Mechanical regulation of cancer cell apoptosis and autophagy: Roles of bone morphogenetic protein receptor, Smad1/5, and p38 MAPK

AbstractMechanical forces induced by interstitial fluid flow in and surrounding tissues and by blood/lymphatic flow in vessels may modulate cancer cell invasion and metastasis and anticancer drug delivery. Our previous study demonstrated that laminar flow-induced shear stress induces G2/M arrest in tumor cells. However, whether shear stress modulates final cell fate remains unclear. In this study, we investigated the role of flow-induced shear stress in modulating the survival of four human tumor cell lines, i.e., Hep3B hepatocarcinoma cells, MG63 osteosarcoma cells, SCC25 oral squamous carcinoma cells, and A549 carcinomic alveolar basal epithelial cells. Laminar shear stress (LSS) ranging from 0.5 to 12dyn/cm2 induced death of these four tumor cell lines. In contrast to LSS at 0.5dyn/cm2, oscillatory shear stress (OSS) at 0.5±4dyn/cm2 cannot induce cancer cell death. Both LSS and OSS had no effect on human normal hepatocyte, lung epithelial, and endothelial cells. Application of LSS to these four cell lines increased the percentage of cells stained positively for annexin V–FITC, with up-regulations of cleaved caspase-8, -9, and -3, and PARP. In addition, LSS also induced Hep3B cell autophagy, as detected by acidic vesicular organelle formation, LC3B transformation, and p62/SQSTM1 degradation. By transfecting with small interfering RNA, we found that the shear-induced apoptosis and autophagy are mediated by bone morphogenetic protein receptor type (BMPR)-IB, BMPR-specific Smad1 and Smad5, and p38 mitogen-activated protein kinase in Hep3B cells. Our findings provide insights into the molecular mechanisms by which shear stress induces apoptosis and autophagy in tumor cells

Prevalence of PIK3CA mutations in Taiwanese patients with breast cancer: a retrospective next-generation sequencing database analysis

BackgroundBreast cancer is the most common cancer type that affects women. In hormone receptor–positive (HR+), human epidermal growth factor receptor 2−negative (HER2–) advanced breast cancer (ABC), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) is the most frequently mutated gene associated with poor prognosis. This study evaluated the frequency of PIK3CA mutations in the Taiwanese breast cancer population.MethodologyThis is a retrospective study; patient data were collected for 2 years from a next-generation sequencing database linked to electronic health records (EHRs). The primary endpoint was the regional prevalence of PIK3CA mutation. The secondary endpoints were to decipher the mutation types across breast cancer subtype, menopausal status, and time to treatment failure after everolimus (an mTOR inhibitor) or cyclin-dependent kinase 4/6 (CDK4/6) inhibitor treatment.ResultsPIK3CA mutations were identified in 278 of 728 patients (38%). PIK3CA mutations were reported in 43% of patients with HR−/HER2+ subtype and 42% of patients with HR+/HER2– postmenopausal status. A lower prevalence of PIK3CA mutations was observed in triple-negative (27%) and HR+/HER2– premenopausal patients (29%). The most common mutation was at exon 20 (H1047R mutation, 41.6%), followed by exon 9 (E545K mutation, 18.9% and E542K mutation, 10.3%). Among patients treated with CDK4/6 inhibitors, the median time to treatment failure was 12 months (95% CI: 7-21 months) in the PIK3CA mutation cohort and 16 months (95% CI: 11-23 months) in the PIK3CA wild-type cohort, whereas patients receiving an mTOR inhibitor reported a median time to treatment failure of 20.5 months (95% CI: 8-33 months) in the PIK3CA mutation cohort and 6 months (95% CI: 2-9 months) in the PIK3CA wild-type cohort.ConclusionA high frequency of PIK3CA mutations was detected in Taiwanese patients with breast cancer, which was consistent with previous studies. Early detection of PIK3CA mutations might influence therapeutic decisions, leading to better treatment outcomes

Women with endometriosis have higher comorbidities: Analysis of domestic data in Taiwan

