Functional and Molecular Characterization of the Role of CTCF in Human Embryonic Stem Cell Biology

The CCCTC-binding factor CTCF is the only known vertebrate insulator protein and has been shown to regulate important developmental processes such as imprinting, X-chromosome inactivation and genomic architecture. In this study, we examined the role of CTCF in human embryonic stem cell (hESC) biology. We demonstrate that CTCF associates with several important pluripotency genes, including NANOG, SOX2, cMYC and LIN28 and is critical for hESC proliferation. CTCF depletion impacts expression of pluripotency genes and accelerates loss of pluripotency upon BMP4 induced differentiation, but does not result in spontaneous differentiation. We find that CTCF associates with the distal ends and internal sites of the co-regulated 160 kb NANOG-DPPA3-GDF3 locus. Each of these sites can function as a CTCF-dependent enhancer-blocking insulator in heterologous assays. In hESCs, CTCF exists in multisubunit protein complexes and can be poly(ADP)ribosylated. Known CTCF cofactors, such as Cohesin, differentially co-localize in the vicinity of specific CTCF binding sites within the NANOG locus. Importantly, the association of some cofactors and protein PARlation selectively changes upon differentiation although CTCF binding remains constant. Understanding how unique cofactors may impart specialized functions to CTCF at specific genomic locations will further illuminate its role in stem cell biology.     

Dynamics of lipid accumulation by the fat body of Rhodnius prolixus: the involvement of lipophorin binding sites

In insects, lipids are stored in the fat body, mainly as triacylglycerol (TAG). In Rhodnius prolixus, a hematophagous hemipteran, lipids are accumulated after blood meal to be used later on. In adult females, at the second day after feeding, the amount of TAG was 57+/-17 microg/fat body, it increased almost five times and at fourth day it was 244+/-35 microg/fat body. TAG content remained constant until day 13, but it then decreased and, at day 20th it was very low (31+/-4.9 microg/fat body). Radiolabeled free fatty acid was used to follow lipid accumulation by the fat body, as it was previously shown that, in R. prolixus, injected free fatty acids associate with lipophorin, a major hemolymphatic lipoprotein. (3)H-palmitic acid was injected into the hemocoel of R. prolixus females. It disappeared from the hemolymph very rapidly, and radioactivity was incorporated by the fat body. Sixty minutes after injection, radioactivity in the fat body was found mainly in TAGs. The capacity of the fat body to incorporate fatty acids from the hemolymph varied according to the days after blood meal, and it was maximal around the fourth day. Lipophorin binding to specific sites in fat body membrane preparations also showed variation at different days. When membranes obtained from insects at the second, fifth and tenth days were compared, binding was highest at fifth day after feeding.     

Nonparametric and semiparametric group sequential methods for comparing accuracy of diagnostic tests

SUMMARY: Comparison of the accuracy of two diagnostic tests using the receiver operating characteristic (ROC) curves from two diagnostic tests has been typically conducted using fixed sample designs. On the other hand, the human experimentation inherent in a comparison of diagnostic modalities argues for periodic monitoring of the accruing data to address many issues related to the ethics and efficiency of the medical study. To date, very little research has been done on the use of sequential sampling plans for comparative ROC studies, even when these studies may use expensive and unsafe diagnostic procedures. In this article we propose a nonparametric group sequential design plan. The nonparametric sequential method adapts a nonparametric family of weighted area under the ROC curve statistics (Wieand et al., 1989, Biometrika 76, 585-592) and a group sequential sampling plan. We illustrate the implementation of this nonparametric approach for sequentially comparing ROC curves in the context of diagnostic screening for nonsmall-cell lung cancer. We also describe a semiparametric sequential method based on proportional hazard models. We compare the statistical properties of the nonparametric approach with alternative semiparametric and parametric analyses in simulation studies. The results show the nonparametric approach is robust to model misspecification and has excellent finite-sample performance.     

Preparation of an (−)-ephedrine intermediate through asymmetric reduction of 1-phenyl-1,2-propanedione by anaerobically pre-treated baker’s yeast

The influence of using an anaerobically pre-treated baker’s yeast on the reduction of (R)-1-hydroxy-1-phenyl-2-propanone (2) and (S)-2-hydroxy-1-phenyl-1-propanone (4) was investigated in comparison with non-pre-treated baker’s yeast reduction (control experiments). We observed that there is no significant difference between the anaerobically pre-treated yeast and the control experiment on the reduction rates of 2. On the other hand, the rate of reduction of 4 mediated by the anaerobically pre-treated yeast is much slower than the aerobic experiment. To improve the regioselectivity of reduction of 1-phenyl-1,2-propanedione (1), a baker’s yeast suspension was pre-treated with nitrogen (60 min) followed by oxygen (20 min), to give 2 in 28–31% of yields (96% e.e.) and 3 in 42–62% (>99% e.e.) after 75–90 min of reaction.     

Life at the Energetic Edge: Kinetics of Circumneutral Iron Oxidation by Lithotrophic Iron-Oxidizing Bacteria Isolated from the Wetland-Plant Rhizosphere

Batch cultures of a lithotrophic Fe(II)-oxidizing bacterium, strain BrT, isolated from the rhizosphere of a wetland plant, were grown in bioreactors and used to determine the significance of microbial Fe(II) oxidation at circumneutral pH and to identify abiotic variables that affect the partitioning between microbial oxidation and chemical oxidation. Strain BrT grew only in the presence of an Fe(II) source, with an average doubling time of 25 h. In one set of experiments, Fe(II) oxidation rates were measured before and after the cells were poisoned with sodium azide. These experiments indicated that strain BrT accounted for 18 to 53% of the total iron oxidation, and the average cellular growth yield was 0.70 g of CH₂O per mol of Fe(II) oxidized. In a second set of experiments, Fe(II) was constantly added to bioreactors inoculated with live cells, killed cells, or no cells. A statistical model fitted to the experimental data demonstrated that metabolic Fe(II) oxidation accounted for up to 62% of the total oxidation. The total Fe(II) oxidation rates in these experiments were strongly limited by the rate of Fe(II) delivery to the system and were also influenced by O₂ and total iron concentrations. Additionally, the model suggested that the microbes inhibited rates of abiotic Fe(II) oxidation, perhaps by binding Fe(II) to bacterial exopolymers. The net effect of strain BrT was to accelerate total oxidation rates by up to 18% compared to rates obtained with cell-free treatments. The results suggest that neutrophilic Fe(II)-oxidizing bacteria may compete for limited O₂ in the rhizosphere and therefore influence other wetland biogeochemical cycles.