Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells

Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4–mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4–mediated phosphoproteome, including construction of kinase–substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, δ-catenin, and PPP1Cα in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy.Breast cancer is the most common cancer in women, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Tumor metastasis is the major source of cancer lethality. Cancer stem cells (CSCs) are small-percentage subpopulation within tumors, which are essential for tumor reoccurrence and metastasis (1). G protein-coupled receptor CXCR4 is critical for tumor growth and metastasis and plays important roles in CSC migration, invasion, and proliferation (2). Chemokine stromal cell-derived factor 1 (SDF-1) (CXCL-12) binds to chemokine (C-X-C motif) receptor 4 (CXCR4) and induces SDF-1/CXCR4 signaling. SDF-1 or CXCR4 knockout mice are embryonic lethal. SDF-1 and CXCR4 are vital for tumor angiogenesis and metastasis (3). The large-scale signal transduction and the feedback regulation downstream of SDF-1/CXCR4 signaling in breast CSCs are unknown but critical to understanding the cellular physiology of breast tumor regrowth and metastasis.Protein phosphorylation and dephosphorylation are essential for cellular signal processing (4). Dynamic regulation of reversible, site-specific protein phosphorylation is critical to the signaling networks that regulate CSC self-renewal, differentiation, and metastasis. Protein-reversible phosphorylation has been extensively analyzed in examining one or a few protein phosphorylation events that affect CSC signaling (1). However, the phosphoproteome composed by protein kinase-driven and phosphatase-regulated signaling networks largely controls CSC fate. Therefore, large-scale analysis of differentially regulated protein phosphorylation is central to understanding complex cellular events, such as CSC maintenance and dissemination.To unveil the signal transduction downstream of SDF-1/CXCR4 signaling in CSCs, in this study we have carried out isotope reductive dimethylation and large-scale liquid chromatography tandem mass spectrometry (LC-MS/MS)-based phosphoproteomic profiling and quantification in human breast CSCs upon SDF-1/CXCR4 stimulation. The phosphorylation events presented here include SDF-1/CXCR4–mediated phosphorylation sites in several key kinases and phosphatases, and several important signaling pathways in breast CSCs.     

平喘益气颗粒联合沙美特罗替卡松气雾剂治疗慢性阻塞性肺疾病稳定期的临床研究

目的探讨平喘益气颗粒联合沙美特罗替卡松气雾剂治疗慢性阻塞性肺疾病稳定期的临床疗效。方法选取2017年6月—2018年6月在天津市黄河医院治疗的94例慢性阻塞性肺疾病稳定期患者为研究对象,将患者按随机数表法分为对照组和治疗组,每组各47例。对照组吸入沙美特罗替卡松吸入气雾剂,2揿/次,2次/d。治疗组在对照组基础上口服平喘益气颗粒,2袋/次,3次/d。两组患者连续治疗3个月。观察两组的临床疗效,比较两组的肺功能指标、SGRQ评分、6 min步行距离、免疫功能、炎症因子。结果治疗后,对照组和治疗组的总有效率分别为76.60%、91.49%,两组比较差异有统计学意义(P0.05)。治疗后,两组第1秒用力呼气容积(FEV1)、用力肺活量(FVC)、FEV1/FVC均显著提高,同组治疗前后比较差异有统计学意义(P0.05);且治疗后治疗组FEV1、FVC、FEV1/FVC明显高于对照组,差异有统计学意义(P0.05)。治疗后,两组圣乔治呼吸问卷(SGRQ)评分显著降低,6 min步行距离显著增加,同组治疗前后比较差异有统计学意义(P0.05);且治疗后治疗组SGRQ评分低于对照组,6 min步行距离长于对照组,差异有统计学意义(P0.05)。治疗后,治疗组CD3~+、CD4~+、CD4~+/CD8~+明显高于治疗前,且治疗组CD3~+、CD4~+、CD4~+/CD8~+明显高于对照组,差异有统计学意义(P0.05)。治疗后,两组肿瘤坏死因子-α(TNF-α)、白细胞介素-8(IL-8)水平显著下降,同组治疗前后比较差异有统计学意义(P0.05);且治疗后治疗组炎症因子水平明显低于对照组,差异有统计学意义(P0.05)。结论平喘益气颗粒联合沙美特罗替卡松气雾剂治疗慢性阻塞性肺疾病稳定期具有较好的临床疗效,可改善患者临床症状及肺功能,调节免疫功能及相关炎症因子,提高生活质量,具有一定的临床推广应用价值。     