AbstractEndometriosis, defined by the presence of viable extrauterine endometrial glands and stroma, can grow or bleed cyclically, and possesses characteristics including a destructive, invasive, and metastatic nature. Since endometriosis may result in pelvic inflammation, adhesion, chronic pain, and infertility, and can progress to biologically malignant tumors, it is a long-term major health issue in women of reproductive age. In this review, we analyze the Taiwan domestic research addressing associations between endometriosis and other diseases. Concerning malignant tumors, we identified four studies on the links between endometriosis and ovarian cancer, one on breast cancer, two on endometrial cancer, one on colorectal cancer, and one on other malignancies, as well as one on associations between endometriosis and irritable bowel syndrome, one on links with migraine headache, three on links with pelvic inflammatory diseases, four on links with infertility, four on links with obesity, four on links with chronic liver disease, four on links with rheumatoid arthritis, four on links with chronic renal disease, five on links with diabetes mellitus, and five on links with cardiovascular diseases (hypertension, hyperlipidemia, etc.). The data available to date support that women with endometriosis might be at risk of some chronic illnesses and certain malignancies, although we consider the evidence for some comorbidities to be of low quality, for example, the association between colon cancer and adenomyosis/endometriosis. We still believe that the risk of comorbidity might be higher in women with endometriosis than that we supposed before. More research is needed to determine whether women with endometriosis are really at risk of these comorbidities

Heterogeneous contributions of change in population distribution of body mass index to change in obesity and underweight NCD Risk Factor Collaboration (NCD-RisC)

From 1985 to 2016, the prevalence of underweight decreased, and that of obesity and severe obesity increased, in most regions, with significant variation in the magnitude of these changes across regions. We investigated how much change in mean body mass index (BMI) explains changes in the prevalence of underweight, obesity, and severe obesity in different regions using data from 2896 population-based studies with 187 million participants. Changes in the prevalence of underweight and total obesity, and to a lesser extent severe obesity, are largely driven by shifts in the distribution of BMI, with smaller contributions from changes in the shape of the distribution. In East and Southeast Asia and sub-Saharan Africa, the underweight tail of the BMI distribution was left behind as the distribution shifted. There is a need for policies that address all forms of malnutrition by making healthy foods accessible and affordable, while restricting unhealthy foods through fiscal and regulatory restrictions

