Comprehensive expression profiles of Arabidopsis (Arabidopsis thaliana) MIRNA genes and mature microRNAs (miRs) are currently not available. We established a quantitative real-time polymerase chain reaction platform that allows rapid and sensitive quantification of 177 Arabidopsis primary miR transcripts (pri-miRs). The platform was used to detect phosphorus (P) or nitrogen (N) status-responsive pri-miR species. Several pri-miR169 species as well as pri-miR398a were found to be repressed during N limitation, whereas during P limitation, pri-miR778, pri-miR827, and pri-miR399 species were induced and pri-miR398a was repressed. The corresponding responses of the biologically active, mature miRs were confirmed using specific stem-loop reverse transcription primer quantitative polymerase chain reaction assays and small RNA sequencing. Interestingly, the latter approach also revealed high abundance of some miR star strands. Bioinformatic analysis of small RNA sequences with a modified miRDeep algorithm led to the identification of the novel P limitation-induced miR2111, which is encoded by two loci in the Arabidopsis genome. Furthermore, miR2111, miR169, a miR827-like sequence, and the abundances of several miR star strands were found to be strongly dependent on P or N status in rapeseed (Brassica napus) phloem sap, flagging them as candidate systemic signals. Taken together, these results reveal the existence of complex small RNA-based regulatory networks mediating plant adaptation to mineral nutrient availability.

Next-generation technologies generate an overwhelming amount of gene sequence data. Efficient annotation tools are required to make these data amenable to functional genomics analyses. The Mercator pipeline automatically assigns functional terms to protein or nucleotide sequences. It uses the MapMan 'BIN' ontology, which is tailored for functional annotation of plant 'omics' data. The classification procedure performs parallel sequence searches against reference databases, compiles the results and computes the most likely MapMan BINs for each query. In the current version, the pipeline relies on manually curated reference classifications originating from the three reference organisms (Arabidopsis, Chlamydomonas, rice), various other plant species that have a reviewed SwissProt annotation, and more than 2000 protein domain and family profiles at InterPro, CDD and KOG. Functional annotations predicted by Mercator achieve accuracies above 90% when benchmarked against manual annotation. In addition to mapping files for direct use in the visualization software MapMan, Mercator provides graphical overview charts, detailed annotation information in a convenient web browser interface and a MapMan-to-GO translation table to export results as GO terms. Mercator is available free of charge via http://mapman.gabipd.org/web/guest/app/ Mercator.

Epileptic encephalopathies are a phenotypically and genetically heterogeneous group of severe epilepsies accompanied by intellectual disability and other neurodevelopmental features1-6. Using next generation sequencing, we identified four different de novo mutations in KCNA2, encoding the potassium channel KV1.2, in six patients with epileptic encephalopathy (one mutation recurred three times independently). Four individuals presented with febrile and multiple afebrile, often focal seizure types, multifocal epileptiform discharges strongly activated by sleep, mild-moderate intellectual disability, delayed speech development and sometimes ataxia. Functional studies of the two mutations associated with this phenotype revealed an almost complete loss-of-function with a dominant-negative effect. Two further individuals presented with a different and more severe epileptic encephalopathy phenotype. They carried mutations inducing a drastic gain-of-function effect leading to permanently open channels. These results establish KCNA2 as a novel gene involved in human neurodevelopmental disorders by two different mechanisms, predicting either hyperexcitability or electrical silencing of KV1.2-expressing neurons.

The gastrointestinal microbiome is a complex ecosystem with functions that shape human health. Studying the relationship between taxonomic alterations and functional repercussions linked to disease remains challenging. Here, we present an integrative approach to resolve the taxonomic and functional attributes of gastrointestinal microbiota at the metagenomic, metatranscriptomic and metaproteomic levels. We apply our methods to samples from four families with multiple cases of type 1 diabetes mellitus (T1DM). Analysis of intra-and inter-individual variation demonstrates that family membership has a pronounced effect on the structural and functional composition of the gastrointestinal microbiome. In the context of T1DM, consistent taxonomic differences were absent across families, but certain human exocrine pancreatic proteins were found at lower levels. The associated microbial functional signatures were linked to metabolic traits in distinct taxa. The methodologies and results provide a foundation for future large-scale integrated multi-omic analyses of the gastrointestinal microbiome in the context of host-microbe interactions in human health and disease.

