A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: Comparative expression profiles of mRNAs, microRNAs and proteins among different feeders and conditioned media

<p>Abstract</p> <p>Background</p> <p>Human embryonic stem (hES) cell lines were derived from the inner cell mass of human blastocysts, and were cultured on mouse embryonic fibroblast (MEF) feeder to maintain undifferentiated growth, extensive renewal capacity, and pluripotency. The hES-T3 cell line with normal female karyotype was previously used to differentiate into autogeneic fibroblast-like cells (T3HDF) as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF) for 14 passages.</p> <p>Results</p> <p>A feeder-free culture on Matrigel in hES medium conditioned by the autogeneic feeder cells (T3HDF) was established to maintain the undifferentiated growth of hES-T3 cells (T3/CMHDF) for 8 passages in this investigation. The gene expression profiles of mRNAs, microRNAs and proteins between the undifferentiated T3/HDF and T3/CMHDF cells were shown to be very similar, and their expression profiles were also found to be similar to those of T3/MEF and T3/CMMEF cells grown on MEF feeder and feeder-free Matrigel in MEF-conditioned medium, respectively. The undifferentiated state of T3/HDF and T3/CMHDF as well as T3/MEF andT3/CMMEF cells was evidenced by the very high expression levels of "stemness" genes and low expression levels of differentiation markers of ectoderm, mesoderm and endoderm in addition to the strong staining of OCT4 and NANOG.</p> <p>Conclusion</p> <p>The T3HDF feeder and T3HDF-conditioned medium were able to support the undifferentiated growth of hES cells, and they would be useful for drug development and toxicity testing in addition to the reduced risks of xenogeneic pathogens when used for medical applications such as cell therapies.</p

Characterization of membrane vesicles in Alteromonas macleodii indicates potential roles in their copiotrophic lifestyle

Bacterial membrane vesicles (MVs) are abundant in the oceans, but their potential functional roles remain unclear. In this study we characterized MV production and protein content of six strains of Alteromonas macleodii, a cosmopolitan marine bacterium. Alteromonas macleodii strains varied in their MV production rates, with some releasing up to 30 MVs per cell per generation. Microscopy imaging revealed heterogenous MV morphologies, including some MVs aggregated within larger membrane structures. Proteomic characterization revealed that A. macleodii MVs are rich in membrane proteins related to iron and phosphate uptake, as well as proteins with potential functions in biofilm formation. Furthermore, MVs harbored ectoenzymes, such as aminopeptidases and alkaline phosphatases, which comprised up to 20% of the total extracellular enzymatic activity. Our results suggest that A. macleodii MVs may support its growth through generation of extracellular ‘hotspots’ that facilitate access to essential substrates. This study provides an important basis to decipher the ecological relevance of MVs in heterotrophic marine bacteria

Vegetation response to invasive Tamarix control in southwestern U.S. rivers: a collaborative study including 416 sites

Most studies assessing vegetation response following control of invasive Tamarix trees along southwestern U.S. rivers have been small in scale (e.g., river reach), or at a regional scale but with poor spatial-temporal replication, and most have not included testing the effects of a now widely used biological control. We monitored plant composition following Tamarix control along hydrologic, soil, and climatic gradients in 244 treated and 172 reference sites across six U.S. states. This represents the largest comprehensive assessment to date on the vegetation response to the four most common Tamarix control treatments. Biocontrol by a defoliating beetle (treatment 1) reduced the abundance of Tamarix less than active removal by mechanically using hand and chain-saws (2), heavy machinery (3) or burning (4). Tamarix abundance also decreased with lower temperatures, higher precipitation, and follow-up treatments for Tamarix resprouting. Native cover generally increased over time in active Tamarix removal sites, however, the increases observed were small and was not consistently increased by active revegetation. Overall, native cover was correlated to permanent stream flow, lower grazing pressure, lower soil salinity and temperatures, and higher precipitation. Species diversity also increased where Tamarix was removed. However, Tamarix treatments, especially those generating the highest disturbance (burning and heavy machinery), also often promoted secondary invasions of exotic forbs. The abundance of hydrophytic species was much lower in treated than in reference sites, suggesting that management of southwestern U.S. rivers has focused too much on weed control, overlooking restoration of fluvial processes that provide habitat for hydrophytic and floodplain vegetation. These results can help inform future management of Tamarix-infested rivers to restore hydrogeomorphic processes, increase native biodiversity and reduce abundance of noxious species

Physiology and evolution of nitrate acquisition in Prochlorococcus

Prochlorococcus is the numerically dominant phototroph in the oligotrophic subtropical ocean and carries out a significant fraction of marine primary productivity. Although field studies have provided evidence for nitrate uptake by Prochlorococcus, little is known about this trait because axenic cultures capable of growth on nitrate have not been available. Additionally, all previously sequenced genomes lacked the genes necessary for nitrate assimilation. Here we introduce three Prochlorococcus strains capable of growth on nitrate and analyze their physiology and genome architecture. We show that the growth of high-light (HL) adapted strains on nitrate is ~17% slower than their growth on ammonium. By analyzing 41 Prochlorococcus genomes, we find that genes for nitrate assimilation have been gained multiple times during the evolution of this group, and can be found in at least three lineages. In low-light adapted strains, nitrate assimilation genes are located in the same genomic context as in marine Synechococcus. These genes are located elsewhere in HL adapted strains and may often exist as a stable genetic acquisition as suggested by the striking degree of similarity in the order, phylogeny and location of these genes in one HL adapted strain and a consensus assembly of environmental Prochlorococcus metagenome sequences. In another HL adapted strain, nitrate utilization genes may have been independently acquired as indicated by adjacent phage mobility elements; these genes are also duplicated with each copy detected in separate genomic islands. These results provide direct evidence for nitrate utilization by Prochlorococcus and illuminate the complex evolutionary history of this trait.Gordon and Betty Moore Foundation (Grant GBMF495)National Science Foundation (U.S.) (Grant OCE-1153588)National Science Foundation (U.S.) (Grant DBI-0424599

