Co-expression networks in generation of induced pluripotent stem cells.

We developed an adenoviral vector, in which Yamanaka's four reprogramming factors (RFs) were controlled by individual CMV promoters in a single cassette (Ad-SOcMK). This permitted coordinated expression of RFs (SOX2, OCT3/4, c-MYC and KLF4) in a cell for a transient period of time, synchronizing the reprogramming process with the majority of transduced cells assuming induced pluripotent stem cell (iPSC)-like characteristics as early as three days post-transduction. These reprogrammed cells resembled human embryonic stem cells (ESCs) with regard to morphology, biomarker expression, and could be differentiated into cells of the germ layers in vitro and in vivo. These iPSC-like cells, however, failed to expand into larger iPSC colonies. The short and synchronized reprogramming process allowed us to study global transcription changes within short time intervals. Weighted gene co-expression network analysis (WGCNA) identified sixteen large gene co-expression modules, each including members of gene ontology categories involved in cell differentiation and development. In particular, the brown module contained a significant number of ESC marker genes, whereas the turquoise module contained cell-cycle-related genes that were downregulated in contrast to upregulation in human ESCs. Strong coordinated expression of all four RFs via adenoviral transduction may constrain stochastic processes and lead to silencing of genes important for cellular proliferation

Validation of an experimental setup to study atmospheric heterogeneous ozonolysis of semi-volatile organic compounds

International audienceThere is currently a need for reliable experimental procedures to follow the heterogeneous processing simulating the atmospheric conditions. This work offers an alternative experimental device to study the behaviour of semi-volatile organic compounds (SVOC) that presumably exhibit extremely slow reactivity (e.g. pesticides) towards the atmospheric oxidants such as ozone and OH. Naphthalene was chosen as a test compound since it was widely studied in the past and hence represents a good reference. Prior to ozone exposure, the gaseous naphthalene was adsorbed via gas-solid equilibrium on silica and XAD-4 particles. Then, the heterogeneous reaction of ozone with adsorbed naphthalene was investigated in specially designed flow tube reactors. After the reaction, the remaining naphthalene (adsorbed on particles surface) was extracted, filtered and analyzed by Gas Chromatography-Flame Ionization Detector (GC-FID). Thus, the kinetics results were obtained following the consumption of naphthalene. Using this procedure, the rate constants of heterogeneous ozonolysis of naphthalene (kO3 silica=2.26 (±0.09)×10−17 cm3 molec−1 s−1 and kO3 XAD-4=4.29 (±1.06)×10−19 cm3 molec−1 s−1) were determined for silica and XAD-4 particles, at 25°C and relative humidity <0.7%. The results show that the nature of the particles significantly affects the kinetics and that heterogeneous ozonolysis of naphthalene is faster than its homogeneous ozonolysis in the gas phase

The NRPD1 N-terminus contains a Pol IV-specific motif that is critical for genome surveillance in Arabidopsis

RNA-guided surveillance systems constrain the activity of transposable elements (TEs) in host genomes. In plants, RNA polymerase IV (Pol IV) transcribes TEs into primary transcripts from which RDR2 synthesizes double-stranded RNA precursors for small interfering RNAs (siRNAs) that guide TE methylation and silencing. How the core subunits of Pol IV, homologs of RNA polymerase II subunits, diverged to support siRNA biogenesis in a TE-rich, repressive chromatin context is not well understood. Here we studied the N-terminus of Pol IV’s largest subunit, NRPD1. Arabidopsis lines harboring missense mutations in this N-terminus produce wild-type (WT) levels of NRPD1, which co-purifies with other Pol IV subunits and RDR2. Our in vitro transcription and genomic analyses reveal that the NRPD1 N-terminus is critical for robust Pol IV-dependent transcription, siRNA production and DNA methylation. However, residual RNA-directed DNA methylation observed in one mutant genotype indicates that Pol IV can operate uncoupled from the high siRNA levels typically observed in WT plants. This mutation disrupts a motif uniquely conserved in Pol IV, crippling the enzyme's ability to inhibit retrotransposon mobilization. We propose that the NRPD1 N-terminus motif evolved to regulate Pol IV function in genome surveillance