EV71 virus subunit vaccine development

腸病毒71 型是屬於微小核醣核酸病毒科（picoornaviridae）的其中一種病毒，腸病毒71 型（ EV - 71 ）所引起的臨床症狀中，輕微者會有發燒、出疹、手足口症或咽峽炎 (herpangina) ， 嚴重的話引發肺水腫(pulmonary edema)、無菌性腦膜炎 (aseptic meningitis)及類似小兒麻痺病毒所引起的急性肢體麻痺症狀(acute flaccid paralysis) 。目前，沒有任何疫苗可與治療僅限於支持治療。在這項研究中所以先利用活病毒及去活化的病毒進行免疫，尋找可以產生中和能力的表面抗原決定位(epitope)，並同時利用電腦軟體去預測腸病毒71 型之鞘膜蛋白( capsid protein )抗體的表面決定位，從此二者結果中尋找到候選疫苗之片段。並依據這些資訊，進一步合成胜肽，(1)VP1P1:在鞘膜蛋白VP1 的第152~185 個胺基酸位置，(2)VP1P2則是在鞘膜蛋白第189-225 個胺基酸的位置,(3)VP1ThP2 胜肽:增加一個通用的Th 表面抗原決定位(AKFVAAWTLKA)，這些胜肽再結合人體可用的佐劑，進行乳化作用後已50 μg 免疫，其中ISA51/CpG 和VP1-Th-P2 胜肽也可以產生很高的IgG 效價(106)以及中和保護效價(1:64)顯示這段含有Th的胜肽有做為未來腸病毒疫苗的潛力，另外由於重組蛋白的腸病毒71 型的鞘膜蛋白VP1 也曾被作為候選疫苗，但是由於製備過程需經包涵體(inclusion body)的步驟，因而降底中和性抗體表位的呈現。因此我們利用一般可以增加蛋白溶解度的序列， 此序列為small ubiquitin-related modifier(SUMO)。它可經由類似ubiquitination 的過程而與目標蛋白質上特定的lysine 支鏈形成共價鍵，而修飾目標蛋白質，所以利用此特性將腸病毒71 型之鞘膜蛋白VP1 在DNA 上重新構築成SUMO-VP1 蛋白，再以重組蛋白 rSumoVP1 免疫50 μg 於BALB/c 老鼠，可以產生很高的IgG 效價(106)，以及中和保護效價(1:32)。以重組蛋白rSumoVP1/ ISA51,CpG 免疫小鼠後，拿取小鼠脾臟細胞進行體外試驗，可以表現相對多的增生能力(proliferation assay)，以及產生IFN-r(803 pg/ml)，IL-4(6.65 pg/ml)，IL-2(203 pg/ml)。這表示著rSumoVP1 的免疫是會產生Th1 以及Th2 的反應。 依上述結果得知VP1-Th-P2/ISA51,CpG 和rSumoVP1/ISA51,CpG 此二者可以作為預防腸病毒感染的疫苗配方。Enterovirus 71 (EV71) is a non-enveloped, single-stranded RNA virus within the Picornaviridae family. EV71 has been associated with an array of clinical diseases, ranging from mild exanthemas such as herpangina to aseptic meningitis, encephalitis and pulmonary edema. EV71 is also an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes outbreaks with significant mortality among young children. Currently, there is no vaccine available and treatment is limited to palliative care. The antisera were raised in mice against live virus and inactive virus spanning the capsid protein of EV71. The antisera epitope mapping with 165 overlapping synthetic peptides spanning the capsid protein of EV71.We find one epitope，V43，contains amino acid 211~225 of VP1 region , another epitope， E5， contains amino acid 21-35 of VP4 region .At the same time, we used Immune Epitope Database (IEDB) software to predict epitopes of the capsid protein with enterovirus71. We synthesized long term peptide (VP1-P1,VP1-P2,VP1-Th-P2) formulate with human used adjuvant.ISA51-CpG formulate with VP1-Th-P2 peptide which can produce high IgG antibody titer and neutralization antibody。At the same time, ISA51-CpG formulate with rSumoVP recombinant protein which can produce neutralization antibody。We got cytokine IFN-r(803 pg/ml)，IL-4(6.65 pg/ml) and IL-2(203 pg/ml) of SumoVP1 protein stimulated in Splenocyte assay。We got the result which VP1-Th-P2/ISA51,CpG和rSumoVP1/ISA51,Cp may be the vaccine candidate.目 錄 摘 要......................................................I Summary...................................................II 目 錄……………………………………………………………………III 圖 表 目 錄..............................................VI 縮 寫 字 對 照 表.......................................VIII 第一章 序 論..............................................1 第一節 腸病毒71 型之病毒學概論.............................1 第二節 腸病毒71 型的致病機轉...............................3 第三節 腸病毒71 型的臨床病症...............................4 第四節 腸病毒71 型的流行病學...............................5 第五節 腸病毒71 型疫苗開發之前人研究.......................5 第二章 研究目的與實驗設計..................................7 第三章 實驗材料與方法......................................9 第一節 材料來源............................................9 一. 實驗動物...............................................9 二. 病毒...................................................9 三. 細胞株................................................9 四. 佐劑..................................................9 五. 設計合成長片段胜.肽..................................10 六. 設計合成短片段胜肽...................................10 七. 重組蛋白(recombinant Sumo-VP1)之質體..................10 第二節 實驗方法........................................10 一. 細胞培養.............................................10 二. 病毒毒力半定量測定(Tissue culture Infection Dose 50，TCID50) ...12 三. 腸病毒中和抗體(Neutralization Antibody)測定..........12 四. 