Strongly consistent modified maximum likelihood estimation of U-shaped hazard functions

A hazard function which initially decreases, then drops down to an essentially constant level, and then increases due to aging, often is described as being U-shaped or bathtub-shaped. For such a hazard function h(·), the model interval M is defined as M = m |h(m) = inf h(x), which is an interval over which the hazard rate is constant;Bray et al. (1967) developed the maximum likelihood estimate cx h(·) of a U-shaped hazard function h(·), with cx h(·) certain to equal zero over some open interval, and showed that, if h(·) is V-shaped, then cx h(·) is strongly consistent at all continuity points of h(·), excepting the turning point (i.e., minimizing point, of a V-shaped h(·));Let [alpha](n) be such that both [alpha](n) and n/[alpha](n) tend to infinity with n. This dissertation presents a maximum likelihood estimate h(·) of h(·) that is restricted to attain its minimum value at least [alpha](n) consecutive observations. This estimator h(·) everywhere exceeds zero, and is strongly consistent at the continuity points of h(·). While Bray et al. (1967) make use of the well-known Pooled-Adjacent-Violators algorithm named by Barlow et al. (1972) for monotone estimation, which involves successive revisions at neighboring observation pairs, the algorithmic computation of h(·) proceeds by successive revisions at neighboring observation triplicates

Nuclear export regulation of COP1 by 14-3-3σ in response to DNA damage

Mammalian constitutive photomorphogenic 1 (COP1) is a p53 E3 ubiquitin ligase involved in regulating p53 protein level. In plants, the dynamic cytoplasm/nucleus distribution of COP1 is important for its function in terms of catalyzing the degradation of target proteins. In mammalian cells, the biological consequence of cytoplasmic distribution of COP1 is not well characterized. Here, we show that DNA damage leads to the redistribution of COP1 to the cytoplasm and that 14-3-3σ, a p53 target gene product, controls COP1 subcellular localization. Investigation of the underlying mechanism suggests that COP1 S387 phosphorylation is required for COP1 to bind 14-3-3σ. Significantly, upon DNA damage, 14-3-3σ binds to phosphorylated COP1 at S387, resulting in COP1's accumulation in the cytoplasm. Cytoplasmic COP1 localization leads to its enhanced ubiquitination. We also show that N-terminal 14-3-3σ interacts with COP1 and promotes COP1 nuclear export through its NES sequence. Further, we show that COP1 is important in causing p53 nuclear exclusion. Finally, we demonstrate that 14-3-3σ targets COP1 for nuclear export, thereby preventing COP1-mediated p53 nuclear export. Together, these results define a novel, detailed mechanism for the subcellular localization and regulation of COP1 after DNA damage and provide a mechanistic explanation for the notion that 14-3-3σ's impact on the inhibition of p53 E3 ligases is an important step for p53 stabilization after DNA damage

Antineoplastic effects of an Aurora B kinase inhibitor in breast cancer

<p>Abstract</p> <p>Background</p> <p>Aurora B kinase is an important mitotic kinase involved in chromosome segregation and cytokinesis. It is overexpressed in many cancers and thus may be an important molecular target for chemotherapy. AZD1152 is the prodrug for AZD1152-HQPA, which is a selective inhibitor of Aurora B kinase activity. Preclinical antineoplastic activity of AZD1152 against acute myelogenous leukemia, multiple myeloma and colorectal cancer has been reported. However, this compound has not been evaluated in breast cancer, the second leading cause of cancer deaths among women.</p> <p>Results</p> <p>The antineoplastic activity of AZD1152-HQPA in six human breast cancer cell lines, three of which overexpress HER2, is demonstrated. AZD1152-HQPA specifically inhibited Aurora B kinase activity in breast cancer cells, thereby causing mitotic catastrophe, polyploidy and apoptosis, which in turn led to apoptotic death. AZD1152 administration efficiently suppressed the tumor growth in a breast cancer cell xenograft model. In addition, AZD1152 also inhibited pulmonary metastatic nodule formation in a metastatic breast cancer model. Notably, it was also found that the protein level of Aurora B kinase declined after inhibition of Aurora B kinase activity by AZD1152-HQPA in a time- and dose-dependent manner. Investigation of the underlying mechanism suggested that AZD1152-HQPA accelerated protein turnover of Aurora B via enhancing its ubiquitination.</p> <p>Conclusions</p> <p>It was shown that AZD1152 is an effective antineoplastic agent for breast cancer, and our results define a novel mechanism for posttranscriptional regulation of Aurora B after AZD1152 treatment and provide insight into dosing regimen design for this kinase inhibitor in metastatic breast cancer treatment.</p