A High-Resolution SNP Array-Based Linkage Map Anchors a New Domestic Cat Draft Genome Assembly and Provides Detailed Patterns of Recombination

High-resolution genetic and physical maps are invaluable tools for building accurate genome assemblies, and interpreting results of genome-wide association studies (GWAS). Previous genetic and physical maps anchored good quality draft assemblies of the domestic cat genome, enabling the discovery of numerous genes underlying hereditary disease and phenotypes of interest to the biomedical science and breeding communities. However, these maps lacked sufficient marker density to order thousands of shorter scaffolds in earlier assemblies, which instead relied heavily on comparative mapping with related species. A high-resolution map would aid in validating and ordering chromosome scaffolds from existing and new genome assemblies. Here, we describe a high-resolution genetic linkage map of the domestic cat genome based on genotyping 453 domestic cats from several multi-generational pedigrees on the Illumina 63K SNP array. The final maps include 58,055 SNP markers placed relative to 6637 markers with unique positions, distributed across all autosomes and the X chromosome. Our final sex-averaged maps span a total autosomal length of 4464 cM, the longest described linkage map for any mammal, confirming length estimates from a previous microsatellite-based map. The linkage map was used to order and orient the scaffolds from a substantially more contiguous domestic cat genome assembly (Felis catusv8.0), which incorporated ∼20 × coverage of Illumina fragment reads. The new genome assembly shows substantial improvements in contiguity, with a nearly fourfold increase in N50 scaffold size to 18 Mb. We use this map to report probable structural errors in previous maps and assemblies, and to describe features of the recombination landscape, including a massive (∼50 Mb) recombination desert (of virtually zero recombination) on the X chromosome that parallels a similar desert on the porcine X chromosome in both size and physical location

A Quantitative Systems Pharmacology perspective on the importance of parameter identifiability

There is an inherent tension in Quantitative Systems Pharmacology (QSP) between the need to incorporate mathematical descriptions of complex physiology and drug targets with the necessity of developing robust, predictive and well-constrained models. In addition to this there is no "gold standard" for model development and assessment in QSP. Moreover, there can be confusion over terminology such as model and parameter identifiability; complex and simple models; virtual populations; and other concepts, which leads to potential miscommunication and misapplication of methodologies within modelling communities, both the QSP community and related disciplines. This perspective article highlights the pros and cons of using simple (often identifiable) vs. complex (more physiologically detailed but often non-identifiable) models, as well as aspects of parameter identifiability, sensitivity and inference methodologies for model development and analysis. The paper distills the central themes of the issue of identifiability and optimal model size and discusses open challenges

Vegetation response to invasive Tamarix control in southwestern U.S. rivers: a collaborative study including 416 sites

Most studies assessing vegetation response following control of invasive Tamarix trees along southwestern U.S. rivers have been small in scale (e.g., river reach), or at a regional scale but with poor spatial-temporal replication, and most have not included testing the effects of a now widely used biological control. We monitored plant composition following Tamarix control along hydrologic, soil, and climatic gradients in 244 treated and 172 reference sites across six U.S. states. This represents the largest comprehensive assessment to date on the vegetation response to the four most common Tamarix control treatments. Biocontrol by a defoliating beetle (treatment 1) reduced the abundance of Tamarix less than active removal by mechanically using hand and chain-saws (2), heavy machinery (3) or burning (4). Tamarix abundance also decreased with lower temperatures, higher precipitation, and follow-up treatments for Tamarix resprouting. Native cover generally increased over time in active Tamarix removal sites, however, the increases observed were small and was not consistently increased by active revegetation. Overall, native cover was correlated to permanent stream flow, lower grazing pressure, lower soil salinity and temperatures, and higher precipitation. Species diversity also increased where Tamarix was removed. However, Tamarix treatments, especially those generating the highest disturbance (burning and heavy machinery), also often promoted secondary invasions of exotic forbs. The abundance of hydrophytic species was much lower in treated than in reference sites, suggesting that management of southwestern U.S. rivers has focused too much on weed control, overlooking restoration of fluvial processes that provide habitat for hydrophytic and floodplain vegetation. These results can help inform future management of Tamarix-infested rivers to restore hydrogeomorphic processes, increase native biodiversity and reduce abundance of noxious species

The Nature of Notebooks: How Enlightenment Schoolchildren Transformed the Tabula Rasa

John Locke's comparison of the mind to a blank piece of paper, the tabula rasa, was one of the most recognizable metaphors of the British Enlightenment. Though scholars embrace its impact on the arts, humanities, natural sciences, and social sciences, they seldom consider why the metaphor was so successful. Concentrating on the notebooks made and used by the schoolchildren of Enlightenment Scotland, this essay contends that the answer lies in the material and visual conditions that gave rise to the metaphor's usage. By the time students had finished school, they had learned to conceptualize the pages, the script, and the figures of their notebooks as indispensable learning tools that could be manipulated by scores of adaptable folding, writing, and drawing techniques. In this article, I reveal that historicizing the epistemology and manipulability of student manuscript culture makes it possible to see that the success of Locke's metaphor was founded on its appeal to everyday note-keeping activities performed by British schoolchildren

Aberrant in Vivo T Helper Type 2 Cell Response and Impaired Eosinophil Recruitment in Cc Chemokine Receptor 8 Knockout Mice

Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (−/−) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8−/− mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo