shown as colored circles outside the ends of whiskers. Asterisk sign (*) shows significance levels at <i>p</i> = 0.5 (dunn’s statistics). <i>Gsr</i>: glutathione reductase; <i>Trp53</i>: tumor protein P53; <i>Pparg</i>: peroxisome proliferator-activated receptor gamma.</p

Effect of nanomaterial (NM) exposure variables on methylation of promoters of genes.

<p>Effect of nanomaterial (NM) exposure variables on methylation of promoters of genes.</p

Effect of nanomaterial (NM) exposure on gene promoter methylation.

<p>Bars connect exposure groups with significant methylation difference, a-d): effects of gold nanoparticles (AuNPs) exposure on promoter methylation levels of <i>Atm</i> (a), <i>Cdk</i> (b), <i>Gp x</i>(c), and <i>Gsr</i> (d) genes in lungs. Effect of single and multi-walled carbon nanotubes (SWCNTs, MWCNTs) exposure on gene promoter methylation levels of <i>Atm</i> (e) gene in lungs. In panels, box plot describes the median (line across the box), inter-quartile range and maximum and minimum values (whiskers). Outliers are shown as colored circles outside the ends of whiskers. Asterisk sign (*) shows significance levels at <i>p</i> = 0.5 (dunn’s statistics). <i>Atm</i>: ataxia telangiectasia mutated; <i>Cdk</i>; cyclin-dependent kinase; <i>Gsr</i>: glutathione reductase; <i>Gpx</i>: glutathione peroxidase.</p

Odds ratios (95% CIs) of large for gestational age and macrosomia at birth and overweight at 1–5 years old according to joint status of maternal pre-pregnancy body mass index and gestational weight gain.

<p>Odds ratios (95% CIs) of large for gestational age and macrosomia at birth and overweight at 1–5 years old according to joint status of maternal pre-pregnancy body mass index and gestational weight gain.</p

Neonatal and childhood major outcomes at birth and 1–5 years old according to maternal pre-pregnancy body mass index and gestational weight gain categories.

<p>Data represent mean (SE), or percentage.</p><p>^a, Adjusted for maternal age, family history of diabetes, education, family income, gestational diabetes treatment during pregnancy, gestational weeks of birth and infant feeding.</p><p>^b, Adjusted for above variables and also birth weight for length for gestational age Z score.</p><p>* P <0.05 for Groups of A and B;</p><p>^# P<0.05 for groups of A and C;</p><p>^{^} P<0.05 for groups of B and C;</p><p>^& P for groups of D and E;</p><p>^† P <0.05 for Groups of E and F;</p><p>^‡ P<0.05 for groups of D and F.</p><p>Neonatal and childhood major outcomes at birth and 1–5 years old according to maternal pre-pregnancy body mass index and gestational weight gain categories.</p