Components of the Plasminogen Activation System Promote Engraftment of Porous Polyethylene Biomaterial via Common and Distinct Effects

Rapid fibrovascularization is a prerequisite for successful biomaterial engraftment. In addition to their well-known roles in fibrinolysis, urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) or their inhibitor plasminogen activator inhibitor-1 (PAI-1) have recently been implicated as individual mediators in non-fibrinolytic processes, including cell adhesion, migration, and proliferation. Since these events are critical for fibrovascularization of biomaterial, we hypothesized that the components of the plasminogen activation system contribute to biomaterial engraftment. Employing in vivo and ex vivo microscopy techniques, vessel and collagen network formation within porous polyethylene (PPE) implants engrafted into dorsal skinfold chambers were found to be significantly impaired in uPA-, tPA-, or PAI-1-deficient mice. Consequently, the force required for mechanical disintegration of the implants out of the host tissue was significantly lower in the mutant mice than in wild-type controls. Conversely, surface coating with recombinant uPA, tPA, non-catalytic uPA, or PAI-1, but not with non-catalytic tPA, accelerated implant vascularization in wild-type mice. Thus, uPA, tPA, and PAI-1 contribute to the fibrovascularization of PPE implants through common and distinct effects. As clinical perspective, surface coating with recombinant uPA, tPA, or PAI-1 might provide a novel strategy for accelerating the vascularization of this biomaterial

Fish assemblage change following the structural restoration of a degraded stream

Decades of anthropogenic pressure have harmed riverscapes throughout North America by degrading habitats and water quality and can result in the extirpation of sensitive aquatic taxa. Local stream restoration projects have increased in frequency, but monitoring is still infrequent. In 2010, Kickapoo Creek in East Central Illinois was subjected to a stream restoration project that included implementation of artificial riffles, riprap, scouring keys, and riparian vegetation. We monitored the restoration efforts for 6years after the restoration through annual sampling efforts at restored and reference sites to determine changes in habitat and fish assemblage using standard habitat sampling and electrofishing techniques. We observed distinct temporal and spatial shifts in physico-chemical parameters along with changes in fish community structure. Although biotic integrity remained moderately low in reference assemblages, restored reaches showed 3-year delay in response to restoration, with biotic integrity positively linked to additional instream habitat and altered channel morphology. Larger substrate sizes, submerged terrestrial vegetation, and newly formed scour pools along with reduced siltation were found in the restored sites, in contrast to the reference sites. These changes resulted in increased species diversity, reduced number of opportunistic species and consequently an overall increase in health of fish communities. We also observed recruitment of habitat specialists and increase in species with reproductive strategies that rely on complex substrates. The results of this study highlight some of the complex dynamics driving reach-scale restoration projects. We demonstrate the usefulness of structural restoration as a management tool to increase biotic integrity through long-term alteration of critical habitat. The delay in the response of species to the restoration efforts emphasizes the need for long-term continuous temporal and spatial monitoring

Additive Manufacturing Under Lunar Gravity and Microgravity

Mankind is setting to colonize space, for which the manufacturing of habitats, tools, spare parts and other infrastructure is required. Commercial manufacturing processes are already well engineered under standard conditions on Earth, which means under Earth’s gravity and atmosphere. Based on the literature review, additive manufacturing under lunar and other space gravitational conditions have only been researched to a very limited extent. Especially, additive manufacturing offers many advantages, as it can produce complex structures while saving resources. The materials used do not have to be taken along on the mission, they can even be mined and processed on-site. The Einstein-Elevator offers a unique test environment for experiments under different gravitational conditions. Laser experiments on selectively melting regolith simulant are successfully conducted under lunar gravity and microgravity. The created samples are characterized in terms of their geometry, mass and porosity. These experiments are the first additive manufacturing tests under lunar gravity worldwide

Sources of Nonnative Centrarchids in the Upper Colorado River Revealed by Stable Isotope and Microchemical Analyses of Otoliths

