Deltex1 Is a Target of the Transcription Factor NFAT that Promotes T Cell Anergy

SummaryThe molecular process underlying T cell anergy is incompletely understood. Deltex1 (DTX1) is a Notch target with unknown physiological function. Here we show that Dtx1 was a transcription target of nuclear factor of activated T cells (NFAT) and participated in T cell anergy. DTX1 protein was upregulated during T cell anergy, and transgenic expression of Dtx1 attenuated T cell activation. DTX1 inhibited T cell activation by both E3-dependent and E3-independent mechanisms. In addition, DTX1 suppressed T cell activation in the absence of its Notch-binding domain. Importantly, DTX1 regulated the expression of two anergy-associated molecules, growth arrest and DNA-damage-inducible 45 β (Gadd45β) and Cbl-b. DTX1 interacted with early growth response 2 (Egr-2) for optimum expression of Cbl-b. Furthermore, deficiency of DTX1 augmented T cell activation, conferred resistance to anergy induction, enhanced autoantibody generation, and increased inflammation. DTX1 therefore represents a component downstream of calcium-NFAT signaling that regulates T cell anergy

The study of DEHP degradation by Pseudomonas alcaligenes A25 and analysis of its extracellular esterase.

鄰苯二甲酸酯 (Phthalates，PAEs) 為鄰苯二甲酸 (Phthalic acid) 酯化後生成的一類化合物，又統稱為鄰苯二甲基酯類，一般揮發性低，且穩定性高，為無色的黏稠油狀液體，對於水的溶解度極低。而PAEs經常被當作塑化劑 (plasticizers) 使用，塑化劑為塑膠添加物以改良塑膠的特性，能提供塑膠彈性、透明度、耐用性及增加使用壽命等功能，但塑化劑及塑膠製品之間並非共價鍵結合，因此若塑膠經陽光曝曬、高溫加熱或接觸有機溶液，便有機會使塑膠中的塑化劑釋放到環境中，除此之外塑膠老化也會加快塑化劑析出的進程。 在台灣行政院保護署的調查當中，發現台灣河川底泥及魚體所含最大宗的PAE即為DEHP (附錄二)。而在2011年時台灣發生塑化劑食品安全事件，不肖業者將廉價的DEHP當作食品添加劑代替合法的起雲劑添加到食品當中，經研究證明DEHP的毒性要比三聚氰胺毒上3.5至20倍，DEHP被認為是潛在的致癌劑、環境賀爾蒙及代謝干擾物，勢必會嚴重影響民眾的健康，因此解決環境中汙染物DEHP成為一項重要的課題。 　　本實驗發現產鹼假單孢菌A25 (Pseudomonas alcaligenes A25) 具有降解DEHP的能力，且分解效率佳，以高效液相色譜法分析DEHP的降解曲線，在低濃度 (100 ppm) 的DEHP條件下約24小時即可降解76%的DEHP，在高濃度 (600 ppm) 的DEHP條件下約48小時可以降解87%的DEHP，而經192小時可以降解98%的DEHP。又經實驗測試得知Pseudomonas alcaligenes A25經DEHP的誘導可以產生胞外酵素，該胞外酵素經過三丁酸甘油酯瓊脂培養基及酵素活性膠體染色的測驗中確認具有酯解酵素活性，在酵素活性測試中得知最佳反應溫度為40℃、酸鹼值為8.0，同時測試不同介面活性劑對於此酵素的活性影響。Pseudomonas alcaligenes A25為一菌種可以有效率的降解DEHP，且產生之降解酵素為胞外蛋白質易於大量收集，將來或許可應用於處理環境中的塑化劑汙染物DEHP。Phthalates, or phthalate esters, are esters of phthalic acid and are with characteristics of general low volatility and high stability, as a colorless viscous oily liquid, very low solubility in water. Phthalates are mainly used as plasticizers, substances added to plastics to increase their flexibility, transparency, durability and longevity. There is no covalent bond between the phthalates and plastics. So if the plastic by exposure to sunlight, high temperature heating or organic solvents, it has the opportunity to make the plasticizers released into the environment. Moreover, plastic aging will accelerate this process. The studies by EPA Taiwan found that DEHP is most abundant phthalate in Taiwan river sediment and fish body (Appendix. 2). In 2011 Taiwan plasticizer food safety incidents occur, unscrupulous industry use cheap DEHP as a food additive instead of a legal Cloudy agent added to food products. The study shows that the DEHP toxicity is more than melamine 3.5 to 20 times. DEHP is considered a potential carcinogen, environmental hormone and metabolic disruptors and is bound to seriously affect the health of the people. Thus, to solve environmental pollutants, DEHP becomes an important issue. Our study found that Pseudomonas alcaligenes A25 have the ability to degrade DEHP with good degradation efficiency. We analyze the degradation rate of DEHP by High performance liquid chromatography. At low concentrations (100 ppm) of DEHP about 24 hours to degrade 76% of DEHP, and at high concentrations (600 ppm) for about 48 hours to degrade 87% of DEHP. The strain A25 could degrade 98% DEHP within 192 hours. We found that strain A25 produce extracellular enzyme by DEHP induction. The extracellular enzyme after tributyrin agar medium and esterase activity staining tests confirmed having esterase activity. The optimal DEHP degradation conditions were 40℃ and pH 8.0. The addition of different nutrient sources and surfactants will affect degradation of DEHP. P. alcaligenes A25 as a newly discovered specie can efficiently degrade DEHP and produces extracellular esterase for DEHP degradation. It has the potential to degrade DEHP, perhaps can be applied to treatment of environmental pollutants DEHP

Major Contribution of Caspase-9 to Honokiol-Induced Apoptotic Insults to Human Drug-Resistant Glioblastoma Cells

Temozolomide (TMZ)-induced chemoresistance to human glioblastomas is a critical challenge now. Our previous studies showed that honokiol, a major bioactive constituent of Magnolia officinalis (Houpo), can kill human glioblastoma cells and suppresses glioblastoma growth. This study was further aimed to evaluate the effects of honokiol on human drug-resistant glioblastoma cells and the possible mechanisms. The results by data mining in the cancer genome atlas (TCGA) database and immunohistochemistry displayed that expression of caspase-9 mRNA and protein in human glioblastomas was induced. Human TMZ-resistant U87-MG-R9 glioblastoma cells were selected and prepared from human drug-sensitive U87-MG cells. Compared to human drug-sensitive U87-MG cells, TMZ did not affect viability of U87-MG-R9 glioblastoma cells. Interestingly, treatment with honokiol suppressed proliferation and survival of human drug-resistant glioblastoma cells in concentration- and time-dependent manners. Compared to caspase-8 activation, honokiol chiefly increased activity of caspase-9 in U87-MG-R9 cells. Successively, levels of cleaved caspase-3 and activities of caspase-3 and caspase-6 in human TMZ-tolerant glioblastoma cells were augmented following honokiol administration. In parallel, honokiol triggered DNA fragmentation of U87-MG-R9 cells. Accordingly, treatment of human TMZ-resistant glioblastoma cells with honokiol induced cell apoptosis but did not affect cell necrosis. Fascinatingly, suppressing caspase-9 activity using its specific inhibitors repressed honokiol-induced caspase-6 activation, DNA fragmentation, and cell apoptosis. Taken together, this study has shown the major roles of caspase-9 in transducing honokiol-induced mitochondria-dependent apoptosis in human drug-resistant glioblastoma cells. Thus, honokiol may be clinically applied as a drug candidate for treatment of glioblastoma patients with chemoresistance

Data analyses of honokiol-induced autophagy of human glioma cells in vitro and in vivo

This article contains raw and processed data related to a research, “Honokiol induces autophagic cell death in malignant glioma through reactive oxygen species-mediated regulation of the p53/PI3K/Akt/mTOR signaling pathway” (C.J. Lin, T.L. Chen, Y.Y. Tseng, G.J. Wu, M.H. Hsieh, Y.W. Lin, R.M. Chen, 2016) [1]. Data were obtained by immunoblotting analyses of light chain 3 (LC3)-II, beclin-1, Akt, and mTOR in human glioma U87 MG cells and mouse glioma tissues treated with honokiol, an active constituent extracted from the bark of Magnolia officinalis, “Honokiol induces autophagy of neuroblastoma cells through activating the PI3K/Akt/mTOR and endoplasmic reticular stress/ERK1/2 signaling pathways and suppressing cell migration” (P.S. Yeh, W. Wang, Y.A. Chang, C.J. Lin, J.J. Wang, R.M. Chen, 2016) [2]. The processed data show the effects of honokiol on induction of autophagy in human glioma U87 MG cells by analyzing levels of LC3-II, p62, and bectin-1, “Honokiol-induced apoptosis and autophagy in glioblastoma multiforme cells” (K.H. Chang, M.D Yan, C.J. Yao, P.C. Lin, G.M. Lai, 2013) [3]. In addition, chloroquine, a lysosomal inhibitor, was administered to the cells to further confirm honokiol-induced cell autophagy. Sequentially, mice with gliomas were created and treated with honokiol. Amounts of phosphorylated and non-phosphorylated Akt and mTOR in glioma tissues were analyzed to determine the possible mechanisms of honokiol-induced autophagy

Topoisomerase II Inhibitors Can Enhance Baculovirus-Mediated Gene Expression in Mammalian Cells through the DNA Damage Response

BacMam is an insect-derived recombinant baculovirus that can deliver genes into mammalian cells. BacMam vectors carrying target genes are able to enter a variety of cell lines by endocytosis, but the level of expression of the transgene depends on the cell line and the state of the transduced cells. In this study, we demonstrated that the DNA damage response (DDR) could act as an alternative pathway to boost the transgene(s) expression by BacMam and be comparable to the inhibitors of histone deacetylase. Topoisomerase II (Top II) inhibitor-induced DDR can enhance the CMV-IE/enhancer mediated gene expression up to 12-fold in BacMam-transduced U-2OS cells. The combination of a Top II inhibitor, VM-26, can also augment the killing efficiency of a p53-expressing BacMam vector in U-2OS osteosarcoma cells. These results open a new avenue to facilitate the application of BacMam for gene delivery and therapy

