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The renal Fanconi syndrome is a defect of proximal tubular function causing aminoaciduria and low-molecular-weight proteinuria. Dent's disease and Lowe syndrome are defined X-linked forms of Fanconi syndrome; there is also an autosomal dominant idiopathic form (ADIF), phenotypically similar to Dent's disease though its gene defect is still unknown. To assess whether their respective gene products are ultimately involved in a common reabsorptive pathway for proteins and low-molecular-mass endogenous metabolites, we compared renal Fanconi urinary proteomes and metabonomes with normal (control) urine using mass spectrometry and 1H-NMR spectroscopy, respectively. Urine from patients with low-molecular-weight proteinuria secondary to ifosfamide treatment (tubular proteinuria; TP) was also analyzed for comparison. All four of the disorders studied had characteristic proteomic and metabonomic profiles. Uromodulin was the most abundant protein in normal urine, whereas Fanconi urine was dominated by albumin. 1H-NMR spectroscopic data showed differences in the metabolic profiles of Fanconi urine vs. normal urine, due mainly to aminoaciduria. There were differences in the urinary metabolite and protein compositions between the three genetic forms of Fanconi syndrome: cluster analysis grouped the Lowe and Dent's urinary proteomes and metabonomes together, whereas ADIF and TP clustered together separately. Our findings demonstrate a distinctive "polypeptide and metabolite fingerprint" that can characterize the renal Fanconi syndrome; they also suggest that more subtle and cause-specific differences may exist between the different forms of Fanconi syndrome that might provide novel insights into the underlying mechanisms and cellular pathways affected.
- kidney
- urine
- mass spectrometry
there is increasing interest in applying "-omics" technologies to discover new biomarkers and to explore pathophysiological processes (39). There is also greater use of these methods in combination to obtain a multilayered description of diseases at different levels of biomarker organization (17, 46). In the present study, we have combined proteomic and metabonomic methods to try and obtain deeper insights into the molecular processes underlying the renal Fanconi syndrome (FS), a disorder that can exist in several forms and can arise from different genetic mutations.
Dent's disease and Lowe syndrome are X-linked genetic forms of the renal FS with impaired proximal tubular function. An autosomal dominant idiopathic form (ADIF) has also been described (5). The defect in the proximal tubule results in a failure to reabsorb a variety of filtered substances, particularly low-molecular-weight proteins (LMWP) and peptides, leading to their increased excretion in urine with other substances like glucose, uric acid, phosphate, and amino acids. Dent's disease typically presents with renal stones, nephrocalcinosis, and reduced renal function; it is due to mutations in CLCN5 on the X chromosome, which encodes the chloride channel ClC-5. This channel is expressed mainly in kidney proximal tubular cells, which can endocytose filtered polypeptides and proteins (13, 45, 47, 57). This endocytic process is receptor mediated (12, 19, 41) and involves the multiligand receptor proteins megalin and cubilin. The role of ClC-5, together with a V-ATPase proton pump, is thought to be to acidify the early endosomes, which is necessary to dissociate the receptor-ligand complex as part of the normal reabsorptive uptake of proteins and polypeptides from tubular fluid, as well as the regulated endocytosis of some plasma membrane transport proteins (2). Disturbed endosomal acidification in proximal tubular cells may underlie the impaired reabsorption and increased urinary losses of solutes and LMWP that occur in FS (32, 37). Lowe syndrome (also known as oculocerebrorenal syndrome) is a rarer X-linked multisystem disorder that is associated with mental retardation, lens cataracts, glaucoma, muscular hypotonia, growth defects, and renal failure (with LMWP); it is due to a mutation in OCRL1 (8), which encodes a phosphatidylinositol (PtdIns)-(4,5)-bisphosphate (P2) 5-phosphatase present in the trans-Golgi network and in early endosomes (48). This enzyme belongs to the a PIP2 5-phosphatase type II group and hydrolyzes PtdIns(4,5)P2 to PtdIns(4)P (60). Both its substrate and product are associated with clathrin-mediated trafficking: the former predominantly at the plasma membrane and the latter at the Golgi (11). PtdIns-(4,5)-bisphosphate (P2) 5-phosphatase is the major hydrolyzing enzyme in normal human kidney proximal tubule cells and is absent in cells grown from Lowe syndrome patients, in which PtdIns(4,5)P2 accumulates (43, 59). How loss of PtdIns-(4,5)-bisphosphate (P2) 5-phosphatase activity in Lowe syndrome causes the eye, brain, and kidney defects that occur is still unclear. As in Dent's disease, receptor-mediated endocytosis is affected, although a direct link between OCRL and defective endocytosis has not so far been demonstrated. The gene defect in ADIF, which is phenotypically very similar to Dent's disease, has not been defined so far, although it has been mapped to a large segment of chromosome 15 (33). If the final common pathway for these genetic forms of FS is receptor-mediated endocytosis, then their gene products should interact in some way and this might be reflected in their respective urinary proteomes and/or metabonomes, which was the reasoning behind this exploratory study.
Thus we describe a qualitative and quantitative comparison of the urinary proteomes and metabonomes of Lowe syndrome, Dent's disease, ADIF, tubular proteinuria (TP), and normal subjects (with normal renal function) (35). Two different comparative proteomic strategies using mass spectrometry (MS) were adopted to enhance the number and confidence of protein identification (31, 34, 58). Two-dimensional gel electrophoresis (2DE) (36, 49) was used first to compare Dent's, Lowe, and normal urinary protein patterns. The identities of separated proteins present in gel spots were determined by peptide mass fingerprinting using matrix-assisted laser desorption ionization (MALDI) time-of-flight MS (16). In a parallel investigation, the resulting peptide products of digested proteins (with an internal standard; IS) were then sequenced and quantified by online liquid chromatography tandem MS (LC-ESI-MS/MS) (21).
We also used 1H-NMR-based metabonomics, a well-established and highly reproducible analytic technique, which has been used successfully in the diagnosis and monitoring of various diseases and toxicities, and also for biomarker discovery (6). Metabolic profiling in this way can facilitate interpreting multiparametric responses in the complex metabolic profiles of biofluids to genetic or pathophysiological stimuli (40). Altered metabolite excretion in urine is also a consequence of genetic mutation in FS patients, and so 1H-NMR-based metabonomics may complement proteomic methods to increase the range of urinary compounds that can be analyzed.
The results of our metabonomic and proteomic studies were found to be self-consistent and indicated that the molecular composition of FS urine is very different from that of normal subjects, as previously reported (19). However, we also found differences in the protein and metabolite compositions between the different forms of FS studied, which suggest that different reabsorptive pathways and mechanisms are differentially affected and that this analytic approach may help in elucidating the underlying defect(s).
We studied eight male patients with X-linked Dent's disease, all with previously characterized mutations of CLCN-5; seven male patients and one female with Lowe syndrome, all characterized by mutations in OCRL; and eight normal subjects (41). In addition, three ADIF male patients and two male subjects with ifosfamide-induced TP, but without full-blown FS, were also studied. All patients were between 35 and 50 yr old.
Control urine designated as "normal" was collected from eight male subjects with no history of renal disease and aged between 40 and 50 yr. Patients and normal volunteers gave informed consent for the collection and analysis of their urine; 50 ml of early morning urine were collected, rapidly frozen in liquid nitrogen, and stored at −80°C until analyzed.
Urine samples were concentrated and separated from organic salts by solid-phase extraction (SPE) using a reverse-phase (RP) C18 POROS R2 20 (Applied Biosystem, Warrington, UK) as the stationary phase. The protein concentration of extracted proteins was determined using the standard Bradford assay (4). To determine both qualitative and quantitative differences, two different analytic proteomic strategies using MS were adopted (23, 42).
2D-gel separation of Lowe syndrome and Dent's disease urinary proteins followed by peptide mass fingerprinting.
