DEUS: an R package for accurate small RNA profiling based on differential expression of unique sequences.

SUMMARY: Despite their fundamental role in various biological processes, the analysis of small RNA sequencing data remains a challenging task. Major obstacles arise when short RNA sequences map to multiple locations in the genome, align to regions that are not annotated or underwent post-transcriptional changes which hamper accurate mapping. In order to tackle these issues, we present a novel profiling strategy that circumvents the need for read mapping to a reference genome by utilizing the actual read sequences to determine expression intensities. After differential expression analysis of individual sequence counts, significant sequences are annotated against user defined feature databases and clustered by sequence similarity. This strategy enables a more comprehensive and concise representation of small RNA populations without any data loss or data distortion. AVAILABILITY AND IMPLEMENTATION: Code and documentation of our R package at http://ibis.helmholtz-muenchen.de/deus/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BMI-Independent Effects of Gestational Diabetes on Human Placenta.

Purpose: Recently, alterations in maternal lipid metabolism were associated with gestational diabetes mellitus (GDM). However, detailed plasma lipid profiles and their relevance for placental and fetal metabolism are currently not understood. Methods: Maternal and placental lipid profiles were characterized in women with GDM and women with normal glucose tolerance (NGT). Inflammatory gene expression was compared in placentas and primary term trophoblasts between the groups. In addition, trophoblasts were stimulated with nonesterified fatty acids (NEFAs), and effects on gene expression were quantified. Finally, placental macrophage content and cord blood concentrations of inflammatory parameters and NEFAs were compared between women with GDM and women with NGT with similar body mass index (BMI). Results: Palmitate and stearate levels were elevated in both maternal plasma and placental tissue of women with GDM. Placental GDM-associated elevations of IL6, IL8, and TLR2 expression were reflected in trophoblasts derived from women with GDM. Stimulation of primary trophoblasts with palmitate led to increased mRNA expression and protein release of the cytokine IL6 and the chemokine IL8. In line with this, elevated amounts of CD68-positive cells were quantified in the placental tissue of women with GDM. No GDM-associated elevations in a range of inflammatory parameters and NEFAs in cord blood of NGT vs GDM neonates was found. Conclusions: GDM, independently of BMI, altered maternal plasma NEFAs and the placental lipid profile. GDM was associated with trophoblast and whole-placenta lipoinflammation; however, this was not accompanied by elevated concentrations of inflammatory cytokines or NEFAs in neonatal cord blood.

A computational biology approach of a genome-wide screen connected miRNAs to obesity and type 2 diabetes.

OBJECTIVE: Obesity and type 2 diabetes (T2D) arise from the interplay between genetic, epigenetic, and environmental factors. The aim of this study was to combine bioinformatics and functional studies to identify miRNAs that contribute to obesity and T2D. METHODS: A computational framework (miR-QTL-Scan) was applied by combining QTL, miRNA prediction, and transcriptomics in order to enhance the power for the discovery of miRNAs as regulative elements. Expression of several miRNAs was analyzed in human adipose tissue and blood cells and miR-31 was manipulated in a human fat cell line. RESULTS: In 17 partially overlapping QTL for obesity and T2D 170 miRNAs were identified. Four miRNAs (miR-15b, miR-30b, miR-31, miR-744) were recognized in gWAT (gonadal white adipose tissue) and six (miR-491, miR-455, miR-423-5p, miR-132-3p, miR-365-3p, miR-30b) in BAT (brown adipose tissue). To provide direct functional evidence for the achievement of the miR-QTL-Scan, miR-31 located in the obesity QTL Nob6 was experimentally analyzed. Its expression was higher in gWAT of obese and diabetic mice and humans than of lean controls. Accordingly, 10 potential target genes involved in insulin signaling and adipogenesis were suppressed. Manipulation of miR-31 in human SGBS adipocytes affected the expression of GLUT4, PPARγ, IRS1, and ACACA. In human peripheral blood mononuclear cells (PBMC) miR-15b levels were correlated to baseline blood glucose concentrations and might be an indicator for diabetes. CONCLUSION: Thus, miR-QTL-Scan allowed the identification of novel miRNAs relevant for obesity and T2D.

Maternal whole blood cell miRNA-340 is elevated in gestational diabetes and inversely regulated by glucose and insulin.

The number of pregnancies complicated by gestational diabetes (GDM) is increasing worldwide. To identify novel characteristics of GDM, we studied miRNA profiles of maternal and fetal whole blood cells (WBCs) from GDM and normal glucose tolerant (NGT) pregnant women matched for body mass index and maternal age. After adjustment for maternal weight gain and pregnancy week, we identified 29 mature micro-RNAs (miRNAs) up-regulated in GDM, one of which, i.e., miRNA-340, was validated by qPCR. mRNA and protein expression of PAIP1, a miRNA-340 target gene, was found down-regulated in GDM women, accordingly. In lymphocytes derived from the mothers' blood and treated in vitro, insulin increased and glucose reduced miRNA-340 expression. In fetal cord blood samples, no associations of miRNA-340 with maternal GDM were observed. Our results provide evidence for insulin-induced epigenetic, i.e., miRNA-dependent, programming of maternal WBCs in GDM.

Early hypermethylation of hepatic Igfbp2 results in its reduced expression preceding fatty liver in mice.

Obesity and ectopic fat disposition are risk factors for metabolic disease. Recent data indicate that IGFBP2 expression in liver is epigenetically inhibited during hepatic steatosis. The aim of this study was to investigate if epigenetic de-regulation of hepatic Igfbp2 occurs already early in life and is associated with increased risk for diet-induced obesity (DIO) during adolescence. Male C57BL/6J mice received a high-fat diet. After 3 weeks on this diet (age of 6 weeks), DIO-susceptible (responder, Resp) and DIO-resistant (non-responder, nResp) mice were identified by early weight gain. At the age of 6 weeks, Resp mice exhibited elevated blood glucose (p < 0.05), plasma insulin (p < 0.01), HOMA-IR and leptin/adiponectin ratio, whereas liver triglycerides were identical but significantly increased (p < 0.01) in Resp mice at 20 weeks of age. Igfbp2 expression was reduced in young Resp compared with nResp mice (p < 0.01), an effect that correlated with elevated DNA methylation of intronic CpG2605 (p < 0.01). The epigenetic inhibition of Igfbp2 was stable over time and preceded DIO and hepatosteatosis in adult mice. In vitro studies demonstrated that selective methylation of CpG2605 significantly reduced reporter activity by ∼85%, indicating that Igfbp2 expression is modulated by methylation. In human whole blood cells, methylation of IGFBP2 at the homologous CpG site was increased in obese men with impaired glucose tolerance. In conclusion, our data show that increased methylation of hepatic Igfbp2 during infancy predicts the development of fatty liver later in life and is linked to deterioration of glucose metabolism.