口腔专科护士数码摄影规范化培训的探索与实践

目的探讨口腔专科护士数码摄影规范化培训的方式及应用效果。方法参考口腔摄影专业相关教材,制定口腔专科护士数码摄影规范化培训模式,选取在广西医科大学附属口腔医院首次进行正畸治疗的错颌畸形患者252例,分三个阶段对该院42名在职注册口腔专科护士进行3个月的数码摄影规范化培训,对护士口腔数码摄影规范化培训前后照片质量、患者满意度、护士岗位胜任能力培训效果进行分析。结果口腔专科护士数码摄影规范化培训后拍摄时长短于规范化培训前;且培训后的照片质量评分及合格率、患者满意度、专业知识、专业技术、专业能力、综合素质评分均高于培训前,差异具有统计学意义。结论数码摄影规范化培训可以提高口腔专科摄影质量和临床护理工作效率,达到护患双赢的目的,有助于提升口腔专科护士的岗位胜任能力。     

Lower dosage of recombinant tissue-type plasminogen activator (rt-PA) in the treatment of acute pulmonary embolism: A systematic review and meta-analysis

Background and Objective

According to US Food and Drugs Administration (FDA), 2 hour recombinant tissue plasminogen activator (rt-PA) 100 mg infusion is recommended for eligible patients with acute pulmonary embolism (PE). However,there exists evidence implying that a lower dosage of rt-PA can be equally effective but potentially safer compared with rt-PA 100 mg regimen. The aim of this systematic review and meta-analysis is to assess the efficacy and safety of low dose rt-PA in the treatment of acute PE.

Material and Method

We searched Pubmed, EMBASE, the Cochrane library and CBM Literature Database for randomized controlled trials (RCT) focusing on low dose rt-PA for acute PE. Outcomes were described in terms of changes of image tests and echocardiography, major bleeding events, all-cause death, and recurrence of PE.

Results

Five studies (440 patients) were included, three of which compared low dose rt-PA (0.6 mg/kg, maximum 50 mg or 50 mg infusion 2 h) with standard dose (100 mg infusion 2 h). There were more major bleeding events in standard dose rt-PA group than in low dose group (OR 0.33, 95%CI 0.12-0.91;P = 0.94,I² = 0%), while there were no statistical differences in recurrent PE or all cause mortality between these two groups. Two studies compared low dose (0.6 mg/kg, maximum 50 mg/2 min bolus or 10 mg bolus, ≤ 40 mg/2 h) with heparin. There was no significant difference in major bleeding events (OR 0.73, 95% CI 0.14-3.98;P = 0.72), recurrent PE or all cause mortality. No dose-related heterogeneity was found for all the included studies.

Conclusions

The results of this meta-analysis were hypothesis-generating. Based on the limited data, our systematic review suggested that low dose rt-PA had similar efficacy but was safer than standard dose of rt-PA. In addition, compared with heparin, low dose rt-PA didn’t increase the risk of major bleeding for eligible PE patients.     

Hierarchical nanostructured conducting polymer hydrogel with high electrochemical activity

Conducting polymer hydrogels represent a unique class of materials that synergizes the advantageous features of hydrogels and organic conductors and have been used in many applications such as bioelectronics and energy storage devices. They are often synthesized by polymerizing conductive polymer monomer within a nonconducting hydrogel matrix, resulting in deterioration of their electrical properties. Here, we report a scalable and versatile synthesis of multifunctional polyaniline (PAni) hydrogel with excellent electronic conductivity and electrochemical properties. With high surface area and three-dimensional porous nanostructures, the PAni hydrogels demonstrated potential as high-performance supercapacitor electrodes with high specific capacitance (~480 F·g(-1)), unprecedented rate capability, and cycling stability (~83% capacitance retention after 10,000 cycles). The PAni hydrogels can also function as the active component of glucose oxidase sensors with fast response time (~0.3 s) and superior sensitivity (~16.7 μA · mM(-1)). The scalable synthesis and excellent electrode performance of the PAni hydrogel make it an attractive candidate for bioelectronics and future-generation energy storage electrodes.     