構築表現VP1 基因(Construction of vector expressing VP1 gene) .....14 五. 腸病毒VP1 重組蛋白之表現與純化........................16 六. 純化rSumo-VP1 蛋白....................................20 七. 西方墨點法............................................21 八. 酵素免疫分析法（Enzyme-linked immunoassay，ELISA）之抗原表面決定位的測試(Epitope mapping ELISA procedure) ........22 九. 酵素免疫分析法（Enzyme-linked immunoassay，ELISA）之血清抗體(serum titer)的測試...................................23 十. 細胞激素丙型干擾素，介白素-2，介白素-4(IFN-γ，IL-2，IL-4) 酵素免疫分析法.........................................23 十一. 抗原乳化作用(antigen formulation) ..................26 十二. 免疫血清之製備......................................27 十三. 脾臟前處裡..........................................27 十四. 脾臟細胞增生試驗....................................28 十五. 脾臟細胞釋放細胞激素實驗............................28 十六. 腸病毒感染動物實驗(in vivo).........................29 第四章 實驗結果...........................................30 第一節 Whole virus 腸病毒71 型免疫小鼠之抗體.............30 第二節 B 細胞表面抗原決定位之測定.........................30 第三節 Th 細胞表面抗原決定位之測定........................31 第四節 軟體預測其表面抗原位置.............................31 第五節 分析VP1-P1 與VP1-P2 胜肽與佐劑乳化、免疫後之關係...32 第六節 腸病毒感染動物實驗之攻毒試驗( challenge test)......33 第七節 構築VP1 基因及表現.................................33 第八節 rVP1 重組蛋免疫小鼠及抗體之表現....................34 第九節 rSumoVP1 重組蛋白表現與純化........................34 第十節 rSumoVP1 重組蛋免疫小鼠及抗體之表現................34 第十一節 探討rSumoVP1 重組蛋免疫小鼠後細胞性免疫之表現...35 第十二節 腸病毒感染動物實驗之攻毒試驗(challenge test)....36 第五章 討 論......................................................................................................37 第六章 結 論......................................................................................................40 參 考 文 獻....................................................41 附錄及附表..........................................................................................................71 圖 表 目 錄 圖一 不同週數血清之抗腸病毒71 型抗體濃度..............................................47 圖二 抗腸病毒71 型不同週數血清之中和抗體濃度......................................48 圖三 抗腸病毒71 抗體之B 細胞表面抗原決定位.......................................49 圖四 抗腸病毒71 型之Th 細胞表面抗原決定位..........................................51 圖五 腸病毒71 型鞘膜蛋白之預測模擬表面抗原機率(Emini Surface Accessibility Prediction profile) ...................................................................53 圖六 不同週數之抗VP1-P2 胜肽血清抗體.....................................................55 圖七. 酵素免疫分析法偵測去活化腸病毒之血清抗體力價...........................56 圖八 抗VP1-P2 胜肽血清抗體之表面抗原決定位確認...............................57 圖九 抗VP1-P2 胜肽不同週數血清之中和抗體濃度.....................................58 圖十 抗VP1-P2 小鼠對腸病毒71 型之攻毒試驗( challenge test)..................59 圖十一 VP1 基因挑選......................................................................................60 圖十二 重組蛋白rVP1 之大小..........................................................................61 圖十三 不同週數之血清抗體............................................................................62 圖十四 rSumoVP1 重組蛋白之純化.................................................................63 圖十五 不同週數血清之抗體濃度....................................................................64 圖十六 不同週數血清之中和抗體濃度............................................................65 圖十七 IgG 抗體亞型分布趨勢.........................................................................66 圖十八 脾臟細胞增生試驗................................................................................67 圖十九 細胞激素酵素免疫分析法(cytokine ELISA)偵測培養四天後之細胞 激素濃度.......................................................................................................69 圖二十 腸病毒感染動物實驗之攻毒試驗( challenge test) ..............................70 附圖一 腸病毒71 型基因架構圖......................................................................71 附圖二 FMDV 基因構造圖................................................................................72 附圖三 鞘膜蛋白短片段胜序列之設計圖........................................................73 附表一 鞘膜蛋白(capsid protein-VP1)之合成胜肽..........................................74 附表二 鞘膜蛋白(capsid protein)之合成胜肽...................................................75 附圖四 構築腸病毒重組蛋白rVP1 基因之示意圖..........................................8

