Stem cell fate is influenced by a number of factors and interactions that require robust control for safe and effective regeneration of functional tissue. Coordinated interactions with soluble factors, other cells, and extracellular matrices define a local biochemical and mechanical niche with complex and dynamic regulation that stem cells sense. Decellularized tissue matrices and synthetic polymer niches are being used in the clinic, and they are also beginning to clarify fundamental aspects of how stem cells contribute to homeostasis and repair, for example, at sites of fibrosis. Multifaceted technologies are increasingly required to produce and interrogate cells ex vivo, to build predictive models, and, ultimately, to enhance stem cell integration in vivo for therapeutic benefit.

DNA hybridization arrays simultaneously measure the expression level for thousands of genes. These measurements provide a "snapshot" of transcription levels within the cell. A major challenge in computational biology is to uncover, from such measurements, gene/protein interactions and key biological features of cellular systems. In this paper, we propose a new framework for discovering interactions between genes based on multiple expression measurements. This framework builds on the use of Bayesian networks for representing statistical dependencies. A Bayesian network is a graph-based model of joint multivariate probability distributions that captures properties of conditional independence between variables. Such models are attractive for their ability to describe complex stochastic processes and because they provide a clear methodology for learning from (noisy) observations. We start by showing how Bayesian networks can describe interactions between genes. We then describe a method for recovering gene interactions from microarray data using tools for learning Bayesian networks. Finally, we demonstrate this method on the S. cerevisiae cell-cycle measurements of Spellman et al. (1998).

Contractile myocytes provide a test of the hypothesis that cells sense their mechanical as well as molecular microenvironment, altering expression, organization, and/or morphology accordingly. Here, myoblasts were cultured on collagen strips attached to glass or polymer gels of varied elasticity. Subsequent fusion into myotubes occurs independent of substrate flexibility. However, myosin/actin striations emerge later only on gels with stiffness typical of normal muscle (passive Young's modulus, E approximately 12 kPa). On glass and much softer or stiffer gels, including gels emulating stiff dystrophic muscle, cells do not striate. In addition, myotubes grown on top of a compliant bottom layer of glass-attached myotubes (but not softer fibroblasts) will striate, whereas the bottom cells will only assemble stress fibers and vinculin-rich adhesions. Unlike sarcomere formation, adhesion strength increases monotonically versus substrate stiffness with strongest adhesion on glass. These findings have major implications for in vivo introduction of stem cells into diseased or damaged striated muscle of altered mechanical composition.

SILVA (from Latin silva, forest, http://www.arb-silva.de) is a comprehensive resource for up-to-date quality-controlled databases of aligned ribosomal RNA (rRNA) gene sequences from the Bacteria, Archaea and Eukaryota domains and supplementary online services. SILVA provides a manually curated taxonomy for all three domains of life, based on representative phylogenetic trees for the small- and large-subunit rRNA genes. This article describes the improvements the SILVA taxonomy has undergone in the last 3 years. Specifically we are focusing on the curation process, the various resources used for curation and the comparison of the SILVA taxonomy with Greengenes and RDP-II taxonomies. Our comparisons not only revealed a reasonable overlap between the taxa names, but also points to significant differences in both names and numbers of taxa between the three resources.

Substrate stiffness is emerging as an important physical factor in the response of many cell types. In agreement with findings on other anchorage-dependent cell lineages, aortic smooth muscle cells are found to spread and organize their cytoskeleton and focal adhesions much more so on "rigid" glass or "stiff" gels than on "soft" gels. Whereas these cells generally show maximal spreading on intermediate collagen densities, the limited spreading on soft gels is surprisingly insensitive to adhesive ligand density. Bell-shaped cell spreading curves encompassing all substrates are modeled by simple functions that couple ligand density to substrate stiffness. Although smooth muscle cells spread minimally on soft gels regardless of collagen, GFP-actin gives a slight overexpression of total actin that can override the soft gel response and drive spreading; GFP and GFP-paxillin do not have the same effect. The GFP-actin cells invariably show an organized filamentous cytoskeleton and clearly indicate that the cytoskeleton is at least one structural node in a signaling network that can override spreading limits typically dictated by soft gels. Based on such results, we hypothesize a central structural role for the cytoskeleton in driving the membrane outward during spreading whereas adhesion reinforces the spreading.