Equal amounts (150 μg) of normal, Lowe, and Dent's urinary proteins were loaded on Immobiline DryStrip (pH = 3–10, 18 cm; Amersham GE Healthcare Life Sciences) and focused for 24 h. The second dimension was 12.5% of acrylamide gels, which were subsequently stained using Coomassie brilliant blue (CBB) G-250. Gel runs were in triplicate, and individual (that is, not pooled) urine samples were analyzed. Gels were scanned using a densitometer (Bio-Rad 800), and gel images were analyzed using Melanie software, which allows spot matching and quantitation. To compensate for image differences caused by variations in experimental conditions, spot intensity was expressed as % optical density (OD), defined as (1) where N is the total spot number. To assess the correlation between %OD values of two protein patterns, Student's t-test was used to determine whether the value of their Pearson correlation coefficient R was statistically different from 0 at P < 0.05. All the spots that could be visualized were excised from the gels, in-gel digested with trypsin (Sequencing Grade Modified Trypsin, Porcine, Promega, Madison, WI), and analyzed by MALDI-MS. MALDI mass spectra of 2DE spots were acquired using an Ultraflex time-of-flight (TOF)/TOF instrument (Bruker Daltonics, Bremen, Germany).
LC-ESI-MS/MS analysis of tryptic peptides of different forms of FS urine proteins.
Desalted proteins were also separated by 1D SDS-PAGE. Forty micrograms of extracted proteins from individual samples of each subtype of FS and control were pooled. Pooled samples were representative for each class. Gel lanes corresponding to total urinary proteomes were cut into five sections of approximately equal molecular weight range. Proteins present in these sections were in-gel digested using standard protocols (18). Peptides derived from trypsin digestion of normal, Dent's, Lowe, ADIF, and TP urine proteins were spiked with 2 pmol of a protein internal standard (IS; carbonic anhydrase, which was added to the gel pieces just before in-gel digestion), and separated by RP-HPLC. Peptides eluted from the C18 RP column were mass analyzed and sequenced online by a hybrid quadrupole-time of flight (Q-Tof) mass spectrometer (Micromass) equipped with a nanoelectrospray (nanoESI) ion source.
Ions were automatically selected for MS/MS by the automatic switching program in MassLynx 4.0 when their intensity in the MS survey scan was >25 ion counts/s. MS/MS spectra were smoothed (mean smooth at 2 channels twice) and centroided (at 80% top). Mascot used uninterpreted MS/MS data to interrogate the NCBI protein database restricted to mammalian entries. Allowed mass accuracies were 100 ppm and 150 millimass units for parent and fragment ions, respectively. Protein hits were accepted when they had statistically significant Mascot scores (P < 0.05) and at least two peptides matched the protein entry.
Quantification was derived by chromatographic peak integration as described previously (1, 10, 20, 25). We used an in-house computer program to extract peak areas and heights, which were taken as peptide ion intensity readings. Ion intensities were normalized for each analyte peptide ion to those of the IS; final values were normalized to the addition of the total peak heights for each particular gel fraction.
Hierarchical "cluster analysis" of quantitative LC-MS/MS data was performed using Cluster and Treeview (24) by complete linkage clustering and the Pearson correlation (uncentered) similarity metric method for both genes and arrays. Data were log2-converted before cluster analysis.
1H-NMR spectroscopy.
In contrast to proteomic analysis, preparation of biofluids for metabonomics is more straightforward, requiring only the addition of buffer to urine samples. Thawed samples were centrifuged at 13,000 g for 10 min; 200-μl aliquots were then added to 96-well plates containing 200 μl H2O and 200-μl solution of 0.2 M phosphate buffer (pH 7.4), 1 mM sodium trimethylsilyl [2,2,3,3-2H4]propionate (TSP), and 3 mM NaN3 in 20% D2O. Spectra from individual (not pooled) samples were acquired on a 600-MHz Bruker DRX spectrometer fitted with a 5-mm flow injection probe connected to a Bruker Efficient Sample Transferring (BEST) system (Bruker Biospin, Karlsruhe, Germany) employing a Gilson 215 liquid handler (Gilson, Middleton, WI). 1D 1H-NMR spectra were acquired with a pulse sequence of the form d1 − π/2x − t1 − π/2x − tm − π/2x, where π/2x represents a 90° hard pulse along the x-axis, d1 is a relaxation delay, t1 is a short delay (4 μs), and tm is the mixing time (100 ms). The resonance of H2O (approximately δ4.7) was selectively irradiated during d1 and tm. For each sample, 128 transients were collected into 32k data points. Before Fourier transformation, the free-induction decay (FID) was multiplied by an exponential line-broadening factor of 1 Hz. Spectra were phased in XWINNMR (Bruker) and referenced to the TSP resonance (δ0.00) using in-house software (MetaSpectra v3.0, O Cloarec, Imperial College London, London, UK).
1H-NMR spectral processing and multivariate analysis.
1H-NMR data acquired from complex biofluids like urine (and especially from human blood plasma) are information rich. Each metabolite gives rise to several peaks in the spectra, with each peak varying in multiplicity (singlets, doublets, triplets, etc.), and some peaks overlapping with neighboring peaks. Analysis of such complex data sets is made possible by the application of unbiased statistical (chemometric) techniques, such as principal components analysis (PCA) and orthogonal projections to latent structures by partial least squares discriminant analysis (O-PLS-DA). An NMR spectrum is a plot of intensities (y-axis) over frequency (x-axis), with each molecule giving rise to one or more peaks with a specific frequency and intensity that are directly proportional to their respective concentrations in the sample. For chemometric analysis, we consider each frequency coordinate (each point on the x-axis) as a variable having a value defined by the intensity at that point. In general, it is a feature of NMR spectroscopy that from one spectrum to the next a given metabolite will have peaks at the same frequency, although there is still minor peak position variation for some metabolites, because of differences in salt content and/or pH from one sample to the next.
Before chemometric analysis, spectral data regions corresponding to residual water (δ4.68 − δ4.93) and urea (δ5.53 − δ6.16) were removed to eliminate variations associated with presaturation. The data were then normalized such that the total integral of the remainder of each spectrum was a constant. PCA and O-PLS-DA (14, 50) models were constructed using MetaSpectra. Statistical TOtal Correlation SpectroscopY (STOCSY) was performed on the data set to detect statistical correlations between resonances within the spectra (15) and to facilitate assignment. Visualizing these correlated resonances was facilitated by back projecting the unit variance as a colored heat map on the O-PLS coefficient plot.
Database searches.
Raw data from mass spectra were submitted to Mascot, a software search engine designed for high-throughput and automated interpretation of mass spectrometric data derived from polypeptides (44). This software was used to interrogate the NCBI database, as described in Supplemental Information S1 (all supplementary material is available in the online version of this article on the AJP-Renal Physiology web site).
Analysis of urinary polypeptides of Dent's disease and Lowe syndrome by 2D gel electrophoresis.
We compared the protein patterns present in the urine of Lowe and Dent's patients with normal individuals by 2DE. Equal amounts (150 μg) of normal, Lowe, and Dent's urinary proteins were loaded on IPG strips. After electrophoretic separation and CBB G-250 staining, all visualized spots were excised from each of the three gels displayed in Fig. 1, and the identities of these spots were determined by MALDI-MS. Labeled spots shown in Fig. 1 correspond to those proteins identified that returned significant "hits," as listed in Table 1. Identification details and spectra are described in more detail in Supplemental Information S2. Most of the proteins identified are of plasma origin and are normally reabsorbed by receptor-mediated endocytosis in the proximal tubule.
Several proteins, such as complement factor D, zinc-α-2-glycoprotein, apolipoprotein IV, vitamin and prosthetic group carriers like vitamin D binding protein, retinol binding protein, transferrin hemopexin, and growth factors [such as pigment epithelium-derived factor], were found in significantly higher amounts in Lowe and Dent's urine; they represented a larger proportion of the urinary proteome compared with normal urine. In contrast, uromodulin and kininogen, proteins of renal origin that are not involved in receptor-mediated endocytosis, were reduced or absent in FS urine. The findings in Dent's urine confirm our earlier reported observations (19).
