Single-Cell Transcriptome Profiling of Human Pancreatic Islets in Health and Type 2 Diabetes.

Hormone-secreting cells within pancreatic islets of Langerhans play important roles in metabolic homeostasis and disease. However, their transcriptional characterization is still incomplete. Here, we sequenced the transcriptomes of thousands of human islet cells from healthy and type 2 diabetic donors. We could define specific genetic programs for each individual endocrine and exocrine cell type, even for rare δ, γ, ε, and stellate cells, and revealed subpopulations of α, ß, and acinar cells. Intriguingly, δ cells expressed several important receptors, indicating an unrecognized importance of these cells in integrating paracrine and systemic metabolic signals. Genes previously associated with obesity or diabetes were found to correlate with BMI. Finally, comparing healthy and T2D transcriptomes in a cell-type resolved manner uncovered candidates for future functional studies. Altogether, our analyses demonstrate the utility of the generated single-cell gene expression resource.

Ligand Binding Mechanism in Steroid Receptors: From Conserved Plasticity to Differential Evolutionary Constraints.

Steroid receptor drugs have been available for more than half a century, but details of the ligand binding mechanism have remained elusive. We solved X-ray structures of the glucocorticoid and mineralocorticoid receptors to identify a conserved plasticity at the helix 6-7 region that extends the ligand binding pocket toward the receptor surface. Since none of the endogenous ligands exploit this region, we hypothesized that it constitutes an integral part of the binding event. Extensive all-atom unbiased ligand exit and entrance simulations corroborate a ligand binding pathway that gives the observed structural plasticity a key functional role. Kinetic measurements reveal that the receptor residence time correlates with structural rearrangements observed in both structures and simulations. Ultimately, our findings reveal why nature has conserved the capacity to open up this region, and highlight how differences in the details of the ligand entry process result in differential evolutionary constraints across the steroid receptors.

Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures.

The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy.

Genome sequencing reveals a phage in Helicobacter pylori.

UNLABELLED: Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population; in a subset of this population, its presence is associated with development of severe disease, such as gastric cancer. Genomic analysis of several strains has revealed an extensive H. pylori pan-genome, likely to grow as more genomes are sampled. Here we describe the draft genome sequence (63 contigs; 26× mean coverage) of H. pylori strain B45, isolated from a patient with gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The major finding was a 24.6-kb prophage integrated in the bacterial genome. The prophage shares most of its genes (22/27) with prophage region II of Helicobacter acinonychis strain Sheeba. After UV treatment of liquid cultures, circular DNA carrying the prophage integrase gene could be detected, and intracellular tailed phage-like particles were observed in H. pylori cells by transmission electron microscopy, indicating that phage production can be induced from the prophage. PCR amplification and sequencing of the integrase gene from 341 H. pylori strains from different geographic regions revealed a high prevalence of the prophage (21.4%). Phylogenetic reconstruction showed four distinct clusters in the integrase gene, three of which tended to be specific for geographic regions. Our study implies that phages may play important roles in the ecology and evolution of H. pylori. IMPORTANCE: Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population, and while most of the infected individuals do not develop disease, H. pylori infection doubles the risk of developing gastric cancer. An abundance and diversity of viruses (phages) infect microbial populations in most environments and are important mediators of microbial diversity. Our finding of a 24.6-kb prophage integrated inside an H. pylori genome and the observation of circular integrase gene-containing DNA and phage-like particles inside cells upon UV treatment demonstrate that we have discovered a viable H. pylori phage. The additional finding of integrase genes in a large proportion of screened isolates of diverse geographic origins indicates that the prevalence of prophages may have been underestimated in H. pylori. Since phages are important drivers of microbial evolution, the discovery should be important for understanding and predicting genetic diversity in H. pylori.

Adenosine kinase deficiency disrupts the methionine cycle and causes hypermethioninemia, encephalopathy, and abnormal liver function.

Four inborn errors of metabolism (IEMs) are known to cause hypermethioninemia by directly interfering with the methionine cycle. Hypermethioninemia is occasionally discovered incidentally, but it is often disregarded as an unspecific finding, particularly if liver disease is involved. In many individuals the hypermethioninemia resolves without further deterioration, but it can also represent an early sign of a severe, progressive neurodevelopmental disorder. Further investigation of unclear hypermethioninemia is therefore important. We studied two siblings affected by severe developmental delay and liver dysfunction. Biochemical analysis revealed increased plasma levels of methionine, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy) but normal or mildly elevated homocysteine (Hcy) levels, indicating a block in the methionine cycle. We excluded S-adenosylhomocysteine hydrolase (SAHH) deficiency, which causes a similar biochemical phenotype, by using genetic and biochemical techniques and hypothesized that there was a functional block in the SAHH enzyme as a result of a recessive mutation in a different gene. Using exome sequencing, we identified a homozygous c.902C>A (p.Ala301Glu) missense mutation in the adenosine kinase gene (ADK), the function of which fits perfectly with this hypothesis. Increased urinary adenosine excretion confirmed ADK deficiency in the siblings. Four additional individuals from two unrelated families with a similar presentation were identified and shown to have a homozygous c.653A>C (p.Asp218Ala) and c.38G>A (p.Gly13Glu) mutation, respectively, in the same gene. All three missense mutations were deleterious, as shown by activity measurements on recombinant enzymes. ADK deficiency is a previously undescribed, severe IEM shedding light on a functional link between the methionine cycle and adenosine metabolism.

Translational database selection and multiplexed sequence capture for up front filtering of reliable breast cancer biomarker candidates.