Use of In Vivo Complementation in \u3cem\u3eMycobacterium tuberculosis\u3c/em\u3e to Identify a Genomic Fragment Associated with Virulence

Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra

Pendekatan Lean Manufacturing Untuk Meningkatkan Efisiensi Dalam Proses Produksi Dengan Menggunakan Value Stream Mapping Pada CV. Indospice

CV. INDOSPICE merupakan Perusahaan yang bergerak pada produksi pala, untuk terus mengoptimalkan kinerja produktifitasnya dan meningkatkatkan laba Perusahaan dengan berusaha menurunkan biaya, meningkatkan kualitas dan tepat waktu dalam pengiriman ke pelanggan. Penelitian ini bertujuan untuk mengetahui berbagai bentuk pemborosan (waste) apa saja yang sering terjadi sehingga dapat meningkatkan efisiensi produksi, karena itu diperlukan suatu pendekatan lean manufacturing. Lean Manufacturing merupakan sebuah pendekatan untuk meminimisasi pemborosan yang terjadi dalam proses produksi melalui value stream mapping untuk meningkatkan efisiensi. Metode yang digunakan dalam penelitian ini adalah deskriptif yang dilakukan dengan meneliti analisa pekerjaan dan aktifitas pada suatu obyek. Hasil penelitian menunjukan bahwa dalam proses produksi yang terjadi masih terdapat bentuk pemborosan berupa proses yang berlebih dan penggunaan mesin yang belum optimal. Untuk itu perlu upaya untuk meningkatkan efisisen berupa penambahan mesin penggiling pala dan pengadaan teknologi modern agar pengerjaan menjadi lebih cepat

Activation of Liver FGF21 in hepatocarcinogenesis and during hepatic stress

BACKGROUND: FGF21 is a promising intervention therapy for metabolic diseases as fatty liver, obesity and diabetes. Recent results suggest that FGF21 is highly expressed in hepatocytes under metabolic stress caused by starvation, hepatosteatosis, obesity and diabetes. Hepatic FGF21 elicits metabolic benefits by targeting adipocytes of the peripheral adipose tissue through the transmembrane FGFR1-KLB complex. Ablation of adipose FGFR1 resulted in increased hepatosteatosis under starvation conditions and abrogation of the anti-obesogenic action of FGF21. These results indicate that FGF21 may be a stress responsive hepatokine that targets adipocytes and adipose tissue for alleviating the damaging effects of stress on the liver. However, it is unclear whether hepatic induction of FGF21 is limited to only metabolic stress, or to a more general hepatic stress resulting from liver pathogenesis and injury. METHODS: In this survey-based study, we examine the nature of hepatic FGF21 activation in liver tissues and tissue sections from several mouse liver disease models and human patients, by quantitative PCR, immunohistochemistry, protein chemistry, and reporter and CHIP assays. The liver diseases include genetic and chemical-induced HCC, liver injury and regeneration, cirrhosis, and other types of liver diseases. RESULTS: We found that mouse FGF21 is induced in response to chemical (DEN treatment) and genetic-induced hepatocarcinogenesis (disruptions in LKB1, p53, MST1/2, SAV1 and PTEN). It is also induced in response to loss of liver mass due to partial hepatectomy followed by regeneration. The induction of FGF21 expression is potentially under the control of stress responsive transcription factors p53 and STAT3. Serum FGF21 levels correlate with FGF21 expression in hepatocytes. In patients with hepatitis, fatty degeneration, cirrhosis and liver tumors, FGF21 levels in hepatocytes or phenotypically normal hepatocytes are invariably elevated compared to normal health subjects. CONCLUSION: FGF21 is an inducible hepatokine and could be a biomarker for normal hepatocyte function. Activation of its expression is a response of functional hepatocytes to a broad spectrum of pathological changes that impose both cellular and metabolic stress on the liver. Taken together with our recent data, we suggest that hepatic FGF21 is a general stress responsive factor that targets adipose tissue for normalizing local and systemic metabolic parameters while alleviating the overload and damaging effects imposed by the pathogenic stress on the liver. This study therefore provides a rationale for clinical biomarker studies in humans

Identification of Prognostic Genes for Recurrent Risk Prediction in Triple Negative Breast Cancer Patients in Taiwan