Just on visual inspection of the 2D gels, it is apparent that the proteomic patterns of the Dent's and Lowe FS urine are very similar (Fig. 1). The actual values of the gel spots' %ODs (of the proteins shown in bold and in the same order as listed in Table 1) for Dent's vs. Lowe correlate significantly (P < 0.05) (see Fig. 2B) in contrast to their correlation with normal urine (P > 0.05) (see Fig. 2, A and C).
Analysis of urinary polypeptides in four different forms of FS by LC-ESI-MS/MS.
To determine whether other forms of the FS also have similar patterns of urinary proteins, we used a label-free quantitative proteomics approach (peak area with internal standard) that allows exhaustive quantitative cross-comparison of an unlimited number of proteomes (20). This strategy (Fig. 3A) involves separating pooled protein mixtures in parallel by 1D-SDS-PAGE. After staining, gel lanes were cut into five sections (fractions) of equal molecular weight, regardless of stain intensity. Proteins present in these gel sections were digested using trypsin, and the peptides produced were sequenced and quantified by LC-MS/MS. To illustrate how this analysis was done, see Fig. 3B, which illustrates some examples of the peptides present in fraction 4 derived from hemopexin, uromodulin, and carbonic anhydrase (which is the IS added to gel pieces before in-gel digestion). Peptides were selected for MS/MS and quantified as described in materials and methods. Figure 3B shows that hemopexin was more abundant in FS urine, whereas uromodulin was more abundant in control urine. Nine peptide ions of the IS were identified. By normalizing all protein-derived peptides to the IS peptide ions, it was possible to use the signal intensities in LC-MS to derive quantitative information from MS across samples (20). Figure 3C shows some examples of proteins that were quantified in this way in fraction 4. Quantification details of proteins identified are listed in Supplementary Table A.
Comparative analysis of three forms of the FS, two cases of TP, and control urinary proteomes confirmed our previous findings (19, 21, 22) and the data presented before (Figs. 1 and 2), indicating that the proteomes in FS are quantitatively and qualitatively different from control. A good example is shown in Fig. 4, in which uromodulin is seen to be the most abundant protein in control urine, whereas it is albumin in FS urine. Other quantitative differences between control and FS urine are apparent in Fig. 4.
The ratios of the amounts of FS urinary proteins relative to the same protein in normal urine were similar among the different forms of the FS, with no obvious differences in the overall patterns by visual inspection (Fig. 4). However, an unbiased and unsupervised (i.e., assumes no a priori knowledge about the origin of the sample) analysis of the data (using the "clustering" tools designed for analysis of gene microarray data) (see above and legend to Fig. 5) clustered Lowe and Dent's urinary proteomes together, whereas ADIF and TP were clustered separately and together (Fig. 5). All FS proteomes clustered separately from control. These results suggest (and confirm) that the composition of the Lowe and Dent's proteomes is very similar, yet subtly different from ADIF and TP urinary proteomes, which are more similar to each other.
NMR analysis of FS and control urine.
1H-NMR analysis of FS and control urine by NMR spectroscopy can detect and analyze the chemical fraction in urine that cannot be detected by standard MS -based proteomics. To complement the proteomics data, we looked for differences among the metabolite compositions in the different forms of FS and made comparisons with control. Nineteen urine samples in total were analyzed by 1H-NMR spectroscopy. The samples were obtained from four patients with Dent's disease, five with Lowe syndrome, three with ADIF, two with isolated TP, and five normal subjects. A representative 1H-NMR spectrum from each of the five subclasses is shown in Fig. 6. The analysis of such complicated spectra is greatly facilitated by chemometric methods, such as PCA and O-PLS-DA (51). All spectra were initially subject to PCA, an unsupervised pattern recognition method that considers each data point in the frequency dimension as an independent variable. PCA presents an overview of the data that can reveal grouping of observations, trends, and outliers in a data set. A PCA model was constructed on 18 samples from this data set (one was removed due to contamination). Figure 7A shows that normal subjects were clearly separated from FS patients. There was also clustering of the metabolic profiles of urine from patients with Dent's disease and Lowe syndrome and patients with TP and ADIF. Within the first three PCs (comprising 69.8% of the total variance in the dataset), all five subclasses were mutually exclusive. To eliminate the possibility that paracetamol metabolites were influencing the separation, PCA models were also constructed for restricted regions of the spectra known predominantly to contain resonances from amino acids (i.e., δ0.5 to δ2.0). Again, control subjects were clearly separated from FS patients and, within FS subtypes, Lowe and Dent's patients separated from ADIF and TP (data not shown).
Further chemometric analysis was carried out on the 1H-NMR spectra using O-PLS-DA (50). This is a supervised statistical approach that takes into account the class of sample being analyzed. Constructing O-PLS-DA models from full-resolution urinary 1H-NMR spectra allows identification of the metabolites most significant in discriminating between two or more classes of samples to a level of accuracy unobtainable with bucketed spectra (29). Figure 7, B–E, presents O-PLS coefficient plots for four pairwise models from subclasses or groups of subclasses. These plots have been color-coded to display the relative significance of each variable to the discrimination, with red-orange colors being most significant, and blue-green colors being least significant. The sign and intensity of each variable indicate its covariance with the respective sample class. In Fig. 7B, data from Dent's and Lowe patients were grouped together and compared with controls. The O-PLS coefficient plot identified several resonances significant in discriminating the two classes, such as a doublet at δ1.48, and multiplets at δ1.71 and δ1.91, in Dent's and Lowe patients, and singlets at δ2.34 and δ4.44. To facilitate assignment of these, STOCSY analysis (15) was performed on the maxima of each group of highly significant data points (corresponding to the apex of an NMR signal). This allowed identification of several potential biomarkers for each FS subtype. Controls exhibited higher levels of N-methylnicotinic acid and a molecule with a spectral pattern consisting of a singlet at δ2.35 and an apparent doublet of doublets centered at δ7.25, the latter being p-cresol sulfate. Dent's and Lowe patients had higher levels of the neutral amino acids alanine and glycine and the basic amino acid lysine. Urinary profiles of Dent's and Lowe patients were directly compared to identify specific differences between these two types of FS. Figure 7C shows the coefficient plot from the O-PLS-DA model, highlighting resonances more significant in the discrimination. Assignment tables combined with STOCSY revealed these to be hippurate and creatinine in Dent's disease, while relatively higher concentrations of alanine, glycine, lysine, and lactate were more likely to be found in urine from patients with Lowe syndrome. However, it should be noted that the cross-validation parameter Q2 is low (0.11), limiting its interpretability, attributable to the small sample size and the relatively small difference between these two disease classes.
An O-PLS-DA model was also constructed to compare urine from patients with ADIF with controls. Figure 7D shows the O-PLS coefficient plot for this model, showing many highly significant (red) positive regions. STOCSY analysis and assignment tables showed these belong mainly to glucose, along with the neutral branched-chain amino acids valine and leucine compared with controls. Visual inspection of the data also showed ADIF patients had considerably lower levels of citrate in their urine compared with controls; but this was not detected by O-PLS analysis due to the significant peak-positional variation in the citrate resonances. Finally, ADIF patients were directly compared with the grouped Dent's and Lowe patients' urine. The O-PLS coefficient plot, shown in Fig. 7E, showed specific and significant differences in the metabolic profiles: urine spectra from ADIF patients were significantly higher in glucose, valine and, to a lesser extent, leucine, while lysine was more significant in discriminating Dent's and Lowe patients as a group.
