LEGAL EDUCATION IN GERMANY

<div><p>Predicting the subcellular localization of proteins conquers the major drawbacks of high-throughput localization experiments that are costly and time-consuming. However, current subcellular localization predictors are limited in scope and accuracy. In particular, most predictors perform well on certain locations or with certain data sets while poorly on others. Here, we present PSI, a novel high accuracy web server for plant subcellular localization prediction. PSI derives the wisdom of multiple specialized predictors via a joint-approach of group decision making strategy and machine learning methods to give an integrated best result. The overall accuracy obtained (up to 93.4%) was higher than best individual (CELLO) by ∼10.7%. The precision of each predicable subcellular location (more than 80%) far exceeds that of the individual predictors. It can also deal with multi-localization proteins. PSI is expected to be a powerful tool in protein location engineering as well as in plant sciences, while the strategy employed could be applied to other integrative problems. A user-friendly web server, PSI, has been developed for free access at <a href="http://bis.zju.edu.cn/psi/" target="_blank">http://bis.zju.edu.cn/psi/</a>.</p></div

Model for combination of group voting and neural network.

<p>Raw prediction results from 11 predictors first entered ranking system to give prediction for each subcellular location of elevated-accuracy. Then neural network took group-voting output as input for further calculation and adjustment. Final prediction results were given through neural network.</p

Determination of the model topological structure.

<p>(a) Performance under different model structures with full data as input. Blue lines denote the training set and red lines denote the test set. Peak for prediction was on 10 neurons, 1 hidden layer. (b) Stepwise-selection performance was evaluated by AUROC. Obvious enhancement took place in step 2, 3 and 4. Peak was in step 9, with best community consisting of cello, Wolf PSORT, MultiLoc, mPloc, YLoc and iPsort. (c) With selected community of predictors, model topological structure was determined using the same method in (a). Peak was on 10 neurons, 1 hidden layer. (d) Model structure evaluation for combination of group-voting and neural network. Results from group-voting were taken as input for neural network. Peak was on 10 neurons, 1 hidden layer. The best results are boxed in dash.</p

Characterization of Adult α- and β-Globin Elevated by Hydrogen Peroxide in Cervical Cancer Cells That Play A Cytoprotective Role Against Oxidative Insults

<div><h3>Objectives</h3><p>Hemoglobin (Hgb) is the main oxygen and carbon dioxide carrier in cells of erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. However, Hgb is also expressed in nonerythroid cells. In the present study, the expression of Hgb in human uterine cervix carcinoma cells and its role in cervical cancer were investigated.</p> <h3>Methodology</h3><p>The expression level of Hgb in cervical cancer tissues was assessed by quantitative reverse transcriptase-PCR (qRT-PCR). We applied multiple methods, such as RT-PCR, immunoblotting, and immunohistochemical analysis, to confirm Hgb expression in cervical cancer cells. The effects of ectopic expression of Hgb and Hgb mutants on oxidative stress and cell viability were investigated by cellular reactive oxygen species (ROS) analysis and lactate dehydrogenase (LDH) array, respectively. Both Annexin V staining assay by flow cytometry and caspase-3 activity assay were used, respectively, to evaluate cell apoptosis.</p> <h3>Results</h3><p>qRT-PCR analysis showed that Hgb-α- (HBA1) and Hgb-β-globin (HBB) gene expression was significantly higher in cervical carcinoma than in normal cervical tissues, whereas the expression of hematopoietic transcription factors and erythrocyte specific marker genes was not increased. Immunostaining experiments confirmed the expression of Hgb in cancer cells of the uterine cervix. Hgb mRNA and protein were also detected in the human cervical carcinoma cell lines SiHa and CaSki, and Hgb expression was up-regulated by hydrogen peroxide-induced oxidative stress. Importantly, ectopic expression of wild type HBA1/HBB or HBA1, rather than mutants HBA1^H88R/HBB^H93R unable to bind hemo, suppressed oxidative stress and improved cell viability.</p> <h3>Conclusions</h3><p>The present findings show for the first time that Hgb is expressed in cervical carcinoma cells and may act as an antioxidant, attenuating oxidative stress-induced damage in cervical cancer cells. These data provide a significant impact not only in globin biology but also in understanding of cervical cancer pathogenesis associated with oxidative stress.</p> </div

Effect of HBA1/HBB, HBA1 and HBA1^H88R/HBB^H93R overexpression on H₂O₂ levels and superoxide anion in SiHa cells undergoing oxidative stress.