Discrepancies in the prognosis of triple negative breast cancer exist between Caucasian and Asian populations. Yet, the gene signature of triple negative breast cancer specifically for Asians has not become available. Therefore, the purpose of this study is to construct a prediction model for recurrence of triple negative breast cancer in Taiwanese patients. Whole genome expression profiling of breast cancers from 185 patients in Taiwan from 1995 to 2008 was performed, and the results were compared to the previously published literature to detect differences between Asian and Western patients. Pathway analysis and Cox proportional hazard models were applied to construct a prediction model for the recurrence of triple negative breast cancer. Hierarchical cluster analysis showed that triple negative breast cancers from different races were in separate sub-clusters but grouped in a bigger cluster. Two pathways, cAMP-mediated signaling and ephrin receptor signaling, were significantly associated with the recurrence of triple negative breast cancer. After using stepwise model selection from the combination of the initial filtered genes, we developed a prediction model based on the genes SLC22A23, PRKAG3, DPEP3, MORC2, GRB7, and FAM43A. The model had 91.7% accuracy, 81.8% sensitivity, and 94.6% specificity under leave-one-out support vector regression. In this study, we identified pathways related to triple negative breast cancer and developed a model to predict its recurrence. These results could be used for assisting with clinical prognosis and warrant further investigation into the possibility of targeted therapy of triple negative breast cancer in Taiwanese patients

Strongly consistent modified maximum likelihood estimation of U-shaped hazard functions

A hazard function which initially decreases, then drops down to an essentially constant level, and then increases due to aging, often is described as being "U-shaped" or "bathtub-shaped." For such a hazard function h(·), the model interval M is defined as M = m |h(m) = inf h(x), which is an interval over which the hazard rate is constant;Bray et al. (1967) developed the maximum likelihood estimate cx h(·) of a U-shaped hazard function h(·), with cx h(·) certain to equal zero over some open interval, and showed that, if h(·) is "V-shaped," then cx h(·) is strongly consistent at all continuity points of h(·), excepting the "turning point" (i.e., minimizing point, of a "V-shaped" h(·));Let [alpha](n) be such that both [alpha](n) and n/[alpha](n) tend to infinity with n. This dissertation presents a maximum likelihood estimate h(·) of h(·) that is restricted to attain its minimum value at least [alpha](n) consecutive observations. This estimator h(·) everywhere exceeds zero, and is strongly consistent at the continuity points of h(·). While Bray et al. (1967) make use of the well-known "Pooled-Adjacent-Violators" algorithm named by Barlow et al. (1972) for monotone estimation, which involves successive revisions at neighboring observation pairs, the algorithmic computation of h(·) proceeds by successive revisions at neighboring observation triplicates.</p