The majority of plants are able to form the arbuscular mycorrhizal (AM) symbiosis in association with AM fungi. During symbiosis development, plant cells undergo a complex reprogramming resulting in profound morphological and physiological changes. MicroRNAs (miRNAs) are important components of the regulatory network of plant cells. To unravel the impact of miRNAs and miRNA-mediated mRNA cleavage on root cell reprogramming during AM symbiosis, we carried out highthroughput (Illumina) sequencing of small RNAs and degradome tags of Medicago truncatula roots. This led to the annotation of 243 novel miRNAs. An increased accumulation of several novel and conserved miRNAs in mycorrhizal roots suggest a role of these miRNAs during AM symbiosis. The degradome analysis led to the identification of 185 root transcripts as mature miRNA and also miRNA*-mediated mRNA cleavage targets. Several of the identified miRNA targets are known to be involved in root symbioses. In summary, the increased accumulation of specific miRNAs and the miRNA-mediated cleavage of symbiosis-relevant genes indicate that miRNAs are an important part of the regulatory network leading to symbiosis development.Small silencing RNAs are a complex group of short RNAs with sizes in the range of approximately 20 to 25 nucleotides in length that can regulate gene expression at the transcriptional and posttranscriptional levels (Bartel, 2004;He and Hannon, 2004). In recent years, it has become evident that two main classes of small silencing RNAs, short interfering RNAs and microRNAs (miRNAs), are of great importance in plants. Small RNAs are involved in developmental processes, hormonal signaling, organ polarity, RNA metabolism, and abiotic and

The rate of caesarean section delivery (CSD) is increasing worldwide. It remains unclear whether disruption of mother-to-neonate transmission of microbiota through CSD occurs and whether it affects human physiology. Here we perform metagenomic analysis of earliest gut microbial community structures and functions. We identify differences in encoded functions between microbiomes of vaginally delivered (VD) and CSD neonates. Several functional pathways are over-represented in VD neonates, including lipopolysaccharide (LPS) biosynthesis. We link these enriched functions to individual-specific strains, which are transmitted from mothers to neonates in case of VD. The stimulation of primary human immune cells with LPS isolated from early stool samples of VD neonates results in higher levels of tumour necrosis factor (TNF-α) and interleukin 18 (IL-18). Accordingly, the observed levels of TNF-α and IL-18 in neonatal blood plasma are higher after VD. Taken together, our results support that CSD disrupts mother-to-neonate transmission of specific microbial strains, linked functional repertoires and immune-stimulatory potential during a critical window for neonatal immune system priming.

The budding yeast Saccharomyces cerevisiae is important for human food production and as a model organism for biological research. The genetic diversity contained in the global population of yeast strains represents a valuable resource for a number of fields, including genetics, bioengineering, and studies of evolution and population structure. Here, we apply a multiplexed, reduced genome sequencing strategy (restriction site−associated sequencing or RAD-seq) to genotype a large collection of S. cerevisiae strains isolated from a wide range of geographical locations and environmental niches. The method permits the sequencing of the same 1% of all genomes, producing a multiple sequence alignment of 116,880 bases across 262 strains. We find diversity among these strains is principally organized by geography, with European, North American, Asian, and African/S. E. Asian populations defining the major axes of genetic variation. At a finer scale, small groups of strains from cacao, olives, and sake are defined by unique variants not present in other strains. One population, containing strains from a variety of fermentations, exhibits high levels of heterozygosity and a mixture of alleles from European and Asian populations, indicating an admixed origin for this group. We propose a model of geographic differentiation followed by human-associated admixture, primarily between European and Asian populations and more recently between European and North American populations. The large collection of genotyped yeast strains characterized here will provide a useful resource for the broad community of yeast researchers.

The epileptic encephalopathies are a clinically and aetiologically heterogeneous subgroup of epilepsy syndromes. Most epileptic encephalopathies have a genetic cause and patients are often found to carry a heterozygous de novo mutation in one of the genes associated with the disease entity. Occasionally recessive mutations are identified: a recent publication described a distinct neonatal epileptic encephalopathy (MIM 615905) caused by autosomal recessive mutations in the SLC13A5 gene. Here, we report eight additional patients belonging to four different families with autosomal recessive mutations in SLC13A5. SLC13A5 encodes a high affinity sodium-dependent citrate transporter, which is expressed in the brain. Neurons are considered incapable of de novo synthesis of tricarboxylic acid cycle intermediates; therefore they rely on the uptake of intermediates, such as citrate, to maintain their energy status and neurotransmitter production. The effect of all seven identified mutations (two premature stops and five amino acid substitutions) was studied in vitro, using immunocytochemistry, selective western blot and mass spectrometry. We hereby demonstrate that cells expressing mutant sodium-dependent citrate transporter have a complete loss of citrate uptake due to various cellular loss-of-function mechanisms. In addition, we provide independent proof of the involvement of autosomal recessive SLC13A5 mutations in the development of neonatal epileptic encephalopathies, and highlight teeth hypoplasia as a possible indicator for SLC13A5 screening. All three patients who tried the ketogenic diet responded well to this treatment, and future studies will allow us to ascertain whether this is a recurrent feature in this severe disorder.