Biomarker identification is of utmost importance for the development of novel diagnostics and therapeutics. Here we make use of a translational database selection strategy, utilizing data from the Human Protein Atlas (HPA) on differentially expressed protein patterns in healthy and breast cancer tissues as a means to filter out potential biomarkers for underlying genetic causatives of the disease. DNA was isolated from ten breast cancer biopsies, and the protein coding and flanking non-coding genomic regions corresponding to the selected proteins were extracted in a multiplexed format from the samples using a single DNA sequence capture array. Deep sequencing revealed an even enrichment of the multiplexed samples and a great variation of genetic alterations in the tumors of the sampled individuals. Benefiting from the upstream filtering method, the final set of biomarker candidates could be completely verified through bidirectional Sanger sequencing, revealing a 40 percent false positive rate despite high read coverage. Of the variants encountered in translated regions, nine novel non-synonymous variations were identified and verified, two of which were present in more than one of the ten tumor samples.

Characterization of species-related differences in the pharmacology of tachykinin NK receptors 1, 2 and 3.

Tachykinin NK receptors (NKRs) differ to a large degree among species with respect to their affinities for small molecule antagonists. The aims of the present study were to clone NKRs from gerbil (NK2R and NK3R) and dog (NK1R, NK2R and NK3R) in which the sequence was previously unknown and to investigate the potency of several NKR antagonists at all known human, dog, gerbil and rat NKRs. The NKR protein coding sequences were cloned and expressed in CHO cells. The inhibitory concentrations of selective and non-selective NKR antagonists were determined by inhibition of agonist-induced mobilization of intracellular Ca2+. Receptor homology models were constructed based on the rhodopsin crystal structure to investigate and identify the antagonist binding sites and interaction points in the transmembrane (TM) regions of the NKRs. Data collected using the cloned dog NK1R confirmed that the dog NK1R displays similar pharmacology as the human and the gerbil NK1R, but differs greatly from the mouse and the rat NK1R. Despite species-related amino acid (AA) differences located close to the antagonist binding pocket of the NK2R, they did not affect the potency of the antagonists ZD6021 and saredutant. Two AA differences located close to the antagonist binding site of NK3R likely influence the NK3R antagonist potency, explaining the 3-10-fold decrease in potency observed for the rat NK3R. For the first time, detailed pharmacological experiments in vitro with cloned NKRs demonstrate that not only human, but also dog and gerbil NKR displays similar antagonist pharmacology while rat diverges significantly with respect to NK1R and NK3R.

Flow cytometry for enrichment and titration in massively parallel DNA sequencing.

Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols.

Functional genomic and metabolic studies of the adaptations of a prominent adult human gut symbiont, Bacteroides thetaiotaomicron, to the suckling period.

The adult human gut microbiota is dominated by two divisions of Bacteria, the Bacteroidetes and the Firmicutes. Assembly of this community begins at birth through processes that remain largely undefined. In this report, we examine the adaptations of Bacteroides thetaiotaomicron, a prominent member of the adult distal intestinal microbiota, during the suckling and weaning periods. Germ-free NMRI mice were colonized at birth from their gnotobiotic mothers, who harbored this anaerobic Gram-negative saccharolytic bacterium. B. thetaiotaomicron was then harvested from the ceca of these hosts during the suckling period (postnatal day 17) and after weaning (postnatal day 30). Whole genome transcriptional profiles were obtained at these two time points using custom B. thetaiotaomicron GeneChips. Transcriptome-based in silico reconstructions of bacterial metabolism and gas chromatography-mass spectrometry and biochemical assays of carbohydrate utilization in vivo indicated that in the suckling gut B. thetaiotaomicron prefers host-derived polysaccharides, as well as mono- and oligosaccharides present in mother's milk. After weaning, B. thetaiotaomicron expands its metabolism to exploit abundant, plant-derived dietary polysaccharides. The bacterium's responses to postnatal alterations in its nutrient landscape involve expression of gene clusters encoding environmental sensors, outer membrane proteins involved in binding and import of glycans, and glycoside hydrolases. These expression changes are interpreted in light of a phylogenetic analysis that revealed unique expansions of related polysaccharide utilization loci in three human alimentary tract-associated Bacteroidetes, expansions that likely reflect the evolutionary adaptations of these species to different nutrient niches.

Message from a human gut symbiont: sensitivity is a prerequisite for sharing.

Microbial genome sequencing projects are beginning to provide insights about the molecular foundations of human-bacterial symbioses. The intestine contains our largest collection of symbionts, where members of Bacteroides comprise approximately 25% of the microbiota in adults. The recently defined proteome of a prominent human intestinal symbiont, Bacteroides thetaiotaomicron, contains an elaborate environmental-sensing apparatus. This apparatus includes an unprecedented number of extracytoplasmic function (ECF) sigma-factors, and a large collection of novel hybrid two-component systems composed of membrane-spanning periplasmic proteins with histidine kinase, phosphoacceptor, response regulator receiver and DNA-binding domains. These sensors are linked to the organism's large repertoire of genes involved in acquiring and processing dietary polysaccharides ('the glycobiome'). This arrangement illustrates how a successful symbiont has evolved strategies for detecting and responding to conditions in its niche so that it can sustain beneficial relationships with its microbial and human partners.

A genomic view of the human-Bacteroides thetaiotaomicron symbiosis.

The human gut is colonized with a vast community of indigenous microorganisms that help shape our biology. Here, we present the complete genome sequence of the Gram-negative anaerobe Bacteroides thetaiotaomicron, a dominant member of our normal distal intestinal microbiota. Its 4779-member proteome includes an elaborate apparatus for acquiring and hydrolyzing otherwise indigestible dietary polysaccharides and an associated environment-sensing system consisting of a large repertoire of extracytoplasmic function sigma factors and one- and two-component signal transduction systems. These and other expanded paralogous groups shed light on the molecular mechanisms underlying symbiotic host-bacterial relationships in our intestine.