Insects are the most speciose group of animals, but the phylogenetic relationships of many major lineages remain unresolved. We inferred the phylogeny of insects from 1478 protein-coding genes. Phylogenomic analyses of nucleotide and amino acid sequences, with site-specific nucleotide or domain-specific amino acid substitution models, produced statistically robust and congruent results resolving previously controversial phylogenetic relations hips. We dated the origin of insects to the Early Ordovician [~479 million years ago (Ma)], of insect flight to the Early Devonian (~406 Ma), of major extant lineages to the Mississippian (~345 Ma), and the major diversification of holometabolous insects to the Early Cretaceous. Our phylogenomic study provides a comprehensive reliable scaffold for future comparative analyses of evolutionary innovations among insects.

High-throughput genome-wide molecular assays, which probe cellular networks from different perspectives, have become central to molecular biology. Probabilistic graphical models are useful for extracting meaningful biological insights from the resulting data sets. These models provide a concise representation of complex cellular networks by composing simpler submodels. Procedures based on well-understood principles for inferring such models from data facilitate a model-based methodology for analysis and discovery. This methodology and its capabilities are illustrated by several recent applications to gene expression data.

Vesicles are microscopic sacs that enclose a volume with a molecularly thin membrane. The membranes are generally self-directed assemblies of amphiphilic molecules with a dual hydrophilic-hydrophobic character. Biological amphiphiles form vesicles central to cell function and are principally lipids of molecular weight less than 1 kilodalton. Block copolymers that mimic lipid amphiphilicity can also self-assemble into vesicles in dilute solution, but polymer molecular weights can be orders of magnitude greater than those of lipids. Structural features of vesicles, as well as properties including stability, fluidity, and intermembrane dynamics, are greatly influenced by characteristics of the polymers. Future applications of polymer vesicles will rely on exploiting unique property-performance relations, but results to date already underscore the fact that biologically derived vesicles are but a small subset of what is physically and chemically possible.

High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript's level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a 'core' module of antiviral genes is expressed very early by a few 'precocious' cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced 'peaked' inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.

We report the interaction between a human centromere antigen and an alphoid DNA, a human centromeric satellite DNA, which consists of 170-bp repeating units. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract is selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension defines the 17-bp segment on the alphoid DNA that is required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17-bp motif are recovered by streptavidin agarose and immunoblotted, the 80-kD centromere antigen (CENP-B) is detected. DNA binding experiments for proteins immunoprecipitated with anticentromere serum, separated by gel electrophoresis, and transferred to a membrane strongly suggest that the 80-kD antigen specifically binds to the DNA fragment with the 17-bp motif. The 17-bp motif is termed the "CENP-B box." Alphoid monomers with the CENP-B box are found in all the known alphoid subclasses, with varying frequencies, except the one derived from the Y chromosome so far cloned. These results imply that the interaction of the 80-kD centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere.

In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.

We sequenced the genomes of a ∼7,000-year-old farmer from Germany and eight ∼8,000-year-old hunter-gatherers from Luxembourg and Sweden. We analysed these and other ancient genomes with 2,345 contemporary humans to show that most present-day Europeans derive from at least three highly differentiated populations: west European hunter-gatherers, who contributed ancestry to all Europeans but not to Near Easterners; ancient north Eurasians related to Upper Palaeolithic Siberians, who contributed to both Europeans and Near Easterners; and early European farmers, who were mainly of Near Eastern origin but also harboured west European hunter-gatherer related ancestry. We model these populations' deep relationships and show that early European farmers had ∼44% ancestry from a 'basal Eurasian' population that split before the diversification of other non-African lineages.

We have converted nanoscale mechanical energy into electrical energy by means of piezoelectric zinc oxide nanowire (NW) arrays. The aligned NWs are deflected with a conductive atomic force microscope tip in contact mode. The coupling of piezoelectric and semiconducting properties in zinc oxide creates a strain field and charge separation across the NW as a result of its bending. The rectifying characteristic of the Schottky barrier formed between the metal tip and the NW leads to electrical current generation. The efficiency of the NW-based piezoelectric power generator is estimated to be 17 to 30%. This approach has the potential of converting mechanical, vibrational, and/or hydraulic energy into electricity for powering nanodevices.

