Automated maintenance of embryonic stem cell cultures

Embryonic stem cell (ESC) technology provides attractive perspectives for generating unlimited numbers of somatic cells for disease modeling and compound screening. A key prerequisite for these industrial applications are standardized and automated systems suitable for stem cell processing. Here we demonstrate that mouse and human ESC propagated by automated culture maintain their mean specific growth rates, their capacity for multi-germlayer differentiation, and the expression of the pluripotency-associated markers SSEA-1/Oct-4 and Tra-1-60/Tra-1-81/Oct-4, respectively. The feasibility of ESC culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.     

Hunting and Plant Community Dynamics in Tropical Forests: A Synthesis and Future Directions

This synthesis builds on the preceding articles of this Special Section and has three goals. We first review the nascent literature that addresses indirect effects of hunting for tropical forest plant communities. Next, we highlight the potential indirect effects of hunting for other groups of organisms. Our final goal is to consider what could be done to ameliorate the demographic threats to harvest-sensitive game species caused by unsustainable hunting. Three conclusions are possible at this time concerning the impact of hunting for tropical forest plant communities: (1) Hunting tends to reduce seed movement for animal-dispersed species with very large diaspores; (2) Hunting reduces seed predation by granivorous vertebrates for species with large seeds; and (3) Hunting alters the species composition of the seedling and sapling layers. The cascading effects of hunting are already known to affect bruchid beetles and dung beetles and are likely to affect other, nongame taxa. To ameliorate these problems, several lines of research should be further explored to facilitate the development of game management plans including: (1) alternative use of sources of animal protein; (2) income supplementation for local people from sources other than wild meat; (3) outreach and extension activities for communities; (4) recognition and facilitation of the shifting of attitudes towards hunting; (5) implementation of community-based wildlife management programs in regulated-use areas such as extractive reserves; and (6) landscape-scale conservation planning that maximizes the source-sink dynamics of harvested and unharvested game populations and enforces game regulations in strictly protected areas.     

Clinical safety and efficacy of NG440: a novel combination of rho iso-alpha acids from hops, rosemary, and oleanolic acid for inflammatory conditions

In this report, we examine the clinical safety and efficacy of NG440, a phytochemical-based antiinflammatory formula consisting of a combination of rho iso-alpha acids from hops, rosemary, and oleanolic acid. In a previous study, we demonstrated that NG440 significantly decreased pain by 50% in patients with osteoarthritis. Consistent with these data, results from a multicentre trial indicate that NG440 reduced pain scores in patients with joint discomfort, as measured by VAS (visual analog scale) methodology. As demonstrated in an ex vivo clinical study, these effects on pain relief may be due to reduced inflammatory cytokine production including lower prostaglandin E2 formation. Finally, strong data exist to suggest that NG440 is a safe formula for human consumption. Animal toxicity data revealed no adverse effects of NG440 at dosages < or =250 mg.kg-1.day-1 for 21 days. Furthermore, human trial data suggest that NG440 does not negatively impact cardiovascular and gastrointestinal markers normally affected by selective COX-2 enzyme inhibitors, including platelet function, blood pressure, blood cell count, or fecal calprotectin, a measure of gastrointestinal injury. In conclusion, NG440 may serve as a safe and efficacious alternative in some areas where specific COX-2 inhibitors have been traditionally used.     

Differentiation of the anomeric configuration and ring form of glucosyl-glycolaldehyde anions in the gas phase by mass spectrometry: isomeric discrimination between m/z 221 anions derived from disaccharides and chemical synthesis of m/z 221 standards

Mass spectrometry of disaccharides in the negative-ion mode frequently generates product anions of m/z 221. With glucose-containing disaccharides, dissociation of isolated m/z 221 product ions in a Paul trap yielded mass spectra that easily differentiated between both anomeric configurations and ring forms of the ions. These ions were shown to be glucosyl-glycolaldehydes through chemical synthesis of their standards. By labeling the reducing carbonyl oxygen of disaccharides with 18O to mass discriminate between monosaccharides, it was established that the m/z 221 ions are comprised solely of an intact nonreducing sugar with a two-carbon aglycon derived from the reducing sugar, regardless of the disaccharide linkage position. This enabled the anomeric configuration and ring form of the ion to be assigned and the location of the ion to the nonreducing side of a glycosidic linkage to be ascertained. Detailed studies of experimental factors necessary for reproducibility in a Paul trap demonstrated that the unique dissociation patterns that discriminate between the isomeric m/z 221 ions could be obtained from month-to-month in conjunction with an internal energy-input calibrant ion that ensures reproducible energy deposition into isolated m/z 221 ions. In addition, MS/MS fragmentation patterns of disaccharide m/z 341 anions in a Paul trap enabled linkage positions to be assigned, as has been previously reported with other types of mass spectrometers.     