Development of microfluidic module for cell lysis

The cellular components such as proteins, DNA, lipids are valuable in modern biotechnology. However, the cell lysis process is necessary for acquiring those materials. In this study, we were inspired form the electroporation technology to develop a microfluidic module that use the pulse waveform for inducing the polarisable particles on the cell membrane to lysis. The microchip with a comb electrodes on glass substrate and PDMS microstructure are fabricated by photolithography. Erythrocyte, yeast and Bacillus were carefully pretreated respectively accroding to their instric characteristics then shocked with designed AC pulse waveform between comb electrodes for cell lysis. The cell wall free erythrocyte can be directly lysed by electrical shock by our desiged AC pulse waveform with the optimal condition at 40 Vpp, 100 k～900 kHz in 5 sec. However, yeast can not be lysed by electrical shock directly because of its cell wall. Therefore, we adopted enzymes prior to the electrical lysis procedure for weakening the cell wall structure. The optimal condition that achieves 91% yeast lysis is electrical shock with designed pulse waveform at 75 Vpp, 50 Hz in 30 sec after incubated with enzymes for 10 min. Electrophoresis results confirm that Bacillus with peptidoglycan cell wall can be weakend by lysozyme and subsequently electrical shock by our module to accelerate the lysis process. The microfluidic module we demonstrated in this study shows the potential that may be integrated with other seperation and analysis modules in the furture to achieve the objective of lab-on-a-chip.細胞內的各種物質諸如蛋白質、DNA、脂質等，在生物技術上都有非常廣泛的用途，但要取得胞內物質常需先將細胞體之細胞膜及細胞壁破壞掉使內容物流出。本研究啟蒙於基因工程常用的電穿孔轉形技術，利用脈衝波誘導細胞膜上極性分子流動變化導致破裂，發展出一套用於裂解細胞的微流體晶片模組。本研究利用微影製程方式，構築出一組齒梳狀電極和搭配的微通道結構形成晶片主體。所使用的細胞樣品有紅血球、酵母菌、枯草桿菌等等，依照不同標的經過適當的前處理後，利用齒梳狀的電極以設計的特殊交流電波形施以高電位差脈衝電溶細胞。缺乏細胞壁的動物細胞紅血球可利用電擊直接將細胞體裂解，最適電溶條件為：使用本研究設計的特殊交流脈衝波形以電位差40 Vpp，頻率100 k～900 kHz之間電擊5秒可達接近100%的破裂效果。酵母菌因為有堅硬的細胞壁保護，需要先用酵素弱化細胞壁的構造再進行電擊，最適電溶條件為：先使用酵素zymolyase處理10分鐘，再用本研究設計的特殊交流脈衝波形以75 Vpp、50 Hz電擊30秒，可達91%的裂解率。而枯草桿菌同樣有細胞壁保護，先用溶菌酶弱化細胞壁後再用本研究模組進行電擊也能加速菌體的裂解。本研究所開發的微流體模組，可望在未來與分離分析的生物晶片模組相結合，達成實驗室平台晶片的最終目標。謝誌 中文摘要 I 英文摘要 II 總目錄 III 表目錄 VI 圖目錄 VII 第一章、前言 1 1.1 生物晶片簡介 1 1.2 細胞在生物工程上的應用 2 1.3 細胞壁簡介 3 1.3.1 細胞壁的功用 3 1.3.2 細胞壁的構造 4 1.4 真菌及其細胞壁 4 1.4.1 酵母菌簡介 5 1.4.2 酵母菌的細胞壁 7 1.5 細菌簡介 7 1.5.1 革蘭氏陽性菌簡介 8 1.5.1.1 革蘭氏陽性菌的細胞壁 8 1.5.2 革蘭氏陰性菌簡介 9 1.5.2.1 革蘭氏陰性菌的細胞壁 10 1.6 常見的破壁方法 10 1.7 紅血球簡介 13 1.7.1 紅血球的細胞膜 14 1.8 電穿孔法的原理與應用 15 1.8.1 電穿孔的原理 15 1.9 研究目的 17 1.10 研究架構 18 第二章 材料與方法 19 2.1 實驗流程 19 2.2 實驗材料與設備 19 2.2.1 菌種 19 2.2.2 藥品及試劑 19 2.2.2.1 藥品 19 2.2.2.2 培養基的配置 21 2.2.2.3 各種試劑 22 2.2.2.4 光阻液 24 2.2.3 儀器設備 24 2.3 晶片的製作 25 2.3.1 光罩的設計與製作 25 2.3.1.1 正光阻用光罩 26 2.3.1.2 負光阻用光罩 27 2.3.2 晶片的清洗 28 2.3.2.1 清洗流程 28 2.3.3 微影製程 28 2.3.3.1於晶片上製作正光阻 31 2.3.3.2於晶片上製作負光阻 31 2.3.4 以PDMS翻模製作微通道結構 32 2.3.5.2 金屬層去除 33 2.4 樣品的製備與前處理 34 2.4.1 冷凍乾燥管之開管 34 2.4.2 菌種的活化與保存 34 2.4.2.1 Saccharomyces cerevisiae BCRC 21679 34 2.4.2.2 Bacillus subtilis DB104 34 2.4.2.3 凍管的製備 35 2.4.3 樣品的前處理 35 2.4.3.1 紅血球前處理 35 2.4.3.2 Saccharomyces cerevisiae前處理 35 2.4.3.3 Bacillus subtilis DB104前處理 36 2.5 細胞電溶實驗 36 2.5.1 波型設計 36 2.5.2 細胞電溶 37 2.5.2.1 紅血球及酵母菌的細胞電溶 37 2.5.2.2 B. subtilis DB104的細胞電溶 38 2.6 細胞電溶結果檢測 38 2.6.1 直接觀察 38 2.6.2 蛋白質之偵測 38 2.6.2.1 Tricine-聚丙烯醯胺膠體電泳膠片的製作 39 2.6.2.2 Tricine-SDS-PAGE電泳步驟 39 2.6.2.3 Tricine-SDS-PAGE膠片染色 40 2.7 超音波破菌 40 第三章 結果與討論 41 3.1 晶片的設計與構築 41 3.2 細胞電溶 44 3.3 電擊波形之設計 44 3.4 紅血球的電溶實驗 47 3.4.1 紅血球的電溶條件測試 54 3.5 酵母菌的電溶 58 3.5.1 酵母菌的弱化測試 60 3.5.2 酵母菌的電溶條件測試 63 3.5.2.1 電擊頻率測試 63 3.5.2.2 電擊電位差測試 67 3.5.2.2 電擊電位差測試 71 3.5.3 Zymolyase對於S. cerevisiae的影響 76 3.6 細菌的電溶 78 3.6.1 細菌的電溶結果檢測 78 3.6.2 蛋白質的偵測 79 第四章、結論與展望 86 參考文獻 8