Applying -omics science to physiology and clinical medicine faces several practical problems, some of which are still technical, but a critical one (similar to that in molecular genetics) is how well defined or characterized ("phenotyped") are the animal models or patient groups under study. Only if robust subclasses are defined can valid comparisons be made between normal, disease and disease variants, and potential biomarkers identified. Moreover, data analysis strategies are also needed that emphasize differences in patterns, rather than in discrete measurements of single variables, as the greater the number of parameters defining a disease, the greater the reliability of any diagnostic test. Therefore, the aims of this study were twofold: 1) to provide and validate an innovative and more comprehensive analysis and characterization of urine composition by combining proteomic and metabonomic techniques; and 2) to test the hypothesis that in defined FS, urine profiling in this way might provide mechanistic insights, or clues, as to the underlying defects and pathophysiology, as well as a means to refine the different FS phenotypes.
Several qualitative and quantitative differences in the proteomic and metabonomic profiles of the different genetic forms of FS patients compared with normal subjects were found using MS and NMR. A preliminary study was conducted on Dent's disease and Lowe syndrome urinary proteins with two different proteomic methods, 2DE and LC-MS/MS. This was done because the physiochemical heterogeneity of proteins (which is the basis of their diverse biological functions) makes a single analytic approach inadequate for a comprehensive analysis of all proteins in a given sample (28). As an alternative to 2DE, we used liquid chromatography for protein or proteolytic product separation of FS and normal urine. With this strategy, polypeptide detection is less discriminatory of the physicochemical properties of the analyte. For example, proteins such as megalin, apolipoprotein A-II, apolipoprotein C-III, β-2-microglobulin precursor, cystatin C, cystatin M precursor, immunoglobulins, insulin-like growth factor binding protein 6, pro-epidermal growth factor precursor, and osteopontin were not detected by 2DE.
Our results show that the urine protein patterns of patients with Dent's disease and Lowe syndrome are very similar. We found that the plasma proteins normally reabsorbed from the glomerular filtrate along the first part of the proximal tubule by receptor-mediated endocytosis represent a larger proportion of the FS urinary proteome compared with normal urine. These results indicate that tubular reabsorption of plasma-derived proteins is similarly reduced in Dent's disease and Lowe syndrome. In contrast to normal urine, proteins of renal origin are decreased in the urine of patients with these forms of FS. Thus the FS urinary proteome is dominated by proteins of plasma origin, and proteins derived from the kidney itself (and are present in normal urine) make up a smaller proportion of the FS urinary proteome. These findings confirm our original observations in Dent's disease only (19) and indicate that in this respect Lowe syndrome and Dent's disease are indistinguishable.
1H-NMR offers a robust and information-rich analytic approach that can complement proteomic data (38). By combining this highly reproducible form of spectroscopy with the chemometric methods of PCA and O-PLS-DA, a more general comparison of the different sources of FS urine and normal urine can be made, and potential biomarkers for each type of FS identified. Consistent with the proteomic findings, the metabonomic analysis showed that the metabolic profiles of urine from Dent's and Lowe patients were very similar and quite different from normal urine. The differences were due mainly to increased amounts of the amino acids lysine (basic) and alanine (neutral) in the urine of FS patients, and decreased amounts of p-cresol sulfate and N-methyl nicotinic acid. This is in keeping with early descriptions of Lowe syndrome, in which basic and neutral (but not branch-chained) aminoaciduria was a recognized feature (9). The decrease in N-methyl nicotinic acid excretion suggests cation transporter dysfunction in the proximal tubule (7, 52), while the decreased p-cresol sulfate, a clostridial metabolite, could be due to altered metabolism by gut microflora (56). It is perhaps relevant here that some forms of FS are associated with different effects on amino acid transport in the kidney and intestine (3), which may influence gut microflora metabolism. Indeed, ClC-5 has been detected in the rat intestine (53), but its function there, or its presence in humans, is unknown. Alternatively, it may be that altered renal function in FS patients results in inefficient clearing of p-cresol sulfate, a well-known uremic toxin (54, 55). However, whether this is a nonspecific reflection of impaired renal function or is more specific to tubular dysfunction in FS is unclear at present.
In both Dent's disease and Lowe syndrome, urinary megalin, the receptor thought to be largely responsible for protein and polypeptide reabsorption in the proximal tubule, is reduced or absent in urine (41), which is again in keeping with our finding of almost identical urinary proteomes and metabonomes, and is thus a common underlying defect in receptor-mediated endocytosis. The products of the gene defects in Dent's disease and Lowe syndrome could be closely involved in the same reabsorptive pathway, and one that may be common to other forms of FS. To explore this concept, we analyzed the urine composition of a phenotypically similar autosomal dominant idiopathic form of FS (ADIF) and compared it with Dent's disease, Lowe syndrome, ifosfamide-induced TP, and normal subjects.
ADIF is another form of inherited FS, clinically similar to Dent's disease (5), but its underlying gene defect is autosomal and still unknown (33). Although proteomic analysis, as for Dent's disease and Lowe syndrome, confirmed that the ADIF urinary proteome is different from control and broadly similar to Dent's and Lowe proteomes (with no clearly distinct proteins or peptides), the urinary metabonome of ADIF showed several qualitative differences in composition, with glucose and valine (neutral) identified as potentially distinguishing biomarkers for this form of FS (Fig. 7D). By cluster analysis the ADIF and TP urinary proteomes appeared to be quite similar, yet different from Dent's and Lowe proteomes (Fig. 5). An earlier 1H-NMR study of urine from patients treated with ifosfamide reported glycosuria and amino aciduria, with concomitant decreases in hippurate and citrate (26, 27). We observed similar metabolite excretion patterns for ifosfamide-induced TP, except there was no apparent increase in histidine in the urine of our patients. In grouping ADIF and TP together, and Dent's and Lowe together, both proteomic and metabonomic analyses were consistent. The discriminators in ADIF were glucose and valine, while in Dent's and Lowe syndrome it was lysine.
Although no specific mechanism(s) has been be identified by this combined analysis, and our interpretation is both limited and somewhat speculative, taking the proteomic and metabonomic data together, the results do suggest that the molecular defects and pathways affected in ADIF may be different from those shared by Dent's disease and Lowe syndrome, and more similar to those occurring in ifosfamide-induced TP. Moreover, our results do confirm our original proteomic findings in Dent's disease and show that the urinary protein and metabolite patterns in Dent's disease and Lowe syndrome are almost identical, qualitatively and quantitatively, and it is likely that their respective gene products are closely involved in the same proximal tubular reabsorptive pathways. Indeed, the recent and confirmed report that a significant number of adult patients with a Dent's-like (predominantly renal) phenotype had mutations of OCRL1, and not CLCN5 (30), is consistent with our findings and this interpretation.
Finally, in this small study we have demonstrated how urinary proteomics and metabonomics can be combined effectively to provide a more complete analysis of urine and that these analytic methods are consonant. By extending urinary-omics in this way (and with the development of high-throughput sampling methods), especially in well-defined and characterized renal disorders, or by comparing clinically similar disorders (as in FS), potential biomarkers and novel mechanistic insights may be revealed.
A. Vilasi, P. R. Cutillas, and R. J. Unwin thank the St. Peter's Trust for Kidney, Bladder, and Prostate Research for financial support. A. D. Maher and S. F. M. Zirah received financial support from the European Union FP6 MolPAGE project.
↵* A. Vilasi, P. R. Cutillas, and A. D. Maher contributed equally to this work.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society