An automatic method to calculate heart rate from zebrafish larval cardiac videos

Abstract Background Zebrafish is a widely used model organism for studying heart development and cardiac-related pathogenesis. With the ability of surviving without a functional circulation at larval stages, strong genetic similarity between zebrafish and mammals, prolific reproduction and optically transparent embryos, zebrafish is powerful in modeling mammalian cardiac physiology and pathology as well as in large-scale high throughput screening. However, an economical and convenient tool for rapid evaluation of fish cardiac function is still in need. There have been several image analysis methods to assess cardiac functions in zebrafish embryos/larvae, but they are still improvable to reduce manual intervention in the entire process. This work developed a fully automatic method to calculate heart rate, an important parameter to analyze cardiac function, from videos. It contains several filters to identify the heart region, to reduce video noise and to calculate heart rates. Results The proposed method was evaluated with 32 zebrafish larval cardiac videos that were recording at three-day post-fertilization. The heart rate measured by the proposed method was comparable to that determined by manual counting. The experimental results show that the proposed method does not lose accuracy while largely reducing the labor cost and uncertainty of manual counting. Conclusions With the proposed method, researchers do not have to manually select a region of interest before analyzing videos. Moreover, filters designed to reduce video noise can alleviate background fluctuations during the video recording stage (e.g. shifting), which makes recorders generate usable videos easily and therefore reduce manual efforts while recording

Composition and Activity of N2-Fixing Microorganisms in Mangrove Forest Soils

Mangrove forests are considered to be a highly productive ecosystem, but they are also generally nitrogen (N)-limited. Thus, soil N2 fixation can be important for the stability of both mangrove ecosystem functions and upland N supply. This study evaluates the N2 fixation activity and composition of relevant microorganisms in two coastal mangrove forests—the Guandu mangrove in an upstream estuary and the Bali mangrove in a downstream estuary—using the acetylene reduction method, real-time polymerase chain reaction, and next-generation sequencing. The results demonstrated that ambient nitrogenase activity was higher in downstream mangrove forests (13.2–15.6 nmol h−1 g−1 soil) than in upstream mangrove forests (0.2–1.4 nmol h−1 g−1 soil). However, both the maximum potential nitrogenase activity and nitrogenase gene (nifH gene) copy number were found to be higher in the upstream than in the downstream mangrove forests, implying that the nitrogenase activity and diazotrophic abundance may not necessarily be positively correlated. In addition, amended MoO4 (which inhibits the activity of sulfate-reducing bacteria in N2-fixation) yielded low nitrogenase activity, and sulfate-reducing bacteria made up 20–50% of the relative diazotrophic abundance in the mangrove forests, indicating that these bacteria might be the major active diazotrophs in this environment

Barrier abnormalities and keratinocyte-derived cytokine cascade after cessation of long-term topical glucocorticosteroid on hairless mouse skin

Background: Previous studies have shown that topical corticosteroid (TCS) use induces structural abnormalities of the stratum corneum (SC), resulting in permeability barrier disruption. It is well-known that epidermal barrier perturbation induces a cytokine cascade, leading to cutaneous inflammation. Accordingly, we hypothesize that barrier disruption caused by long-term TCS therapy may trigger a cutaneous cytokine cascade, which plays an important role in withdrawal dermatitis (WD) following discontinuation of TCS. The objective of this study was to elucidate the possible mechanism of WD. Methods: Hairless mice were treated once daily with 0.064% betamethasone dipropionate ointment for 6 weeks. After discontinuation of TCS, we examined the transepidermal water loss (TEWL), SC lipids and expression of the cytokines interleukin 1-alpha (IL1-α) and tumor necrosis factor-alpha (TNF-α) and their downstream signaling pathway in the following 2 weeks. Results: We observed upregulation of IL1-α, TNF-α, inhibitor of nuclear factor kappa-B kinase subunits alpha and beta (IKK1, IKK2) and nuclear factor kappa-B (NF-κB) in the epidermis, accompanied by a significantly higher TEWL after TCS cessation. These cytokines gradually disappeared with concomitant normalization of TEWL after 1 week. Only negligible amounts of the aforementioned cytokines were observed in the dermis. Furthermore, concurrent application of petrolatum during TCS treatment decreased barrier impairment and production of cytokines. Conclusion: An epidermis-derived cytokine cascade was observed following TCS-induced barrier disruption, which is similar to that from permeability barrier insults by acetone or tape stripping. The study suggests that concurrent application of skin care products during TCS treatment improves barrier homeostasis, and should become a standard practice to alleviate TCS-induced WD