Developing a Novel Gene-Delivery Vector System Using the Recombinant Fusion Protein of Pseudomonas Exotoxin A and Hyperthermophilic Archaeal Histone HPhA

Non-viral gene delivery system with many advantages has a great potential for the future of gene therapy. One inherent obstacle of such approach is the uptake by endocytosis into vesicular compartments. Receptor-mediated gene delivery method holds promise to overcome this obstacle. In this study, we developed a receptor-mediated gene delivery system based on a combination of the Pseudomonas exotoxin A (PE), which has a receptor binding and membrane translocation domain, and the hyperthermophilic archaeal histone (HPhA), which has the DNA binding ability. First, we constructed and expressed the rPE-HPhA fusion protein. We then examined the cytotoxicity and the DNA binding ability of rPE-HPhA. We further assessed the efficiency of transfection of the pEGF-C1 plasmid DNA to CHO cells by the rPE-HPhA system, in comparison to the cationic liposome method. The results showed that the transfection efficiency of rPE-HPhA was higher than that of cationic liposomes. In addition, the rPE-HPhA gene delivery system is non-specific to DNA sequence, topology or targeted cell type. Thus, the rPE-HPhA system can be used for delivering genes of interest into mammalian cells and has great potential to be applied for gene therapy.     

A simple method for measuring the kinetics of the formation of oxygen radicals

Sodium 2, 6-dichloroindophenol (DCIP Na ) was used to measure the kinetics of the formation of oxygen radicals. The oxygen radicals react with DCIP - , resulting in a decrease in DCIP concentration which is monitored by the decrease in A . A method based on this principle was demonstrated with xanthine/xanthine oxidase giving a K = 4.9x10 M. Cu significantly inhibited the enzyme reaction.     

RhoA Is Essential for Maintaining Normal Megakaryocyte Ploidy and Platelet Generation

RhoA plays a multifaceted role in platelet biology. During platelet development, RhoA has been proposed to regulate endomitosis, proplatelet formation, and platelet release, in addition to having a role in platelet activation. These processes were previously studied using pharmacological inhibitors in vitro, which have potential drawbacks, such as non-specific inhibition or incomplete disruption of the intended target proteins. Therefore, we developed a conditional knockout mouse model utilizing the CRE-LOX strategy to ablate RhoA, specifically in megakaryocytes and in platelets to determine its role in platelet development. We demonstrated that deleting RhoA in megakaryocytes in vivo resulted in significant macrothrombocytopenia. RhoA-null megakaryocytes were larger, had higher mean ploidy, and exhibited stiff membranes with micropipette aspiration. However, in contrast to the results observed in experiments relying upon pharmacologic inhibitors, we did not observe any defects in proplatelet formation in megakaryocytes lacking RhoA. Infused RhoA-null megakaryocytes rapidly released platelets, but platelet levels rapidly plummeted within several hours. Our evidence supports the hypothesis that changes in membrane rheology caused infused RhoA-null megakaryocytes to prematurely release aberrant platelets that were unstable. These platelets were cleared quickly from circulation, which led to the macrothrombocytopenia. These observations demonstrate that RhoA is critical for maintaining normal megakaryocyte development and the production of normal platelets.     

Atypical GATA protein TRPS1 plays indispensable roles in mouse two-cell embryo

Zygotic genome activation (ZGA) is one of the most critical events at the beginning of mammalian preimplantation embryo development (PED). The mechanisms underlying mouse ZGA remain unclear although it has been widely studied. In the present study, we identified that tricho-rhino-phalangeal syndrome 1 (TRPS1), an atypical GATA family member, is an important factor for ZGA in mouse PED. We found that the Trps1 mRNA level peaked at the one-cell stage while TRPS1 protein did so at the two/four-cell stage. Knockdown of Trps1 by the microinjection of Trps1 siRNA reduced the developmental rate of mouse preimplantation embryos by approximately 30%, and increased the expression of ZGA marker genes MuERV-L and Zscan4d via suppressing the expression of major histone markers H3K4me3 and H3K27me3. Furthermore, Trps1 knockdown decreased the expression of Sox2 but increased Oct4 expression. We conclude that TRPS1 may be indispensable for zygotic genome activation during mouse PED.     