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But in 2007, Marion was stripped of those medals because of her admission to using performance-enhancing drugs. As a result of her confession, Marion was sentenced and served six months in federal prison for lying to federal investigators. Marion voluntarily returned her five Olympic medals and publicly apologized to her family, friends, and fans all over the world.
As a result, Marion has now committed her life to inspiring others who face life-challenging situations. As a public speaker and soon-to-be published author, Marion candidly, articulately and passionately shares her life story, how she came to grips with her lies and the consequences of her actions, and how she found meaning in all of it. She discusses real issues that we all face: learning to grow through pain, making decisions that will help us far into the future, overcoming failure and discouragement, and applying practical principles that point the way to personal and spiritual breakthrough.
Marion just successfully completed her rookie year in the WNBA. She resides in Austin, Texas with her husband Obadele, and their three children: Monty, seven, Amir, three, and one-year old Eva-Marie.
Susie Jarosch
High Five Agency, www.highfiveagency.com
(206) 852-8524

subset: commoncrawl


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Recorded in Dresden in 2008, Nikolaus Lehnhoff's production of Rigoletto is a ferocious, if uneven affair, that awkwardly positions the opera between religious drama and psychopathology. Lehnhoff roots the tragedy in the dysfunctional relationship between Rigoletto (Zeljko Lucic) and Gilda (Diana Damrau), rather than in the jester's developing conflict with the Duke (Juan Diego Flórez). Yet he also takes on board a comment in the text that this world is hell: the Duke's cronies don demonic disguises to carry out Gilda's abduction, while her final act of self-sacrifice, played out against a Michelangelesque backdrop of the day of judgment, acquires ambivalently redemptive, Christ-like overtones. Some of this strains against the score, since Damrau and Lucic have to pitch their characterisations as more neurotic – therefore less sympathetic – than the music suggests. But vocally they've rarely been bettered, while Flórez, cast against his squeaky-clean image, is insidiously sensual as the Duke. There's powerhouse conducting from Fabio Luisi, too.
Damrau/Flórez/Lucic/Dresden Strate Opera Chorus/Dresden Staatskapelle/Luisi
(Virgin Classics)
(Virgin Classics)


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The Michigan Supreme Court has appointed Stephen P. Carras as chief judge of the 42nd Circuit Court (Midland), the 75th District Court, and the Midland County Probate Court for a term ending December 31, 2013. The appointment is effective immediately. ...Read More »
Yearly Archives: 2013
Real Property – No ownership interest defeats principal residence tax exemption
Petitioner's lease and license arrangements with the corporate owner of real property did not give petitioner an ownership interest upon which to claim a principal residence tax exemption. There is no dispute that petitioner occupied a residence on the property ...Read More »
Professional Responsibility – Court could hear legal malpractice case involving patent claim
The federal courts do not have exclusive jurisdiction to hear a Michigan legal malpractice claim where the underlying case arises from whether defendant law firm properly advised plaintiffs in a patent infringement case. The trial court erred by dismissing the ...Read More »
Probate – Personal representative's reimbursement disapproved
The probate court correctly disapproved a personal representative's self-payment from the decedent's estate for care-giving expenses incurred as the decedent's conservator while he was still alive. The personal representative's claim against the estate could not be satisfied without "a formal ...Read More »
Probate – Emergency appointment of special personal representative denied
The imminent expiration of a negligence action's statute of limitations was not an emergency that would justify appointing a special personal representative and opening an estate for the deceased alleged tortfeasor without providing proper notice to the interested parties. However, ...Read More »
Family Law – Custodial environment finding upheld in custody change case
The trial court correctly determined, after awarding defendant temporary physical custody of the parties' children, that subsequent circumstances supported a finding that neither parent had an established custodial environment. The court's award of joint physical custody and school-year parenting time ...Read More »
Employment – No contract with employer for paid comp time
The trial court correctly dismissed plaintiff's claim against defendant employer for unpaid comp time. There was no evidence defendant contractually agreed to pay comp time. Defendant supplied contract workers, including plaintiff, to Ford Motor Co. Ford was responsible for setting ...Read More »
Justice to speak at law school orientation
Newly appointed Michigan Supreme Court Justice David Viviano will be the keynote speaker at The Thomas M. Cooley Law School's "Professional in Action Orientation" on April 30. Viviano will be speaking on ethics and professionalism in the legal profession. The ...Read More »
'Take control' of your lawyer profile and 'guard it carefully'
DETROIT – Attorneys need to "take control" of their Martindale Hubbell ratings – and make sure the listing accurately reflects who you are as a practitioner. "Develop an image and guard it carefully," advised Farmington Hills attorney Julie Fershtman, who ...Read More »
Dispute over where decedent lived at time of crash settles
A case turning on whether a military man was a "resident" of his stepfather's home has settled for the policy limits of $300,000. The man, who driving when the wreck occurred, was killed and his friend was injured. The Michigan ...
Tagged with: Judge Paul Hamre Michigan Court of Appeals No-Fault Law Van Buren County Circuit CourtRead More »