The dimensional arrow: agreement in directional mapping of dimensions among Mandarin Chinese- and English-speakers

We present evidence that English- and Mandarin-speakers agree about how to map dimensions (e.g., size and clarity) to vertical space and that they do so in a directional way. We first developed visual stimuli for four dimensions—size, clarity, complexity, and darkness—and in each case we varied the stimuli to express a range of the dimension (e.g., there were five total items expressing the range covering big, medium, and small). In our study, English- and Mandarin-speakers mapped these stimuli to an unlabelled vertical scale. Most people mapped dimensional endpoints in similar ways; using size as a standard, we found that the majority of participants mapped the clearest, most complex, and darkest items to the same end of the vertical scale as they mapped the biggest items. This indicates that all four dimensions have a weighted or unmarked end (i.e., all are directional or polar). The strong similarities in polarity across language groups contrasted with group differences on a lexical task, for which there was little cross-linguistic agreement about which comparative words to use to describe stimulus pairs (e.g., “bigger” vs. “smaller”). Thus, we found no evidence in this study that the perception of these dimensions is influenced by language

Oxidative Stress and Nonalcoholic Fatty Liver Disease in Hemodialysis Patients

Introduction. Nonalcoholic fatty liver disease (NAFLD) is becoming more common around the world and it may progress to cirrhosis and liver failure, increasing mortality risk. In hemodialysis (HD) patients, NAFLD may be a novel risk factor for their high cardiovascular mortality. Heightened oxidative stress is highly prevalent in HD patients. However, the relationship between oxidative stress and NAFLD in HD patients is not well defined. Methods. We studied seventy-one stable nondiabetic HD patients. Nineteen patients had the diagnosis of NAFLD by ultrasonography. Blood levels of oxidative stress markers were measured in each patient, including thiobarbituric acid reactive substances (TBARS), free thiols, superoxide dismutase (SOD) activities, and glutathione peroxidase (GPx) activity. The copy numbers of mitochondrial DNA (mtDNA) in peripheral leukocytes were also determined. Demographic, biochemistry, and hemogram data were recorded. The two groups of patients were compared in order to determine the factors associated with NAFLD in HD patients. Findings. Compared to those without NAFLD, nondiabetic HD patients with NAFLD had significantly higher mtDNA copy number and GPx levels. The two groups did not differ significantly in dialysis adequacy, hemoglobin, serum calcium, phosphorus, albumin, liver function tests, or lipid profiles. Regression analysis confirmed mtDNA copy numbers and GPx levels as two independent factors associated with NAFLD. Compared to those with polysulfone, patients dialyzed with cellulose membrane have significantly higher levels of TBARS. However, patients with or without NAFLD did not differ in their use of either dialysis membrane. Discussion. Oxidative stress (represented by antioxidant defense, GPx) and mitochondrial DNA copy numbers are independently associated with fatty liver disease in nondiabetic HD patients. The diagnostic and therapeutic implications of this key observation warrant further exploration

Income inequalities in exposure to endocrine disrupting chemicals among pregnant women in Taiwan

Background: Exposure to endocrine disrupting chemicals (EDCs) is being rigorously studied in associations with various health outcomes, however less attention has been paid to its socio-economic determinants. This study investigated how EDCs exposure levels in pregnant women could differ on individual- and area- level income in Taiwan. Methods: Urinary measurements of phthalates, nonylphenol, bisphenol A, parabens and individual socio-economic variables (income, education, etc.) of pregnant women from Taiwan Maternal and Infant Cohort Study (TMICS) were linked via residence to area-level average annual household income. EDCs concentrations were compared between the four main Taiwan regions (North, Central, South and East) and between individual income groups. Lorenz curves were plotted to describe inequalities in EDCs exposure, and EDCs exposure related to income. Concentration indexes (CIx) were calculated and compared between the four regions. Results: No significant differences between EDCs concentrations adjusted for molar mass and creatinine across individual-level income groups were detected. Exposure inequalities were highest for parabens (CIx = 73.6 %), BPA (CIx = 73.6) and low molecular weight (LMW) phthalates (CIx = 63.3 %). Lorenz curve for LMW phthalates distribution across area-level income was significantly above the equality line (CIx = -21.9 %, p-value < 0.05). Stratification showed significant differences in inequality (p = 0.046) in LMW phthalates exposure across area-level income between South (CIx = -23.1 %), East (CIx = -35.3 %), North (CIx = -3.3 %) and Central (CIx = -6.5 %) regions. Conclusion: Inequalities in LMW phthalates exposure related to area-level income in East and South regions suggest target areas for public health interventions in terms of environmental regulations and health promotion. Using Lorenz curves and CIx to describe inequalities can be useful in case of inadequate sample sizes in costly human biomonitoring studies, however this method needs to be further developed and validated using human biomonitoring datasets in more diverse populations with larger socio-economic inequalities

Cytokine secretion from BMDCs treated with TcdA rRBD or its truncated fragments.

<p>After BMDCs were treated with TcdA rRBD or its truncated fragments at day 6 for 18 h, the culture supernatants were collected and analyzed for cytokine profiles using specific cytokine ELISAs: (A) IL-6, (B) IL12p40, and (C) TNF- α. The symbols * and ** indicate <i>p</i><0.05 and <i>p</i><0.01, respectively.</p