Inhibition of MAPK by prolactin signaling through the short form of its receptor in the ovary and decidua: involvement of a novel phosphatase

Prolactin (PRL) is essential for normal reproduction and signals through two types of receptors, the short (PRL-RS) and long (PRL-RL) form. We have previously shown that transgenic mice expressing only PRL-RS (PRLR(-/-)RS) display abnormal follicular development and premature ovarian failure. Here, we report that MAPK, essential for normal follicular development, is critically inhibited by PRL in reproductive tissues of PRLR(-/-)RS mice. Consequently, the phosphorylation of MAPK downstream targets are also markedly inhibited by PRL without affecting immediate upstream kinases, suggesting involvement of MAPK specific phosphatase(s) in this inhibition. Similar results are obtained in a PRL-responsive ovary-derived cell line (GG-CL) that expresses only PRL-RS. However, we found the expression/activation of several known MAPK phosphatases not to be affected by PRL, suggesting a role of unidentified phosphatase(s). We detected a 27-kDa protein that binds to the intracellular domain of PRL-RS and identified it as dual specific phosphatase DUPD1. PRL does not induce expression of DUDP1 but represses its phosphorylation on Thr-155. We also show a physical association of this phosphatase with ERK1/2 and p38 MAPK. Using an in vitro phosphatase assay and overexpression studies, we established that DUPD1 is a MAPK phosphatase. Dual specific phosphatase inhibitors as well as siRNA to DUPD1, completely prevent PRL-mediated MAPK inhibition in ovarian cells. Our results strongly suggest that deactivation of MAPK by PRL/PRL-RS contributes to the severe ovarian defect in PRLR(-/-)RS mice and demonstrate the novel association of PRL-RS with DUPD1 and a role for this phosphatase in MAPK deactivation.     

池蝶蚌(贝)血细胞显微观察

本文对池蝶蚌血细胞的形态结构及其动态变化进行了研究。根据血细胞形态、大小和密度等特征,把血细胞分为六类：颗粒细胞、透明细胞、浆液细胞、类淋巴细胞、梭形细胞和血栓细胞;并对各种血细胞显微结构予以描述,统计了血细胞胞体大小、细胞核大小、核质比、密度及所占比例,其中颗粒细胞所占比例最大,透明细胞次之,类淋巴细胞最少,颗粒细胞和透明细胞是两种主要的细胞类型,约占总数的82%,担负着最基本的代谢和免疫功能;通过对池蝶蚌血细胞形态的连续观察,发现血细胞存在形态变化现象,推测细胞间可能存在相互转化的情况。     

ITMSQ: A software tool for N‐ and C‐terminal fragment ion pairs based isobaric tandem mass spectrometry quantification

Tandem MS (MS2) quantification using the series of N‐ and C‐terminal fragment ion pairs generated from isobaric‐labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solutionfor N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantitative methods.     

Impact of nonrandom selection mechanisms on the causal effect estimation for two-sample Mendelian randomization methods

Nonrandom selection in one-sample Mendelian Randomization (MR) results in biased estimates and inflated type I error rates only when the selection effects are sufficiently large. In two-sample MR, the different selection mechanisms in two samples may more seriously affect the causal effect estimation. Firstly, we propose sufficient conditions for causal effect invariance under different selection mechanisms using two-sample MR methods. In the simulation study, we consider 49 possible selection mechanisms in two-sample MR, which depend on genetic variants (G), exposures (X), outcomes (Y) and their combination. We further compare eight pleiotropy-robust methods under different selection mechanisms. Results of simulation reveal that nonrandom selection in sample II has a larger influence on biases and type I error rates than those in sample I. Furthermore, selections depending on X+Y, G+Y, or G+X+Y in sample II lead to larger biases than other selection mechanisms. Notably, when selection depends on Y, bias of causal estimation for non-zero causal effect is larger than that for null causal effect. Especially, the mode based estimate has the largest standard errors among the eight methods. In the absence of pleiotropy, selections depending on Y or G in sample II show nearly unbiased causal effect estimations when the casual effect is null. In the scenarios of balanced pleiotropy, all eight MR methods, especially MR-Egger, demonstrate large biases because the nonrandom selections result in the violation of the Instrument Strength Independent of Direct Effect (InSIDE) assumption. When directional pleiotropy exists, nonrandom selections have a severe impact on the eight MR methods. Application demonstrates that the nonrandom selection in sample II (coronary heart disease patients) can magnify the causal effect estimation of obesity on HbA1c levels. In conclusion, nonrandom selection in two-sample MR exacerbates the bias of causal effect estimation for pleiotropy-robust MR methods.     

Auto-adaptive Alpha Allocation: A Strategy to Mitigate Risk on Study Assumptions

In some clinical development programs, there are potential biomarkers with promising but uncertain predictive effect, while the probability of success in the overall population cannot be readily dismissed. It is risky to focus only on the overall population, or just the biomarker subpopulation. In 2009, Chen and Beckman proposed a Bayesian decision framework to optimize the type I error rate (alpha) allocation in a Phase III clinical study with possible predictive subset effect. The utilization of internal data in this framework is of particular interest because it provides an opportunity to mitigate the potential risk of misspecified study assumptions using an auto-adaptive strategy. In this paper, we examine this auto-adaptive strategy in detail through extensive numerical case studies and provide guidance on the appropriate use of partial current trial (internal) data in this data-driven optimization framework. We show that internal data can be used to inform the alpha allocation to hypothesis testing in the overall population and the subgroup. The resulting adaptive testing strategy is robust with respect to the uncertainty in the predictive subgroup effect and biomarker prevalence.     

水鳖蕨配子体系统学特征的培养观察

在光学显微镜下观察了水鳖蕨(Sinephropteris delavayi(Franch.)Mickel)配子体发育的详细过程。水鳖蕨孢子培养6 d左右萌发;原丝体单列细胞,或发育出2~3个分支;片状体生长点不明显,成熟配子体近心形。本文还讨论了其生长点、毛状体及性器的发育特点,结果支持Mickel将水鳖蕨独立成属的观点。     

Soluble expression, purification and functional identification of a disulfide-rich conotoxin derived from Conus litteratus

Conotoxins are a diverse array of small peptides mostly with multiple disulfide bridges. These peptides become an increasing significant source of neuro-pharmacological probes and drugs as a result of the high selectivity for ion channels and receptors. Usually, the analogue of natural conotoxins is produced by means of chemical synthesis. Here, we present a simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression. By fused with thioredoxin and His tag, a novel O-superfamily conotoxin lt7a was successfully expressed in Escherichia coli and purified, resulting in a high yield of recombinant lt7a about 6 mg/l. The purity of target protein is up to 95% as identified by HPLC results. Whole cell patch-clamp recording revealed that the new conotoxin blocked voltage-sensitive sodium channels in rat dorsal root ganglion neurons, indicating it might be a novel microO-conotoxin.     

Effects of caffeine on in vivo and in vitro oocyte maturation in mice

The objective was to investigate, using a mouse model, the effects of caffeine on the number of ovulated oocytes, the rate of oocyte maturation, the susceptibility of oocytes to activating stimuli, spindle morphology, and distribution of cortical granules (CGs). Mice were given caffeine (150 mg/kg body weight ip) at various times relative to hCG (-2, 0, and +2h); in an in vitro study, 1, 5 or 10 mM caffeine was added to the maturation culture. Caffeine had no effect on the quality of oocytes in vivo maturation, but caffeine was detrimental to the quality of oocytes matured in vitro. Further studies are needed to determine caffeine concentration in follicles relative to that in culture medium.