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Save on 3-day or 5-day juice cleanses Up to 30% off at Z.E.N. Foods
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Why You'll Love It: When your body's dragging and you just feel "off", Z.E.N.'s Rejuvenate Cleanse will start turning things around. Perfect for beginners, it's designed for those who desire a gentler cleanse. Filled with healthy, detox-promoting greens and fruits, and protein in liquid form (to fill you up), you'll feel lighter and clearer in just a few days.
How You'll Detox: Each day, you'll drink six pure pressed juices with names like Glow, Bliss, and Serenity, containing fresh ingredients that promote detoxification. There's fennel for its anti-inflammatory properties, carrots to enhance your complexion and strengthen your immune system, cayenne and cinnamon to boost your metabolism, and beets to oxygenate your system and fight the aging process.
Fine Print
- Only 1 per customer, but buy 2 for your friends.
- Delivery is included to: Los Angeles County, Orange County and parts of Ventura County.
- A $13-per-day delivery fee applies to Santa Barbara County, San Diego County and parts of Ventura County.
- Please call to confirm delivery availability.
- Cannot be used in conjunction with other deals.
About Z.E.N. Foods
Redemption Instructions:
- Please call (310) 205-9368 or (818) 784-9368 to place order and provide voucher number at time of order.
- Voucher does not need to be presented at time of delivery.
- A valid ID may be required upon redemption.


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The female cast of Dancing With The Stars posed for glamour magazine in a feature editorial on body image, an issue increasingly more women struggle with.
Dressed in black and white bikinis, the women of Dancing With The Stars posed under direction by Glamour's Suzanne Donaldson.
It's a great topic to be covered, although I'm not really sure what purpose the photos serve. The photos flaunt beautiful imagery of the bodies from The Dancing With The Stars girls, all of which are in impeccably enviable shape. Perhaps we need the Glamour article to put it into context.
Photo Shoots For Body Image
More Stats +/-
30 Views on Body Image
Autonomous Bao Restaurants
Liquid-Vitalizing Water Bottles
Hybrid Bakery-Restaurants
Repurposed Protein Cups


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Dry Ridge Presbyterian Church, 15 Warsaw Avenue, will host a community worship service at 7 p.m. Thursday, April 5.
Crittenden Christian Church and Dry Ridge Christian Church members will also be a part of this service.
"Because of the strong community support of a Christmas Candlelight Service based on scripture and song, it was decided to have a Tenebrae Service, which includes the passion narrative from the gospels of Matthew, Mark, Luke and John amid decreasing candlelight," said Julie Donahue, music director at Dry Ridge Presbyterian Church.
Cherry Grove Pentecostal Church of God
• April 8 - Easter program given by the youth. Easter evening services will be at 6 p.m.
Clarks Creek Baptist
• April 7 - Easter Egg Hunt will be at 2 p.m. at the church.
• April 8 - Sunrise service at 8 a.m. with breakfast after the service, prepared by the men. The Creative Ministry Team will be performing during worship service. Feel free to come worship with us.
Corinth Christian
I like the Book of James. He doesn't beat around the bush. Like in Chapter 3, when James is teaching us about "a small member" of our body that is "a restless evil, full of deadly poison." He is referring to our tongue, of course.
We are all guilty. I know I can hardly get through a day without saying a harsh word to or about someone. And have you ever just stopped to listen to the conversations around us – among friends and neighbors … and yes -- church members. It seems James knew us well.
Cherry Grove Pentecostal Church of God
• April 8 - Easter program given by the youth. Easter evening at 6 p.m.
Clarks Creek Baptist
• April 7 - Easter Egg Hunt will be at 2 p.m. at the church.
• April 8 - Sunrise service at 8 a.m. with breakfast after the service, prepared by the men. The Creative Ministry Team will be performing during worship service. If you don't have a home church, feel free to come worship At Clark's Creek Baptist.
Daniel Wilson found his calling when he was 6-years-old.
"I was saved at an young age," he said. "I just knew at that moment I was going to be a pastor."
Wilson, 30, has been the minister at Dry Ridge Baptist Church for four months, after serving in the evangelism office of the Alabama Baptist State Convention (ABSC). He has been a Southern Baptist pastor for 10 years and has preached at churches in Alabama and Kentucky. He replaced Joseph Kitchens, who moved to preach in Alabama.
Quite possibly, the four weeks of the NCAA tournament are the most anticipated four weeks of the year for Kentucky fans. We cheer and yell; spend inordinate amounts of money to buy tickets, shirts, hats, and jerseys.
We are some of the best fans in the world and as Coach Cal says, "You fans are crazy!"
1100 N. Main Street in Williamstown
Senior staff: Earl and Rebecca Hampton
Number of members: 100
Service times: 10:30 Sundays (also community groups and classes weekly.)
Christ Community
• Rhonda used Romans 8:26-27 for the message. In one's prayer life, a person needs to take time to be quiet before God and yield to the work of the Holy Spirit.
• Sunday evening service is back on schedule.
• Tuesdays – Lunch, Bible study and prayer with the women at 12:30 p.m. For more information, call 859-394-2586.
• Wednesdays evenings - Children's program and adult Bible study.
Easter is one of the best times of the year. Spring is just around the corner. You can buy "Peeps" at the store and there are always eggs to color. There is something about this holiday that propels us into good spirits. Maybe it's the warm weather, baseball season beginning (Go Reds) or the hope of summer coming.
I believe there is a bigger reason for the hope that spring brings to us. This hope is found is something much more profound. The hope of Easter comes in the reality of why we celebrate with so much anticipation.
Copyright © 2017
The Grant County News is your source for local news, sports, events, and information in Dry Ridge, KY and the surrounding area.


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JOHANNESBURG, South Africa — The Lonmin platinum mine where police shot and killed 34 striking miners has resumed operations, a day after the company said workers must return to their jobs or face being fired.
A statement from the company said that one-third of the Marikana mine's 28,000 workers had reported for work Monday morning. A deadline for striking workers to return to work or face dismissal was extended by a day, to Tuesday.
"Lonmin can confirm that work at its Marikana operations resumed today as significant numbers of employees returned to work," the statement said.
"The company can also announce that those illegal strikers who did not return to work this morning will not be dismissed and have been allowed an extra day in light of current circumstances."
President Jacob Zuma on Sunday declared a week of national morning for those killed at the mine.
More from GlobalPost: South Africa's perfect storm
According to police, 34 people were killed and 78 injured when police officers opened fire on striking workers near the mine, located in South Africa's North West province.
Some of the 250 people arrested on charges related to the violence appeared in court today.
The Marikana mine has been the site of a violent turf war between rival unions. In the week ahead of the shooting, 10 people had died in clashes at the mine.
South Africa is the world's biggest platinum producer, accounting for three-quarters of the world's known supplies of the precious metal.
More from GlobalPost: Jacob Zuma orders inquiry into South Africa mine shootings


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Steve Carell has been cast in the lead role in a new, Disney live-action, family adventure-comedy.
The Office star will also produce the upcoming project, titled Brooklyn Family Robinson, according to The Hollywood Reporter.
Finding Nemo co-writer David Reynolds is penning the script, which centres on a father (Carell) who takes his family away from the bustling city life of New York to start anew on a tropical island.
Carell will next appear on the big screen in Foxcatcher.