Nonnative fishes represent a significant impediment to the recovery of imperiled fishes, including those endemic to the Colorado River in the southwestern United States. Efforts to control nonindigenous fish abundance in the upper Colorado River basin have been unsuccessful owing in part to lack of knowledge regarding nonnative fish recruitment sources. We determined the source habitat (floodplain pond versus riverine habitats) for nonnative centrarchid fishes (largemouth bass Micropterus salmoides, green sunfish Lepomis cyanellus, bluegill L. macrochirus, and black crappie Pomoxis nigromaculatus) in the upper Colorado River using stable hydrogen isotopic composition (δD) and strontium:calcium (Sr:Ca) ratios in fish otoliths as natural markers of environmental history. Stable hydrogen isotope analysis revealed that 59% of centrarchids exhibited the otolith core signatures expected for riverine-origin fish, while 22% had emigrated from floodplain ponds and 19% were of uncertain origin. Strontium:calcium ratio data were consistent with the δD assays and indicated that relatively few fish immigrated to the river from high-salinity habitats. Black crappie was the only species that originated primarily from floodplain ponds. Efforts to control the abundance of most of the fishes included in this study should be concentrated in riverine habitats given the hydrologic conditions (below-average river discharge) present during our study. However, the proportion of pond-origin fish increased with fish age, which, coupled with historical river discharge data, suggested that floodplain pond contributions to riverine nonnative fish populations fluctuate with the interannual variations in flow regime and river–pond connectivity. Our results are the first to demonstrate the utility of δD as a natural marker of fish environmental history that will probably provide valuable insights into the management of fish in other environments

A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors†

We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5′ end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators

Growth, Fecundity, and Diets of Newly Established Silver Carp in the Middle Mississippi River

The silver carp Hypophthalmichthys molitrix has spread throughout the Mississippi River drainage. During 2003, we determined its population status and potential impact in the middle Mississippi River (MMR), the conduit between the lower Mississippi River and the upper Mississippi, Missouri, and Illinois rivers. We quantified growth, age structure, fecundity, and diets of silver carp sampled with trammel nets and AC electrofishing in main-channel areas. Mean length at age in the MMR exceeded that of populations in Asia by as much as 26%. Individuals were typically more than 1 year old and 230 mm total length, suggesting that small, young fish were absent. Individuals in this population matured earlier (age 2) than in the species\u27 native range. Regardless of phytoplankton variation (using chlorophyll a as a surrogate) and zooplankton concentration at MMR sites, phytoplankton was consistently most abundant in diets. Silver carp are finding suitable resources within the MMR, allowing individuals to grow rapidly during early life, persist as adults, and successfully disperse upstream

Habitat Associations of Fish Species of Greatest Conservation Need at Multiple Spatial Scales in Wadeable Iowa Streams

Fish and habitat data were collected from 84 wadeable stream reaches in the Mississippi River drainage of Iowa to predict the occurrences of seven fish species of greatest conservation need and to identify the relative importance of habitat variables measured at small (e.g., depth, velocity, and substrate) and large (e.g., stream order, elevation, and gradient) scales in terms of their influence on species occurrences. Multiple logistic regression analysis was used to predict fish species occurrences, starting with all possible combinations of variables (5 large-scale variables, 13 small-scale variables, and all 18 variables) but limiting the final models to a maximum of five variables. Akaike’s information criterion was used to rank candidate models, weight model parameters, and calculate model-averaged predictions. On average, the correct classification rate (CCR = 80%) and Cohen’s kappa (κ = 0.59) were greatest for multiple-scale models (i.e., those including both large-scale and small-scale variables), intermediate for small-scale models (CCR = 75%; κ = 0.49), and lowest for large-scale models (CCR = 73%; κ = 0.44). The occurrence of each species was associated with a unique combination of large-scale and small-scale variables. Our results support the necessity of understanding factors that constrain the distribution of fishes across spatial scales to ensure that management decisions and actions occur at the appropriate scale