Anti-IL-7 receptor-α reverses established type 1 diabetes in nonobese diabetic mice by modulating effector T-cell function

Genetic variation in the IL-7 receptor-α (IL-7R) gene is associated with susceptibility to human type 1 diabetes (T1D). Here we investigate the therapeutic efficacy and mechanism of IL-7Rα antibody in a mouse model of T1D. IL-7Rα antibody induces durable, complete remission in newly onset diabetic mice after only two to three injections. IL-7 increases, whereas IL-7Rα antibody therapy reduces, the IFN-γ-producing CD4(+) (T(H)1) and IFN-γ-producing CD8(+) T cells. Conversely, IL-7 decreases and IL-7Rα antibody enhances the inhibitory receptor Programmed Death 1 (PD-1) expression in the effector T cells. Programmed Death 1 blockade reversed the immune tolerance mediated by the IL-7Rα antibody therapy. Furthermore, IL-7Rα antibody therapy increases the frequency of regulatory T cells without affecting their suppressor activity. The durable efficacy and the multipronged tolerogenic mechanisms of IL-7Rα antibody therapy suggest a unique disease-modifying approach to T1D.     

Genetic variants in human leukocyte antigen/DP-DQ influence both hepatitis B virus clearance and hepatocellular carcinoma development

Recent genome-wide association studies showed that four single-nucleotide polymorphisms (SNPs) in human leukocyte antigen (HLA)-DP (rs3077 and rs9277535) and HLA-DQ (rs2856718 and rs7453920) were associated with chronic hepatitis B virus (HBV) infection in Japanese populations. More than 75% of hepatocellular carcinoma (HCC) patients are attributable to persistent infection of hepatitis B virus (HBV), especially in China. We genotyped these four SNPs in 1,300 HBV-positive HCC patients, 1,344 persistent HBV carriers, and 1,344 persons with HBV natural clearance from Southeast China to further test the associations of HLA-DP/DQ variants and with risk of both HBV clearance and HCC development. Logistic regression analyses showed that HLA-DQ rs2856718 significantly decreased host HCC risk, whereas three SNPs were associated with HBV clearance (HLA-DP rs9277535 as well as HLA-DQ rs7453920 and rs2856718). In addition, HLA-DP rs3077 showed an approaching significant effect on susceptibility to HBV persistent infection and HCC development when considering multiple testing adjustments. Taken together, we report, for the first time, that genetic variants in the HLA-DP and HLA-DQ loci may be marker SNPs for risk of both HBV clearance and HCC development.     