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Pulse in view
Steel Pulse. Back Row: Jerry Johnson, C-Sharp, David Hinds, Keysha McTaggart, Selwyn Brown, Donovan McKitty. Front: Sydney Mills, Amlak TafariGroup photos of reggae legends Steel Pulse are pretty rare, so when they do surface, its worth recording them for posterity. This one was taken just a couple of days ago during their current tour of the United States. It's not exactly a professional shoot but it's as good as it gets. The band will be heading for a series of gigs in Brazil in May. You can find out a lot more about the group here.
Labels: Steel Pulse


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ISRN Immunology
Volume 2013 (2013), Article ID 453561, 5 pages
Circulating CCL5 Levels in Patients with Breast Cancer: Is There a Correlation with Lymph Node Metastasis?
1Multidisciplinary Breast Center, KU Leuven, University Hospitals, Herestraat 49, 3000 Leuven, Belgium
2Department of Oncology, KU Leuven, Surgical Oncology, University Hospitals, Herestraat 49, 3000 Leuven, Belgium
3Laboratory of Experimental Oncology (LEO), Department of Oncology, KU Leuven and Department of General Medical Oncology, University Hospitals Leuven, Leuven Cancer Institute, Herestraat 49, 3000 Leuven, Belgium
4Interuniversity Institute for Biostatistics and Statistical Bioinformatics, KU Leuven, Kapucijnenvoer 35, 3000 Leuven, Belgium
5Department of Pathology, KU Leuven, University Hospitals, Minderbroederstraat 12, 3000 Leuven, Belgium
Received 26 March 2013; Accepted 8 May 2013
Academic Editors: S. Seki, A. Taylor-Robinson, and A. Vicente
Copyright © 2013 Ann Smeets et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Citations to this Article [2 citations]
The following is the list of published articles that have cited the current article.
- Tanusree Paul, Christian Schumann, Stefan Rüdiger, Stefan Boeck, Volker Heinemann, Volker Kächele, Michael Steffens, Catharina Scholl, Vivien Hichert, Thomas Seufferlein, and Julia Carolin Stingl, "Cytokine regulation by epidermal growth factor receptor inhibitors and epidermal growth factor receptor inhibitor associated skin toxicity in cancer patients," European Journal of Cancer, vol. 50, no. 11, pp. 1855–1863, 2014. View at Publisher · View at Google Scholar
- Alfonso Colombatti, "The Inflammatory Chemokine CCL5 and Cancer Progression," Mediators of Inflammation, 2014. View at Publisher · View at Google Scholar


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Standard course
(6 years) A100
In years 1-3 (pre-clinical), students gain a comprehensive grounding in the sciences underlying medicine and obtain a BA degree in Medical Sciences.
Years 4-6 comprise the clinical component and lead to the award of the BM, BCh.
Images courtesy of Shutterstock
Graduate-entry course
(4 years) A101
An accelerated four-year medical course for graduates in applied or experimental sciences
Clinical attachments of up to 10 weeks for medical students from other universities in the UK and overseas in their final two years of study
Highest student satisfaction rate for all UK medical courses in the National Student Survey 2013 and 2014
Best University for medicine in the Times Higher Education World University Rankings 2014-15


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Hip Appeal: Say Goodbye to the Fanny Pack Forever
"Workout Clothes You'll Want to Wear" at Lifescript.com For every fashionista, there are a variety of colors to choose from to match every wardrobe and each item has three pockets: one front zippered pocket, one rear zippered pocket and one interior front-seam pocket. The sizes range from an XS through an XL, which is measured in waist size. The XS starts at a size O and the XL goes up to size 28. A test run Lifescript took the Hip Appeal for a hike up Stone Mountain in Georgia for a test run in hot, humid weather. Stored in the Hip Appeal were a smartphone, a small sunscreen roll-on and lip balm. For the two-hour trail up and down the mountain, the Hip Appeal safely stored those items without any major jostling. Even though it was a hot day, the additional layer of fabric didn't add to the sweat factor too much. It was helpful to have easy access to the camera on my smartphone to take fun photos at the top of the peak. It's a great way to have an outdoor workout that's hands-free without worrying about dropping your expensive technology items or losing your keys. The Hip Appeal did a terrific job of marrying function and fashion. With affordable prices ranging from $25.99-$27.99, it's the perfect item for the fashionable athlete in your household. For more information, check out Hip Appeal.When it comes to workout apparel, fitness buffs want as smooth a line as possible in their yoga pants or shorts. That often doesn't leave room for pockets to store items like smartphones, lip balm and valuables. The designers at Hip Appeal heard everyone's cries and they came up with a chic and flattering alternative to a bulky fanny pack called Hip Appeal. The product is made of a Micro-Poly/Lycra blend of fabrics so it fits snugly around your hips. Read
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A firefighter working for Chief W. Keith Brower Jr. was severely burned and forced to retire.
"Newer homes, because of the amount of synthetics that are used and because of the diminished construction, are actually less safe than an older home built with dimension construction. That's why residential sprinklers have value. They may not always put the fire out, but they can keep the interior of the building from going to flash over, allowing firefighters to escape without getting burned or worse."
Name: Chief W. Keith Brower, Jr.
Date of fire: May 25, 2008
Location: Loudoun County, Virginia
Injury: One of his firefighters was severely burned and forced to retire.
Download Chief Brower's Faces of Fire story (PDF)
Download this video of Chief Brower.
Chief Brower and his firefighters put their lives on the line every day. It's an occupational risk. But when he heard the mayday call as he was approaching a fire one beautiful May weekend in 2008, it felt as if his heart had stopped. The fear that is sparked when a member of his crew is in danger never becomes routine.
"An integral part of my job is to do all that I can to train the members of my department," Brower says. "I feel a sense of personal responsibility to get them home safely to their families and loved ones."
As his firefighters entered the burning house, a fireball erupted on the first floor, trapping them upstairs. The flashover was so intense, their hose line burned in two.
"When the mayday was called, I was approximately seven to eight miles out, and my heart stopped," Brower says. "It literally froze me. It was chilling."
Fortunately, the four firefighters escaped the burning home, but one sustained serious burns and his injuries forced him to retire.
"When I see this firefighter, I don't know what to say," Brower says, "I really feel awkward saying" Hey, how's it going?" because I know how it's going. He's partly incapacitated. He can't do the job he loves."
Part of the Washington, D.C., metropolitan area, Loudoun County, Va., where Chief Brower is stationed, is one of the fastest growing, wealthiest counties in the nation. With population growth of nearly 80 percent in the last decade, prosperous new housing developments are everywhere. While fire standards have improved in home construction, new risks have emerged.
Chief Brower says that installing residential fire sprinklers is especially important in newer homes because modern construction techniques rely on synthetics that burn more quickly, making them more prone to collapse
"We used to have an expectation that we could fight fire in a building for eight hours or so. In other words, the building would burn down before it would fall down," Brower says.
Visit the Faces of Fire page for more information.
Use these fill-in-the-blank tools to help convince decision-makers and members of your community about the life-saving potential of home fire sprinklers.
© Copyright 2016 NFPA. All rights reserved.