基于主成分-聚类-逐步回归分析构建番茄苗期耐铝性综合评价体系

铝毒是限制酸性土壤中作物产量的主要因素之一。番茄(Solanum lycopersicum)是适合在酸性土壤中种植的主要经济作物, 不同品种番茄对铝胁迫的响应存在差异, 因此, 筛选苗期耐铝毒种质对番茄生产及研究具有重要意义。以10个番茄品种为材料, 采用室内土培盆栽, 设置1 000 µmol∙L^-1 AlCl₃·6H₂O处理, 测定反映植物铝胁迫下生长状况的16个形态、生理生化及光合指标。通过主成分分析, 将铝胁迫下番茄幼苗的16个指标转化为5个独立的综合指标, 累积贡献率达90.779%。基于耐铝性综合评价值(A)的系统聚类分析, 将供试种质划分为5类, 第I类为高度耐铝品种Qianxi, 第V类为高度不耐铝品种Puluowangsi。经多元线性逐步回归分析得出番茄苗期耐铝评价方程: y=0.046+0.405X₆+0.515X₁₀-0.207X₁₅+0.028X₃ (R²=0.997), 从16个指标中提取出与A值显著相关(P<0.01)的4个指标: 丙二醛含量(X₃)、净光合速率(X₆)、叶面积(X₁₀)和地下部干重(X₁₅)。利用评价方程可判断不同番茄品种苗期的耐铝性, 使番茄耐铝性鉴定工作快速简便。     

寄生动物

总结归纳了寄生动物的概念和寄生类型,另外对寄生动物适应寄生生活的生物学特性和生活史特征进行了综述。     

Rotavirus infection alters peripheral T-cell homeostasis in children with acute diarrhea

The patterns of gene expression and the phenotypes of lymphocytes in peripheral blood mononuclear cells (PBMC) from children with diarrhea caused by rotavirus and healthy children were compared by using DNA microarray, quantitative PCR, and flow cytometry. We observed increased expression of a number of genes encoding proinflammatory cytokines and interferon or interferon-stimulated proteins and demonstrated activation of some genes involved in the differentiation, maturation, activation, and survival of B lymphocytes in PBMC of patients with rotavirus infection. In contrast, we observed a consistent pattern of lower mRNA levels for an array of genes involved in the various stages of T-cell development and demonstrated a reduction in total lymphocyte populations and in the proportions of CD4 and CD8 T lymphocytes from PBMC of patients. This decreased frequency of T lymphocytes was transient, since the proportions of T lymphocytes recovered to almost normal levels in convalescent-phase PBMC from most patients. Finally, rotavirus infection induced the activation and expression of the early activation markers CD83 and CD69 on a fraction of CD19 B cells and the remaining CD4 and CD8 T lymphocytes in acute-phase PBMC of patients; the expression of CD83 continued to be elevated and was predominantly exhibited on CD4 T lymphocytes in convalescent-phase PBMC. On the basis of these findings at the molecular, phenotypic, and physiologic levels in acute-phase PBMC, we conclude that rotavirus infection induces robust proinflammatory and antiviral responses and B-cell activation but alters peripheral T-cell homeostasis in children.     

