Maternal diabetes and obesity induce marked abnormalities in glucose homeostasis and insulin secretion in the fetus, and are linked to obesity, diabetes, and metabolic disease in the offspring, with specific metabolic characterization based on offspring sex. Gestational diabetes (GDM) has profound effects on the intrauterine milieu, which may reflect and/or modulate the function of the maternal–fetal unit. In order to characterize metabolic factors that affect offspring development, we profiled the metabolome of second trimester amniotic fluid (AF) from women who were subsequently diagnosed with gestational diabetes (GDM) using a targeted metabolomics approach, profiling 459 known biochemicals through gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) assays. Using a nested case-control study design, we identified 69 total biochemicals altered by GDM exposure, while sex-specific analysis identified 44 and 58 metabolites in male and female offspring, respectively. The most significant changes were in glucose, amino acid, glutathione, fatty acid, sphingolipid, and bile acid metabolism with specific changes identified based on the offspring sex. Targeted isotope dilution LC/MS confirmatory assays measured significant changes in docosahexaenoic acid and arachidonic acid. We conclude that the sex-specific alterations in GDM maternal–fetal metabolism may begin to explain the sex-specific metabolic outcomes seen in offspring exposed to GDM in utero.

The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, wild-type C. elegans strains vary dramatically in their response to exogenous RNAi, challenging our characterization of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains, but dampens it further in others. Across strains, we observe variability in expression of known RNAi genes and strain-specific instances of pseudogenization and allelic divergence. Our results support the conclusions that Argonautes share overlapping functions, that germline RNAi incompetence is strain-specific but likely caused by genetic variants at common genes, and that RNAi pathways are evolving rapidly and dynamically. This work expands our understanding of RNAi by identifying conserved and variable pathway components, and it offers new access into characterizing gene function, identifying pathway interactions, and elucidating the biological significance of RNAi.

Aims/HypothesisWe hypothesized that diabetes during pregnancy (DDP) alters genome-wide DNA methylation in placenta resulting in differentially methylated loci of metabolically relevant genes and downstream changes in RNA and protein expression.MethodsWe mapped genome-wide DNA methylation with the Infinium 450K Human Methylation Bead Chip in term fetal placentae from Native American and Hispanic women with DDP using a nested case-control design (n = 17 pairs). RNA expression and protein levels were assayed via RNA-Seq and Western Blot.ResultsGenome-wide DNA methylation analysis revealed 465 CpG sites with significant changes for male offspring, 247 for female offspring, and 277 for offspring of both sexes (p<0.001). Placentae from female offspring were 40% more likely to have significant gains in DNA methylation compared with placentae from male offspring exposed to DDP (p<0.001). Changes in DNA methylation corresponded to changes in RNA and protein levels for 6 genes: PIWIL3, CYBA, GSTM1, GSTM5, KCNE1 and NXN. Differential DNA methylation was detected at loci related to mitochondrial function, DNA repair, inflammation, oxidative stress.Conclusions/InterpretationThese findings begin to explain mechanisms responsible for the increased risk for obesity and type 2 diabetes in offspring of mothers with DDP.

PUF (PUmilio/FBF) RNA-binding proteins recognize distinct elements. In C. elegans, PUF-8 binds to an 8-nt motif and restricts proliferation in the germline. Conversely, FBF-2 recognizes a 9-nt element and promotes mitosis. To understand how motif divergence relates to biological function, we first determined a crystal structure of PUF-8. Comparison of this structure to that of FBF-2 revealed a major difference in a central repeat. We devised a modified yeast 3-hybrid screen to identify mutations that confer recognition of an 8-nt element to FBF-2. We identified several such mutants and validated structurally and biochemically their binding to 8-nt RNA elements. Using genome engineering, we generated a mutant animal with a substitution in FBF-2 that confers preferential binding to the PUF-8 element. The mutant largely rescued overproliferation in animals that spontaneously generate tumors in the absence of puf-8. This work highlights the critical role of motif length in the specification of biological function.

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