MicroRNA-22 Can Reduce Parathymosin Expression in Transdifferentiated Hepatocytes

Pancreatic acinar cells AR42J-B13 can transdifferentiate into hepatocyte-like cells permissive for efficient hepatitis B virus (HBV) replication. Here, we profiled miRNAs differentially expressed in AR42J-B13 cells before and after transdifferentiation to hepatocytes, using chip-based microarray. Significant increase of miRNA expression, including miR-21, miR-22, and miR-122a, was confirmed by stem-loop real-time PCR and Northern blot analyses. In contrast, miR-93, miR-130b, and a number of other miRNAs, were significantly reduced after transdifferentiation. To investigate the potential significance of miR-22 in hepatocytes, we generated cell lines stably expressing miR-22. By 2D-DIGE, LC-MS/MS, and Western blot analyses, we identified several potential target genes of miR-22, including parathymosin. In transdifferentiated hepatocytes, miR-22 can inhibit both mRNA and protein expression of parathymosin, probably through a direct and an indirect mechanism. We tested two computer predicted miR-22 target sites at the 3′ UTR of parathymosin, by the 3′ UTR reporter gene assay. Treatment with anti-miR-22 resulted in significant elevation of the reporter activity. In addition, we observed an in vivo inverse correlation between miR-22 and parathymosin mRNA in their tissue distribution in a rat model. The phenomenon that miR-22 can reduce parathymosin protein was also observed in human hepatoma cell lines Huh7 and HepG2. So far, we detected no major effect on several transdifferentiation markers when AR42J-B13 cells were transfected with miR-22, or anti-miR-22, or a parathymosin expression vector, with or without dexamethasone treatment. Therefore, miR-22 appears to be neither necessary nor sufficient for transdifferentiation. We discussed the possibility that altered expression of some other microRNAs could induce cell cycle arrest leading to transdifferentiation

Estrogen Modulates the Sensitivity of Lung Vagal C Fibers in Female Rats Exposed to Intermittent Hypoxia

Obstructive sleep apnea is mainly characterized by intermittent hypoxia (IH), which is associated with hyperreactive airway diseases and lung inflammation. Sensitization of lung vagal C fibers (LVCFs) induced by inflammatory mediators may play a central role in the pathogenesis of airway hypersensitivity. In females, estrogen interferes with inflammatory signaling pathways that may modulate airway hyperreactivity. In this study, we investigated the effects of IH on the reflex and afferent responses of LVCFs to chemical stimulants and lung inflammation in adult female rats, as well as the role of estrogen in these responses. Intact and ovariectomized (OVX) female rats were exposed to room air (RA) or IH for 14 consecutive days. On day 15, IH enhanced apneic responses to right atrial injection of chemical stimulants of LVCFs (e.g., capsaicin, phenylbiguanide, and α,β-methylene-ATP) in intact anesthetized females. Rats subjected to OVX prior to IH exposure exhibited an augmented apneic response to the same dose of stimulants compared with rats subjected to other treatments. Apneic responses to the stimulants were completely abrogated by bilateral vagotomy or perivagal capsaicin treatment, which blocked the neural conduction of LVCFs. Electrophysiological experiments revealed that in IH-exposed rats, OVX potentiated the excitability of LVCFs to stimulants. Moreover, LVCF hypersensitivity in rats subjected to OVX prior to IH exposure was accompanied by enhanced lung inflammation, which was reflected by elevated inflammatory cell infiltration in bronchoalveolar lavage fluid, lung lipid peroxidation, and protein expression of inflammatory cytokines. Supplementation with 17β-estradiol (E2) at a low concentration (30 μg/ml) but not at high concentrations (50 and 150 μg/ml) prevented the augmenting effects of OVX on LVCF sensitivity and lung inflammation caused by IH. These results suggest that ovarian hormones prevent the enhancement of LVCF sensitivity and lung inflammation by IH in female rats, which are related to the effect of low-dose estrogen

Relationship of teicoplanin MICs to treatment failure in teicoplanin-treated patients with methicillin-resistant Staphylococcus aureus pneumonia