Techniques for the regeneration and genetic transformation of Arabidopsis pumila: an ephemeral plant suitable for investigating the mechanisms for adaptation to desert environments

Arabidopsis pumila is a type of cruciferous ephemeral plant, which in China mainly grows in the desert environments of northern Xinjiang. A. pumila not only has a short growth duration, but also has high photosynthetic efficiency, seed yield, salt tolerance, and drought resistance. It is an ideal species for the study of environmental adaptations in ephemeral plants. We induced callus tissue formation on the roots and hypocotyls of 8-day-old seedlings, and on the leaves and petioles of 4-week-old seedlings, and obtained multiple adventitious shoots on these tissues grown on Murashige and Skoog induction medium supplemented with 0.5 mg/L 6-Benzylaminopurine and 0.1 mg/L α-Naphthalene acetic acid. Young roots, hypocotyls, leaves, and petioles could all induce calluses, but the induction rate was highest on young roots. In addition, the leaves and petioles of 4-week-old seedlings were used as explants, the Δ1-pyrroline-5-carboxylic acid synthase gene 1 of A. pumila controlled by 35S promoter of cauliflower mosaic virus was used as target gene, and hygromycin B was used as screening antibiotic to explore Agrobacterium tumefaciens GV3101 mediated transformation. The results showed that the callus induction rate of petiole explants was the highest when they were treated with Agrobacterium suspension (OD₆₀₀?=?0.6) for 10 min and thenco-cultured in dark for 2 days. The qRT-PCR results showed that the ApP5CS1.1 gene was overexpressed in the transgenic plants. These protocols provide working research methods for exploring the cellular level adaptative mechanisms of this species to desert environments.

     

Genome sequencing and comparison of two nonhuman primate animal models, the cynomolgus and Chinese rhesus macaques

The nonhuman primates most commonly used in medical research are from the genus Macaca. To better understand the genetic differences between these animal models, we present high-quality draft genome sequences from two macaque species, the cynomolgus/crab-eating macaque and the Chinese rhesus macaque. Comparison with the previously sequenced Indian rhesus macaque reveals that all three macaques maintain abundant genetic heterogeneity, including millions of single-nucleotide substitutions and many insertions, deletions and gross chromosomal rearrangements. By assessing genetic regions with reduced variability, we identify genes in each macaque species that may have experienced positive selection. Genetic divergence patterns suggest that the cynomolgus macaque genome has been shaped by introgression after hybridization with the Chinese rhesus macaque. Macaque genes display a high degree of sequence similarity with human disease gene orthologs and drug targets. However, we identify several putatively dysfunctional genetic differences between the three macaque species, which may explain functional differences between them previously observed in clinical studies.     

Aberrant hippocampal subregion networks associated with the classifications of aMCI subjects: a longitudinal resting-state study

Background

Altered hippocampal structure and function is a valuable indicator of possible conversion from amnestic type mild cognitive impairment (aMCI) to Alzheimer''s disease (AD). However, little is known about the disrupted functional connectivity of hippocampus subregional networks in aMCI subjects.

Methodology/Principal Findings

aMCI group-1 (n = 26) and controls group-1 (n = 18) underwent baseline and after approximately 20 months follow up resting-state fMRI scans. Integrity of distributed functional connectivity networks incorporating six hippocampal subregions (i.e. cornu ammonis, dentate gyrus and subicular complex, bilaterally) was then explored over time and comparisons made between groups. The ability of these extent longitudinal changes to separate unrelated groups of 30 subjects (aMCI-converters, n = 6; aMCI group-2, n = 12; controls group-2, n = 12) were further assessed. Six longitudinal hippocampus subregional functional connectivity networks showed similar changes in aMCI subjects over time, which were mainly associated with medial frontal gyrus, lateral temporal cortex, insula, posterior cingulate cortex (PCC) and cerebellum. However, the disconnection of hippocampal subregions and PCC may be a key factor of impaired episodic memory in aMCI, and the functional index of these longitudinal changes allowed well classifying independent samples of aMCI converters from non-converters (sensitivity was 83.3%, specificity was 83.3%) and controls (sensitivity was 83.3%, specificity was 91.7%).

Conclusions/Significance

It demonstrated that the functional changes in resting-state hippocampus subregional networks could be an important and early indicator for dysfunction that may be particularly relevant to early stage changes and progression of aMCI subjects.