BACKGROUND

Recent studies have found many proteins containing regions that do not form well-defined three-dimensional structures in their native states. The study and detection of such disordered regions is important both for understanding protein function and for facilitating structural analysis since disordered regions may affect solubility and/or crystallizability.

RESULTS

We have developed the regional order neural network (RONN) software as an application of our recently developed 'bio-basis function neural network' pattern recognition algorithm to the detection of natively disordered regions in proteins. The results of blind-testing a panel of nine disorder prediction tools (including RONN) against 80 protein sequences derived from the Protein Data Bank shows that, based on the probability excess measure, RONN performed the best.

Vesicles were made from amphiphilic diblock copolymers and characterized by micromanipulation. The average molecular weight of the specific polymer studied, polyethyleneoxide-polyethylethylene (EO40-EE37), is several times greater than that of typical phospholipids in natural membranes. Both the membrane bending and area expansion moduli of electroformed polymersomes (polymer-based liposomes) fell within the range of lipid membrane measurements, but the giant polymersomes proved to be almost an order of magnitude tougher and sustained far greater areal strain before rupture. The polymersome membrane was also at least 10 times less permeable to water than common phospholipid bilayers. The results suggest a new class of synthetic thin-shelled capsules based on block copolymer chemistry.

The pattern of local image velocities on the retina encodes important environmental information. Although humans are generally able to extract this information, they can easily be deceived into seeing incorrect velocities. We show that these 'illusions' arise naturally in a system that attempts to estimate local image velocity. We formulated a model of visual motion perception using standard estimation theory, under the assumptions that (i) there is noise in the initial measurements and (ii) slower motions are more likely to occur than faster ones. We found that specific instantiation of such a velocity estimator can account for a wide variety of psychophysical phenomena.

Real-time polymerase chain reaction (PCR) is a highly sensitive method that can be used for the detection and quantification of microbial populations without cultivating them in anaerobic processes and environmental samples. This work was conducted to design primer and probe sets for the detection of methanogens using a real-time PCR with the TaqMan system. Six group-specific methanogenic primer and probe sets were designed. These sets separately detect four orders (Methanococcales, Methanobacteriales, Methanomicrobiales, and Methanosarcinales) along with two families (Methanosarcinaceae and Methanosaetaceae) of the order Methanosarcinales. We also designed the universal primer and probe sets that specifically detect the 16S rDNA of prokaryotes and of the domain Bacteria and Archaea, and which are fully compatible with the TaqMan real-time PCR system. Target-group specificity of each primer and probe set was empirically verified by testing DNA isolated from 28 archaeal cultures and by analyzing potential false results. In general, each primer and probe set was very specific to the target group. The primer and probe sets designed in this study can be used to detect and quantify the order-level (family-level in the case of Methanosarcinales) methanogenic groups in anaerobic biological processes and various environments.

Genome-wide mapping of nucleosomes has revealed a great deal about the relationships between chromatin structure and control of gene expression, and has led to mechanistic hypotheses regarding the rules by which chromatin structure is established. High-throughput sequencing has recently become the technology of choice for chromatin mapping studies, yet analysis of these experiments is still in its infancy. Here, we introduce a pipeline for analyzing deep sequencing maps of chromatin structure and apply it to data from S. cerevisiae. We analyze a digestion series where nucleosomes are isolated from under- and overdigested chromatin. We find that certain classes of nucleosomes are unusually susceptible or resistant to overdigestion, with promoter nucleosomes easily digested and mid-coding region nucleosomes being quite stable. We find evidence for highly sensitive nucleosomes located within "nucleosome-free regions," suggesting that these regions are not always completely naked but instead are likely associated with easily digested nucleosomes. Finally, since RNA polymerase is the dominant energy-consuming machine that operates on the chromatin template, we analyze changes in chromatin structure when RNA polymerase is inactivated via a temperature-sensitive mutation. We find evidence that RNA polymerase plays a role in nucleosome eviction at promoters and is also responsible for retrograde shifts in nucleosomes during transcription. Loss of RNA polymerase results in a relaxation of chromatin structure to more closely match in vitro nucleosome positioning preferences. Together, these results provide analytical tools and experimental guidance for nucleosome mapping experiments, and help disentangle the interlinked processes of transcription and chromatin packaging.