Background/PurposeThe objective of this study was to determine the predictive value of teicoplanin minimal inhibitory concentrations (MICs) for treatment failure among patients with methicillin-resistant Staphylococcus aureus (MRSA) pneumonia.MethodsIn this study, all patients with ≥1 tracheal aspirates or sputum cultures positive for MRSA admitted to the hospital between April 2011 and September 2011 were reviewed. We enrolled patients who are ≥18 years of age, with a diagnosis of pneumonia, and with a receipt of teicoplanin therapy throughout the course. The relationship between teicoplanin Etest MICs and treatment outcomes of MRSA pneumonia was analyzed to identify the breakpoint of teicoplanin MICs influencing treatment outcomes.ResultsOf the 80 patients enrolled, 31 had a lower teicoplanin MIC level (<2.0 mg/L) and 49 had a higher MIC level (≥2.0 mg/L) for MRSA. The lower MIC group had a higher clinical resolution rate in 14 days [24 (77.4%) vs. 23 (46.9%), p = 0.007] and a lower treatment failure rate at the end of teicoplanin treatment [4 (12.9%) vs. 18 (36.7%), p = 0.020]. A comparison between the treatment success and failure groups showed that the former had a longer duration of teicoplanin use (18.76 ± 10.34vs.12.41 ± 5.65 days; p = 0.014). Results of a multivariate analysis showed that teicoplanin MICs ≥ 2.0 mg/Land shorter duration of teicoplanin therapy were independent risk factors for treatment failure.ConclusionA higher teicoplanin MIC value (≥2.0 mg/L) may predict the treatment failure among patients with teicoplanin-treated MRSA pneumonia

Application of Multiple Sensors in Monitoring Land Subsidence in Central Taiwan

Source: ICHE Conference Archive - https://mdi-de.baw.de/icheArchive

Visfatin mediates malignant behaviors through adipose-derived stem cells intermediary in breast cancer

Adipose-derived stem cells (ADSCs) have been implicated in tumor growth and metastasis in breast cancer. ADSCs exhibit tumor tropism, and are of increasing clinical relevance due to the autologous fat grafting for breast reconstruction. Although we have previously shown that a high level of the adipocytokine visfatin in human breast cancer tissues correlated with tumor progression mediated by cAbl and STAT3, the effects of visfatin in the tumor microenvironment are unclear. To understand how visfatin modulates breast cancer within the tumor-stromal environment, we examined determinants of breast cancer progression using a visfatin-primed ADSCs-tumor co-culture model. ADSCs were isolated from tumor-free adipose tissue adjacent to breast tumors. ADSCs were treated with or without visfatin for 48 h and then collected for co-culture with breast cancer cell line MDA-MB-231 for 72 h in a transwell system. We found that the MDA-MB-231 cells co-cultured with visfatin-treated ADSCs (vADSCs) had higher levels of cell viability, anchorage independent growth, migration, invasion, and tumorsphere formation than that co-cultured with untreated ADSCs (uADSCs). Growth differentiation factor 15 (GDF15) upregulation was found in the co-culture conditioned medium, with GDF15 neutralizing antibody blocking the promoting effect on MDA-MB-231 in co-culture. In addition, a GDF15-induced AKT pathway was found in MDA-MB-231 and treatment with PI3K/AKT inhibitor also reversed the promoting effect. In an orthotopic xenograft mouse model, MDA-MB-231 co-injected with vADSCs formed a larger tumor mass than with uADSCs. Positive correlations were noted between visfatin, GDF15, and phosphor-AKT expressions in human breast cancer specimens. In conclusion, visfatin activated GDF15-AKT pathway mediated via ADSCs to facilitate breast cancer progression

MicroRNA-130a can inhibit hepatitis B virus replication via targeting PGC1 alpha and PPAR gamma

In hepatitis B virus (HBV)-replicating hepatocytes, miR-130a expression was significantly reduced. In a reciprocal manner, miR-130a reduced HBV replication by targeting at two major metabolic regulators PGC1 alpha and PPAR gamma, both of which can potently stimulate HBV replication. We proposed a positive feed-forward loop between HBV, miR-130a, PPAR gamma, and PGC1 alpha. Accordingly, HBV can significantly enhance viral replication by reducing miR-130a and increasing PGC1a and PPAR gamma NF-kappa B/p65 can strongly stimulate miR-130a promoter, while miR-130a can promote NF-kappa B/p65 protein level by reducing PPAR gamma and thus NF-kappa B/p65 protein degradation. We postulated another positive feed-forward loop between miR-130a and NF-kappa B/p65 via PPAR gamma. During liver inflammation, NF-kappa B signaling could contribute to viral clearance via its positive effect on miR-130a transcription. Conversely, in asymptomatic HBV carriers, persistent viral infection could reduce miR-130a and NF-kappa B expression, leading to dampened inflammation and immune tolerance. Finally, miR-130a could contribute to metabolic homeostasis by dual targeting PGC1 alpha and PPAR gamma simultaneously

