Indirect ultrasonication for protein quantification and peptide mass mapping through mass spectrometry-based techniques

We report in this work a fast protocol for protein quantification and for peptide mass mapping that rely on ¹⁸O isotopic labeling through the decoupling procedure. It is demonstrated that the purity and source of trypsin do not compromise the labeling degree and efficiency of the decoupled labeling reaction, and that the pH of the labeling reaction is a critical factor to obtain a significant ¹⁸O double labeling. We also show that the same calibration curve can be used for MALDI protein quantification during several days maintaining a reasonable accuracy, thus simplifying the handling of the quantification process. In addition we demonstrate that ¹⁸O isotopic labeling through the decoupling procedure can be successfully used to elaborate peptide mass maps. BSA was successfully quantified using the same calibration curve in different days and plasma from a freshwater fish, Cyprinus carpio, was used to elaborate the peptide mass maps.     

Trace LC/MS/MS quantitation of 17β‐estradiol as a biomarker for selective estrogen receptor modulator activity in the rat brain

A sensitive LC/MS/MS method has been developed by derivatization of 17β‐estradiol (E2) with dansyl chloride to quantitate 17β‐E2 in female rat serum. The use of E2‐d₅ minimized interferences from endogenous 17β‐E2 in order to achieve a limit of quantitation (LOQ) of 2.5 pg/ml using 150 µl of female rat serum. The recovery of the dansyl derivative was 95% or greater in quality control samples. The intra and interday assay precision was better than 8.2 and 6.2%, respectively, with accuracies ranging from 97 to 101% in the quality control samples. The assay was used for the quantitation of serum E2 as a biomarker for the estrogen receptor (ER) antagonist activity of small molecule SERMs (selective estrogen receptor modulators) in the female rat brain. The study revealed that a statistically significant upregulation of serum 17β‐E2 occurred for rats dosed with SERMs that are known to penetrate the brain and disrupt the hypothalamic‐pituitary‐ovarian (HPO) axis. Variations in 17β‐E2 in ascending dose studies also correlated with the corresponding trends in CYP17a1 levels, an mRNA biomarker for ovarian hyperstimulation. This biomarker assay has provided a useful screen for medicinal chemistry optimization to produce SERMs that do not interfere with negative feedback of estrogens on the brain and for biological hypothesis testing. Copyright © 2009 John Wiley & Sons, Ltd.     

Gene tree correction for reconciliation and species tree inference: Complexity and algorithms

Reconciliation consists in mapping a gene tree T into a species tree S, and explaining the incongruence between the two as evidence for duplication, loss and other events shaping the gene family represented by the leaves of T. When S is unknown, the Species Tree Inference Problem is to infer, from a set of gene trees, a species tree leading to a minimum reconciliation cost. As reconciliation is very sensitive to errors in T, gene tree correction prior to reconciliation is a fundamental task. In this paper, we investigate the complexity of four different combinatorial approaches for deleting misplaced leaves from T. First, we consider two problems (Minimum Leaf Removal and Minimum Species Removal) related to the reconciliation of T with a known species tree S. In the former (latter respectively) we want to remove the minimum number of leaves (species respectively) so that T is “MD-consistent” with S. Second, we consider two problems (Minimum Leaf Removal Inference and Minimum Species Removal Inference) related to species tree inference. In the former (latter respectively) we want to remove the minimum number of leaves (species respectively) from T so that there exists a species tree S such that T is MD-consistent with S. We prove that Minimum Leaf Removal and Minimum Species Removal are APX-hard, even when each label has at most two occurrences in the input gene tree, and we present fixed-parameter algorithms for the two problems. We prove that Minimum Leaf Removal Inference is not only NP-hard, but also W[2]-hard and inapproximable within factor $c\ln n$, where n is the number of leaves in the gene tree. Finally, we show that Minimum Species Removal Inference is NP-hard and W[2]-hard, when parameterized by the size of the solution, that is the minimum number of species removals.     

Improving the non-dominated sorting genetic algorithm using a gene-therapy method for multi-objective optimization

The non-dominate sorting genetic algorithmic-II (NSGA-II) is an effective algorithm for finding Pareto-optimal front for multi-objective optimization problems. To further enhance the advantage of the NSGA-II, this study proposes an evaluative-NSGA-II (E-NSGA-II) in which a novel gene-therapy method incorporates into the crossover operation to retain superior schema patterns in evolutionary population and enhance its solution capability. The merit of each select gene in a crossover chromosome is estimated by exchanging the therapeutic genes in both mating chromosomes and observing their fitness differentiation. Hence, the evaluative crossover operation can generate effective genomes based on the gene merit without explicitly analyzing the solution space. Experiments for nine unconstrained multi-objective benchmarks and four constrained problems show that E-NSGA-II can find Pareto-optimal solutions in all test cases with better convergence and diversity qualities than several existing algorithms.     

人nov基因全长cDNA克隆、测序及其组织表达谱分析

采用特异性mRNA富集技术，从人早期胚胎中得到人nov基因(novH)的cDNA克隆，序列分析表明，该基因cDNA序列由2389个碱基对组成，包含一段长1071bp的开放阅读框(ORF)，3′端含有加尾信号AATAAA和ply(A)尾巴。Northern杂交显示，在Hela细胞和肾上腺组织中有表达的novHmRNA，大小约为2．5kb；多组织RNA点杂交分析结果进一步表明，novH基因在肾上腺和主动脉中的表达水平相对较高，在成人肾、肝、肺和小肠组织中亦有少量的表达。     

Harris公式和ab公式对超形变核转动带的适用性的比较

对A≈190区超变形转动带分别用Harris二参数公式和ab公式进行了系统分析.分析结果表明,与正常变形核动带的情况相似,Harris公式与实验有明显的系统偏离,而ab公式则与实验很接近,可以相当精确和方便地描述超变形转动带.     

Developmental changes of poly(ADP-ribose) polymerase expression in Sarcophaga peregrina

To elucidate the physiological role of poly(ADP-ribose) polymerase (PARP), we studied the levels of PARP mRNA and protein during the developmental stages of Sarcophaga peregrina. PARP mRNA expression changed remarkably throughout the developmental stages. The level of PARP mRNA (the molecular ratio of PARP mRNA to the total RNA) was highest in unfertilized eggs and that of PARP protein (the molecular ratio of PARP protein to the total protein of the crude extract) was high in unfertilized and fertilized eggs and in 1st instar larvae. During the embryogenesis period, the levels of PARP mRNA and protein gradually decreased. The levels of PARP mRNA during larval and pupal periods became less than about 5% of that in unfertilized eggs. After the emergence of adult flies, the levels of PARP mRNA and protein increased both in female and male flies. PARP activity normalized with the total amount of protein in the crude extract changed in parallel to the level of PARP protein throughout the developmental stages. The biological significance of the drastic change of mRNA and protein levels of PARP still remains to be clarified.