Amyloid-β Induces Hepatic Insulin Resistance In Vivo via JAK2

Amyloid-β (Aβ), a natural product of cell metabolism, plays a key role in the pathogenesis of Alzheimer’s disease (AD). Epidemiological studies indicate patients with AD have an increased risk of developing type 2 diabetes mellitus (T2DM). Aβ can induce insulin resistance in cultured hepatocytes by activating the JAK2/STAT3/SOCS-1 signaling pathway. Amyloid precursor protein and presenilin 1 double-transgenic AD mouse models with increased circulating Aβ level show impaired glucose/insulin tolerance and hepatic insulin resistance. However, whether Aβ induces hepatic insulin resistance in vivo is still unclear. Here we show C57BL/6J mice intraperitoneally injected with Aβ42 exhibit increased fasting blood glucose level, impaired insulin tolerance, and hepatic insulin signaling. Moreover, the APPswe/PSEN1dE9 AD model mice intraperitoneally injected with anti-Aβ neutralizing antibodies show decreased fasting blood glucose level and improved insulin sensitivity. Injection of Aβ42 activates hepatic JAK2/STAT3/SOCS-1 signaling, and neutralization of Aβ in APPswe/PSEN1dE9 mice inhibits liver JAK2/STAT3/SOCS-1 signaling. Furthermore, knockdown of hepatic JAK2 by tail vein injection of adenovirus inhibits JAK2/STAT3/SOCS-1 signaling and improves glucose/insulin tolerance and hepatic insulin sensitivity in APPswe/PSEN1dE9 mice. Our results demonstrate that Aβ induces hepatic insulin resistance in vivo via JAK2, suggesting that inhibition of Aβ signaling is a new strategy toward resolving insulin resistance and T2DM.More than 250 million people worldwide were diagnosed with type 2 diabetes mellitus (T2DM) in 2011, and this number is expected to double within the next 20 years (1). Insulin resistance is a key element in the pathogenesis of T2DM, defined as a state of reduced responsiveness to normal circulating levels of insulin in insulin-target liver, skeletal muscle, and adipose tissues (2). Many states give rise to insulin resistance, and all are explained by numerous mechanisms in which insulin signaling is decreased (3). Interestingly, it has been reported that brains from Alzheimer’s disease (AD) patients display impaired insulin signaling (4,5), and some AD patients exhibit impaired glucose metabolism and hyperinsulinemia (6,7). Furthermore, an epidemiological study indicates patients with AD have an increased risk of developing T2DM (8), and experimental studies demonstrate AD model mice also exhibit diabetic phenotype (9,10). These studies together reveal a strong correlation between AD and insulin resistance/T2DM.Amyloid-β (Aβ) is a natural product during cell metabolism and originates from proteolysis of the amyloid precursor protein (APP) by the sequential enzymatic actions of β-site amyloid precursor protein–cleaving enzyme 1 (BACE-1) and γ-secretase (11). According to the amyloid cascade hypothesis, Aβ has an early and vital role in the pathogenesis of AD (11,12). In the central nervous system, Aβ has been reported to impair neuronal synaptic function in early AD by compromising insulin signaling (13–16). Interestingly, Aβ can be detected in the peripheral circulation and tissues (17–19).We have previously reported that Aβ induces insulin resistance in cultured hepatocytes mainly through the JAK2/STAT3/SOCS-1 signaling pathway (10), indicating that Aβ is an inducer of insulin resistance in vitro. On the other hand, animal studies demonstrate that the crossbred mice of APP23 transgenic AD model mice and ob/ob mice showed an accelerated diabetic phenotype (20). Moreover, APPswe/PS1^(A246E) transgenic AD model mice with increased plasma Aβ42 level exhibit impaired glucose tolerance when fed a chow diet (9,21). Consistently, we have recently reported that APPswe/PSEN1dE9 (APP/PS1) transgenic AD model mice with increased plasma Aβ40/42 levels show impaired glucose/insulin tolerance and hepatic insulin signaling, hyperinsulinemia, and upregulation of SOCS-1 and phosphorylated JAK2 and STAT3 in the liver (10). However, it is still possible that the insulin resistance in AD model mice might be due to the overexpression of presenilin 1, APP, and/or APP cleavage products except Aβ. Thus, whether Aβ itself can induce insulin resistance in vivo is yet to be elucidated. In addition, we previously showed that Aβ induces insulin resistance by activating the JAK2/STAT3/SOCS-1 signaling pathway in cultured hepatocytes (10). Whether Aβ also induces hepatic insulin resistance in vivo by activating the JAK2/STAT3/SOCS-1 signaling pathway is still unclear.In this study, we investigated the effect of Aβ on insulin sensitivity in vivo by injection of Aβ42 into wild-type mice and injection of Aβ-neutralizing antibodies or adenovirus expressing JAK2 small interfering (si)RNA into APP/PS1 AD model mice. We found that injection of Aβ42 into C57BL/6J mice induces insulin resistance and activates hepatic JAK2/STAT3/SOCS-1 signaling. Moreover, APP/PS1 mice treated with anti–Aβ-neutralizing antibodies show improved insulin sensitivity and attenuated hepatic JAK2/STAT3/SOCS-1 signaling. Furthermore, knockdown of hepatic JAK2 by adenovirus inhibited JAK2/STAT3/SOCS-1 signaling and improved insulin sensitivity in APP/PS1 mice.