Purification of HBV Particles from Patients' Serum

人類B型肝炎病毒是帶有外套膜的DNA病毒，內有一個二十面體的核心顆粒。在受到病毒感染的肝細胞中，核心顆粒的組裝是在細胞質中發生，組裝的過程中伴隨著包裹前基因體RNA與病毒DNA聚合酶的結合複合體和一些細胞因子像是熱休克蛋白與會磷酸化核心蛋白的磷酸激酶。磷酸激酶的活性在其他種類的肝炎病毒中也有被發現，並有一些研究鑑定出具有此活性之蛋白質。而熱休克蛋白像是Hsp90、Hsp70、p23都有報導說其對於病毒進行protein priming與病毒顆粒組裝是必需的。這些細胞蛋白質似乎在HBV生活史中，都扮演很重要的角色。因此我們很有興趣去找尋在病毒顆粒中，被特定包裹於其中的細胞蛋白質。 為了能取得大量的病毒顆粒，於是我們選擇使用HBV感染者的血清來當做純化病毒的來源。利用不同濃度硫酸銨沉澱與sucrose cushion來去除血清中大量的血清蛋白像是白蛋白(albumin)。再接著使用蔗糖濃度梯度與氯化銫密度梯度，成功的分離出高純度的病毒顆粒，並同時將表面抗原顆粒與鄧氏顆粒分離出來。我們再利用電子顯微鏡和南方墨點法確認病毒顆粒的結構與鄧氏顆粒內的DNA基因體。最後，我們建立了一套能夠從血清中獲得高純度的病毒顆粒純化方法。The hepatitis B virus (HBV) is an enveloped DNA virus with an icosahedral nucleocapsid. In an HBV infected cell, assembly of nucleocapsid occurs in the cytosol and is accompanied by packaging of the viral pregenomic RNA bound to the viral polymerase protein together with cellular protein such as chaperons and protein kinases phosphorylating core protein. This kinase activity is also conserved in other members of hepadnaviridae and plays an important role in viral life cycle. Besides, chaperons like hsp90,hsp70 ,p23 are required for protein priming and encapsidation .Therefore , we are interested in finding out specific cellular proteins incorporated into HBV virions. In order to obtain enough quantity of hepatitis B virus, we chose serum from HBV-infected patients as materials for virus purification. Ammonium sulfate precipitation and sucrose cushion were used to remove high abundant serum proteins, like albumins. After sucrose gradient and cesium chloride gradient, Dane particles and empty particles were obtained with high purity. Then we utilized electron microscopy and Southern-blotting to confirm the structure of particles and virus genome inside Dane particles. Finally, we set up a system for virus purification from serum with high purity.中文摘要……………………………………………………………… .Ⅱ 英文摘要……………………………………………………………… .Ⅲ 目錄…………………………………………………………………… .Ⅳ 表次……………………………………………………………………. Ⅴ 圖次……………………………………………………………………. Ⅵ 壹、序論 B型肝炎病毒簡介…………………………………… .1 B型肝炎病毒的基因與蛋白質……………………… .2 B型肝炎病毒結構…………………………………… .3 B型肝炎病毒的生活史………………………………..4 細胞蛋白與病毒蛋白之交互作用…………………….5 研究目的……………………………………………… 9 貳、材料與方法 大腸桿菌之轉形(Transformation)………………… 10 HBV copy number 標準品之製備………………….. 10 HBV病毒顆粒之純化………………………………… 11 HBV病毒DNA之萃取……………………………….....12 定量即時聚合酶連鎖反應…………………………… 13 TCA(Trichloroacetic acid)沉澱法……………… 14 南方墨點法(Southern blotting)………………… 14 西方墨點法(Western blotting)…………………… 16 銀染法(Silver stain)……………………………… 17 穿透式電子顯微鏡及負染法………………………….17 參、結果 (一) 所需病毒之來源……………………………………………18 (二)硫酸銨沉澱病毒顆粒……………………………………….18 (三)蔗糖濃度梯度分離不同大小的病毒顆粒………………….19 (四)氯化銫密度梯度分離鄧氏顆粒與次病毒顆粒…………….20 (五)證明鄧氏顆粒存在………………………………………….21 肆、討論……………………………………………………………….23 附表…………………………………………………………………….27 附圖…………………………………………………………………… 30 伍、參考文獻………………………………………………………….44 表次 表一、不同硫酸銨濃度中，表面抗原與DNA的含量………28 表二、病毒純化之回收率……………………………………29 圖次 圖一、B型肝炎病毒顆粒種類及結構…………………………………31 圖二、B型肝炎病毒生活史……………………………………………32 圖三、肝炎病毒核心顆粒組裝之假設模型……………………………33 圖四、不同的硫酸銨濃度沉澱血清內的蛋白…………………………34 圖五、用不連續蔗糖濃度梯度純化HBV病毒顆粒…………………..35 圖六、用氯化銫密度梯度分離出次病毒顆粒…………………………36 圖七、氯化銫fraction中的蛋白質分析(Ⅰ)……………………… 37 圖八、用氯化銫密度梯度分離出鄧氏顆粒……………………………38 圖九、氯化銫fraction中的蛋白質分析 (Ⅱ)…………………… .39 圖十、氯化銫fraction中的蛋白質分析 (Ⅲ)………………………40 圖十一、濃縮後的病毒液與病毒蛋白分析……………………………41 圖十二、電子顯微鏡觀察結果…………………………………………42 圖十三、南方墨點法偵測病毒基因體…………………………………4

Resistive Switching of γ-APTES Pertaining to Molecular Structure

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