DNA methylation plays an important role in epigenetic signalling, having an impact on gene regulation, chromatin structure, development and disease. Here, we review the structures and functions of the mammalian DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b, including their domain structures, catalytic mechanisms, localisation, regulation, post-translational modifications and interaction with chromatin and other proteins, summarising data obtained in genetic, cell biology and enzymatic studies. We focus on the question of how the molecular and enzymatic properties of these enzymes are connected to the dynamics of DNA methylation patterns and to the roles the enzymes play in the processes of de novo and maintenance DNA methylation. Recent enzymatic and genome-wide methylome data have led to a new model of genomic DNA methylation patterns based on the preservation of average levels of DNA methylation in certain regions, rather than the methylation states of individual CG sites.

All-optical electrophysiology-spatially resolved simultaneous optical perturbation and measurement of membrane voltage-would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

OBJECTIVE

The colonic microbiota ferment dietary fibres, producing short chain fatty acids. Recent evidence suggests that the short chain fatty acid propionate may play an important role in appetite regulation. We hypothesised that colonic delivery of propionate would increase peptide YY (PYY) and glucagon like peptide-1 (GLP-1) secretion in humans, and reduce energy intake and weight gain in overweight adults.

METHODS

To investigate whether propionate promotes PYY and GLP-1 secretion, a primary cultured human colonic cell model was developed. To deliver propionate specifically to the colon, we developed a novel inulin-propionate ester. An acute randomised, controlled cross-over study was used to assess the effects of this inulin-propionate ester on energy intake and plasma PYY and GLP-1 concentrations. The long-term effects of inulin-propionate ester on weight gain were subsequently assessed in a randomised, controlled 24-week study involving 60 overweight adults.

RESULTS

Propionate significantly stimulated the release of PYY and GLP-1 from human colonic cells. Acute ingestion of 10 g inulin-propionate ester significantly increased postprandial plasma PYY and GLP-1 and reduced energy intake. Over 24 weeks, 10 g/day inulin-propionate ester supplementation significantly reduced weight gain, intra-abdominal adipose tissue distribution, intrahepatocellular lipid content and prevented the deterioration in insulin sensitivity observed in the inulin-control group.

CONCLUSIONS

These data demonstrate for the first time that increasing colonic propionate prevents weight gain in overweight adult humans.

BACKGROUND

NCT00750438.

A new phototransistor based on the mechanically exfoliated single-layer MoS(2) nanosheet is fabricated, and its light-induced electric properties are investigated in detail. Photocurrent generated from the phototransistor is solely determined by the illuminated optical power at a constant drain or gate voltage. The switching behavior of photocurrent generation and annihilation can be completely finished within ca. 50 ms, and it shows good stability. Especially, the single-layer MoS(2) phototransistor exhibits a better photoresponsivity as compared with the graphene-based device. The unique characteristics of incident-light control, prompt photoswitching, and good photoresponsivity from the MoS(2) phototransistor pave an avenue to develop the single-layer semiconducting materials for multifunctional optoelectronic device applications in the future.

The Dnmt3a DNA methyltransferase contains in its N-terminal part a PWWP domain that is involved in chromatin targeting. Here, we have investigated the interaction of the PWWP domain with modified histone tails using peptide arrays and show that it specifically recognizes the histone 3 lysine 36 trimethylation mark. H3K36me3 is known to be a repressive modification correlated with DNA methylation in mammals and heterochromatin in Schizosaccharomyces pombe. These results were confirmed by equilibrium peptide binding studies and pulldown experiments with native histones and purified native nucleosomes. The PWWP-H3K36me3 interaction is important for the subnuclear localization of enhanced yellow fluorescent protein-fused Dnmt3a. Furthermore, the PWWP-H3K36me3 interaction increases the activity of Dnmt3a for methylation of nucleosomal DNA as observed using native nucleosomes isolated from human cells after demethylation of the DNA with 5-aza-2'-deoxycytidine as substrate for methylation with Dnmt3a. These data suggest that the interaction of the PWWP domain with H3K36me3 is involved in targeting of Dnmt3a to chromatin carrying that mark, a model that is in agreement with several studies on the genome-wide distribution of DNA methylation and H3K36me3.