Sperm competition games between sneaks and guards: a comparative analysis using dimorphic male beetles

Sperm competition is widely recognized as a pervasive force of sexual selection. Theory predicts that across species increased risk of sperm competition should favor an increased expenditure on the ejaculate, a prediction for which there is much evidence. Sperm competition games have also been developed specifically for systems in which males adopt the alternative male mating tactics of sneaking copulations or guarding females. These models have not yet been tested in a comparative context, but predict that: across species male expenditure on the ejaculate should increase with increasing probability of a sneak mating; within species, sneaks should have the greater expenditure on the ejaculate; and the disparity in expenditure between sneaks and guards should be greatest in species with moderate risk of a sneak mating, and decline toward parity in species with low or high risk. Beetles in the genus Onthophagus are often characterized by dimorphic male morphologies that reflect the alternative mating tactics of sneak (minor males) and guard (major males). We conducted a comparative analysis across 16 species of male dimorphic onthophagines, finding that testes size increased across species with increasing frequency of the minor male phenotype. Minor males generally had the greater testes size, but across species the disparity between morphs was independent of the frequency of minor males. We present data on testes allometry from two populations of O. taurus that have undergone genetic divergence in the frequency of minor males. Consistent with the comparative analysis, these data support the notion that the relative frequency of sneaks in the population influences male expenditure on the ejaculate.     

Ecological speciation in Gambusia fishes

Although theory indicates that natural selection can facilitate speciation as a by-product, demonstrating ongoing speciation via this by-product mechanism in nature has proven difficult. We examined morphological, molecular, and behavioral data to investigate ecology's role in incipient speciation for a post-Pleistocene radiation of Bahamas mosquitofish (Gambusia hubbsi) inhabiting blue holes. We show that adaptation to divergent predator regimes is driving ecological speciation as a by-product. Divergence in body shape, coupled with assortative mating for body shape, produces reproductive isolation that is twice as strong between populations inhabiting different predator regimes than between populations that evolved in similar ecological environments. Gathering analogous data on reproductive isolation at the interspecific level in the genus, we find that this mechanism of speciation may have been historically prevalent in Gambusia. These results suggest that speciation in nature can result as a by-product of divergence in ecologically important traits, producing interspecific patterns that persist long after speciation events have completed.     

Genomewide association analysis in diverse inbred mice: power and population structure

The discovery of quantitative trait loci (QTL) in model organisms has relied heavily on the ability to perform controlled breeding to generate genotypic and phenotypic diversity. Recently, we and others have demonstrated the use of an existing set of diverse inbred mice (referred to here as the mouse diversity panel, MDP) as a QTL mapping population. The use of the MDP population has many advantages relative to traditional F(2) mapping populations, including increased phenotypic diversity, a higher recombination frequency, and the ability to collect genotype and phenotype data in community databases. However, these methods are complicated by population structure inherent in the MDP and the lack of an analytical framework to assess statistical power. To address these issues, we measured gene expression levels in hypothalamus across the MDP. We then mapped these phenotypes as quantitative traits with our association algorithm, resulting in a large set of expression QTL (eQTL). We utilized these eQTL, and specifically cis-eQTL, to develop a novel nonparametric method for association analysis in structured populations like the MDP. These eQTL data confirmed that the MDP is a suitable mapping population for QTL discovery and that eQTL results can serve as a gold standard for relative measures of statistical power.     

High resolution structure of Deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution

Phytochromes are red/far red light photochromic photoreceptors that direct many photosensory behaviors in the bacterial, fungal, and plant kingdoms. They consist of an N-terminal domain that covalently binds a bilin chromophore and a C-terminal region that transmits the light signal, often through a histidine kinase relay. Using x-ray crystallography, we recently solved the first three-dimensional structure of a phytochrome, using the chromophore-binding domain of Deinococcus radiodurans bacterial phytochrome assembled with its chromophore, biliverdin IXalpha. Now, by engineering the crystallization interface, we have achieved a significantly higher resolution model. This 1.45A resolution structure helps identify an extensive buried surface between crystal symmetry mates that may promote dimerization in vivo. It also reveals that upon ligation of the C3(2) carbon of biliverdin to Cys(24), the chromophore A-ring assumes a chiral center at C2, thus becoming 2(R),3(E)-phytochromobilin, a chemistry more similar to that proposed for the attached chromophores of cyanobacterial and plant phytochromes than previously appreciated. The evolution of bacterial phytochromes to those found in cyanobacteria and higher plants must have involved greater fitness using more reduced bilins, such as phycocyanobilin, combined with a switch of the attachment site from a cysteine near the N terminus to one conserved within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. From analysis of site-directed mutants in the D. radiodurans phytochrome, we show that this bilin preference was partially driven by the change in binding site, which ultimately may have helped photosynthetic organisms optimize shade detection. Collectively, these three-dimensional structural results better clarify bilin/protein interactions and help explain how higher plant phytochromes evolved from prokaryotic progenitors.     

Interleukin-1 (IL-1)-induced TAK1-dependent Versus MEKK3-dependent NFkappaB activation pathways bifurcate at IL-1 receptor-associated kinase modification

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is phosphorylated after it is recruited to the receptor, subsequently ubiquitinated, and eventually degraded upon IL-1 stimulation. Although a point mutation changing lysine 134 to arginine (K134R) in IRAK abolished IL-1-induced IRAK ubiquitination and degradation, mutations of serines and threonines adjacent to lysine 134 to alanines ((S/T)A (131-144)) reduced IL-1-induced IRAK phosphorylation and abolished IRAK ubiquitination. Through the study of these IRAK modification mutants, we uncovered two parallel IL-1-mediated signaling pathways for NFkappaB activation, TAK1-dependent and MEKK3-dependent, respectively. These two pathways bifurcate at the level of IRAK modification. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classical NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through IkappaBalpha phosphorylation and subsequent dissociation from NFkappaB but without IkappaBalpha degradation. These results provide significant insight to our further understanding of NFkappaB activation pathways.