<p>(<b>A</b>) Whole cell lysates from SiHa cells transfected with a control plasmid or HBA1 and HBB expression plasmids were analyzed with an anti-Hb antibody to confirm the overexpression of globin proteins. Control and HBA1/HBB- overexpressing cells were stained with fluorogenic probes to detect intracellular H₂O₂ and superoxide anion and exposed to extracellular H₂O₂ (1 mM) for 10 min. Immunostaining confirmed that the intracellular generation of H₂O₂ (<b>B</b>) and superoxide anion (<b>C</b>) was suppressed in HBA1/HBB-overexpressing cells. Flow cytometry analysis confirmed these results (<b>D</b> and <b>E</b>) and showed that the intracellular levels of H₂O₂ and superoxide anion were higher in cells exposed to extracellular stimuli than in untreated cells (<b>B</b> and <b>C</b>). Quantitative analyses confirmed that the intracellular generation of H₂O₂ (<b>F</b>) and superoxide anion (<b>G</b>) was inhibited in HBA1/HBB-overexpressing cells (black column) more than in control cells (gray column; <i>P</i><0.05). Quantitative analyses confirmed that the intracellular generation of ROS was reduced in HBA1-overexpressing cells (black column, <b>H</b>), but not in HBA1^H88R/HBB^H93R-overexpressing cells (black column, <b>I</b>) more than in control vector-overexpressing cells (gray column). *<i>P</i><0.05; **<i>P</i><0.01; ^N<i>P</i>>0.05.</p

Clinicopathological characteristics of the cervical cancer patients.

<p>FIGO: International Federation of Gynecology and Obstetrics; HPV: Human papillomavirus; NA, Not available.</p

Elevated HBA1 and HBB expression in cervical cancer.

a<p>Fold-change is calculated based on comparison between cervical cancer and normal cervix epithelium.</p

Up-regulation of Hgb expression by oxidative stress.

<p>(<b>A</b>) HBA1 and HBB mRNA expression in SiHa cells was induced by H₂O₂ treatment. SiHa cells were treated with H₂O₂ (1 mM) for 24 h and Hgb mRNA levels were determined by qRT-PCR. Hgb levels in controls were normalized to 1. Data were expressed as mean ± SD (n = 3).^**<i>P</i><0.01. (<b>B</b>) HBA1 and HBB protein expression was induced by H₂O₂ treatment. Whole cell lysates obtained from SiHa cells treated with or without H₂O₂ (1 mM, 48 h) were analyzed for HBA1 and HBB levels. β-actin was used as a loading control. (<b>C</b>) HBA1 and HBB mRNAs were up-regulated by H₂O₂ treatment in HEK293 cells. HEK293 cells were treated with H₂O₂ (1 mM, 36 h). Reverse transcription PCR products were separated on 2% agarose gels and visualized with ethidium bromide. GAPDH was used as a loading control. (<b>D</b>) Dose and time dependency of the response of SiHa cells to H₂O₂ treatment. SiHa cells were treated with a series of concentrations (0, 0.25, 0.5 and 1 mM) of H₂O₂ for 8, 16, 24 or 36 h. Relative HBA1 mRNA level was determined by qRT-PCR. Data were expressed as mean ± SD (n = 3). Values were normalized to the HBA1 level in the control (0 mM, 8 h), which was set to 1. One-way ANOVA test for multiple comparisons was used to analyze the differences between treatments. ^**<i>P</i><0.01; ^***<i>P</i><0.001, when compared to 0 mM treatment.</p

Elevated expression of HBA1 and HBB in human cervical cancer tissues.

<p>(<b>A</b>) To confirm the specificity of the HBA1 and HBB primers for qRT-PCR, expression of HBA1 and HBB was examined in human bone marrow and peripheral blood cells. Retrotranscriptase free (–) as a negative control, H₂O as a system control. Mr, Marker. (<b>B</b>) cDNA from cervical cancer samples was prepared and analyzed for expression of HBA1 and HBB by RT-PCR. PCR products were separated on 2% agarose gels and visualized with ethidium bromide. GAPDH was used as a loading control. H₂O as a system control, T, represents primary tumour samples. The expression levels of HBA1 and HBB were measured by qRT-PCR in 20 human cervical cancer specimens and 10 normal cervical tissue controls. Quantification of the indicated normalized HBA1 and HBB levels (log₁₀) of the total unpaired normal cervix (n = 10) and cervical cancer (n = 20) samples are shown. The tukey boxes represent the upper and lower quartiles divided by the median and whiskers are the largest and smallest values, excluding outliers represented by circles. All differences were statistically significant at <i>P</i><0.05 (<b>C and D</b>).</p

Expression of HBA1 and HBB in cultured cervical cancer cells.

<p>(<b>A</b>) RT-PCR amplification of HBA1 and HBB from SiHa, CaSki cells and human blood RNA. The HBA1 and HBB transcripts were clearly amplified from the human cervical cancer cell lines, SiHa and CaSki. RNA extracted from blood was used as a positive control and GAPDH was detected as a loading control. Amplicon identities were confirmed by sequencing. (<b>B</b>) HBA1 and HBB were analyzed by western blotting. Peripheral blood leukocytes (PBL) were used as a positive control and β-actin was detected as a loading control. (<b>C</b>) Cell lysates from SiHa and CaSki cells were immunoprecipitated and immunoblotted with the indicated antibodies. A clear band of 17 kDa was enriched from SiHa and CaSki lysates by immunoprecipitation using an anti-human Hgb antibody. Non-specific IgG was used as immunoprecipitation control. Cytoplasmic staining of HBA1 and HBB was detected in cervical cancer cells (<b>D</b> and <b>E</b>, <b>F</b> and <b>G</b>). Double-immunostaining with p16^INK4A, a specific marker of cervical cancer for cytology and histological diagnosis, demonstrated that Hgb was expressed by cervical cancer cells (<b>H–J</b>). Inserts are magnified images of selected areas (small squares).</p