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The eagle-eyed amongst you may have spotted the hidden link in our 'You ask, we answer' blog post from
19 July, which took you to an interview J.K. Rowling gave in 2000.
In that interview was some interesting information about a Magical Quill, located in Hogwarts School of Witchcraft and Wizardry. The Magical Quill detects the birth of a magical child and writes their name down in a large book. Every year, Professor McGonagall checks the book and sends owls to the children who are turning eleven, to inform them that they have a place at Hogwarts.
Each day for seven days, from 31 July to 6 August, we will be placing a clue on the Pottermore homepage to help you locate The Magical Quill. If you find it, you'll have the chance to gain early access to Pottermore.
There will be a new clue every day, and each clue will relate to a different book in the Harry Potter series. On Day 1 the clue will relate to Harry Potter and the Philosopher's Stone, on Day 2 the clue will relate to Harry Potter and the Chamber of Secrets, and so forth. The clues will also vary in difficulty. On Days 1-3 the clues will be harder, and on Days 4-7 the clues will be easier.
To find The Magical Quill, you need to solve the clue and add the answer to the end of the web address http://quill.pottermore.com/ This will lead you to a website where The Magical Quill will be located while early access places are available. The Magical Quill's location will change every day.
Clue: How many books are there in the Harry Potter series? Multiply this number by 10.
Answer: 70
Add this number to the end of the website address. (In this case it would be http://quill.pottermore.com/70)
Hit 'Return' to go to the website where The Magical Quill is located.
Once you have found The Magical Quill, you will be redirected to Pottermore.com. If the registration process is still open for that day, you will be able to submit your details to register to access Pottermore early.
It's important to note that finding The Magical Quill does not guarantee you access to registration, as registration will only remain open while the early access places allocated for the relevant day are available.
Don't worry if you don't manage to register first time. There will be plenty of opportunities to find The Magical Quill as we will be revealing a new clue each day from 31 July to 6 August. For those who miss out on early access to Pottermore, please note the site will be open to all from October. Keep visiting Pottermore to find out when registration commences.
We won't be telling you when a clue goes live, so you'll need to keep a careful eye on Pottermore.com over the seven-day period. However, we will let you know, via Pottermore.com and the Insider, when registration for a particular day has come to an end.
Finally, we will not be enabling early access to the site straight away. Once The Magical Quill challenge is over, we will send successful early access registrants a Welcome email, letting them know when they will be able to access Pottermore. We won't be able to let everyone into the Beta site at the same time, so please be patient with us. You may have to wait a few weeks for your Welcome email to arrive.
For more information about The Magical Quill and early registration for Pottermore please visit the Help section of Pottermore.com.
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Why Dwyane Wade Hasn't Become OverratedJanuary 16, 2013
It's easy to look at Miami Heat guard Dwyane Wade's basic statistics and come to the conclusion that he's overrated and declining in talent significantly.
Through 32 games, Wade is on pace to finish with his second-worst scoring season, third-worst rebounding season and worst passing season.
However, those numbers tell far from the full story.
Wade is still very much an elite player.
Wade's total output is down from past years not because he's no longer great, but rather because of new team circumstances.
Due to the addition of Ray Allen this offseason, the 2012-13 Miami Heat feature the best collection of scorers Wade has every played with.
The Heat's need for him to be a prolific scorer has never been lower, which is why he's only taking 14.9 shots per game when his career average is 18.1.
What the Heat do need from Wade is to be efficient when he puts the ball up, and that's exactly what he's done. In fact, Wade is on pace to finish with the highest shooting percentage of his career (50.4 percent).
That's because Wade is playing incredibly smart basketball by picking the right times to attack instead of forcing his shot.
Wade's performance against the Atlanta Hawks in December is just one of the examples from this season in which Wade was able to have a significant impact on the game scoring-wise while not dominating the ball and still setting up his teammates.
Even though Wade is having to once again adjust to a smaller role, as his usage rate has declined for the fourth consecutive season (according to Hoopdata), he's still been the NBA's third-most efficient shooting guard this season with a 22.50 PER.
Also, due to the caliber of his teammates and presumably coach Erik Spoelstra's desire to keep him fresh for the postseason, Wade is only playing 33.2 minutes per game this season.
So, his numbers are understandably declining. But it's not as if Wade is producing far less when on the court compared to years past.
According to Basketball-Reference, Wade is averaging 4.9 rebounds per 36 minutes this season, which matches exactly his per-36-minute career average.
And while his assist numbers this season per 36 minutes fall short of his career average, it's a bit unfair to Wade to compare much of the past with now, considering he had much more distributing responsibility prior to LeBron coming to South Beach.
What would be more indicative of Wade's decline is if he were dishing out fewer assists than he has the previous two seasons with LBJ. Unfortunately for Wade's detractors, though, Wade is averaging 4.7 assists per 36 minutes this year after averaging 4.4 in 2009-10 and 5.0 last season.
Also, Wade's basic statistics ignore his defensive presence. Take a look at how many points the Heat allow per 100 possessions when Wade is on the court compared to when he's not, according to 82games.
Wade is one of the game's premier perimeter defenders, and as the chart suggests, the Heat rely on that ability greatly.
He is once again on pace to finish as the league's top blocking guard, and he displayed that skill particularly well in an early season matchup against the Cleveland Cavaliers.
Take a look at how Wade was able to regroup and block a potentially game-winning shot from Jeremy Pargo.
Still, the desire by many to pronounce Wade as no longer elite remains. To be fair, the reasoning behind that argument is clear.
At age 30, Wade is getting older, so naturally his athletic superiority over his opponents is in decline.
However, he hasn't let it affect his game to the extent many seem to think and is still playing two-way basketball better than almost anyone in the game today. His statistics aren't as flashy as they've been in year's past, so this seems to go unnoticed.
Wade was recently asked if he ever missed being the go-to guy, which is when he was able to log such incredible numbers.
From ESPN's Brian Windhorst:
Before the Heat left on this trip, Wade was asked if he missed the days of taking 20 to 25 shots a game. The days before James and Bosh and being relegated to the third option some nights. Wade's response: "Every day."
Though Wade misses that responsibility, he doesn't act on that feeling. You won't see Wade jacking up shots due to being frustrated with his role. He understands what type of play this team needs from him in order to win.
And that's one of the primary reasons the Heat sit atop the Eastern Conference right now.
Wade deserves a substantial amount of credit for his play this season, not criticism.
No. With how often many seem to want to bury this guy, Wade is underrated.
Note: Statistics are accurate as of January 15.


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Student loan bill runs aground
In response to "Bill on student loan rates blocked" (May 9): Democrats want to use wealthy people's money to keep student loans from doubling. Republicans are blocking this proposal to protect millionaires and billionaires. I note that they never refer to the rich as wealthy, but always as "job creators," and they're right. These hedge fund managers, CEOs and Wall Street bankers create jobs for truckers, longshoremen and ship captains and their crews who haul crates stuffed with job creators' cash to the Cayman Islands and other offshore locations. – Cliff McReynolds, La Jolla
Letters and commentary policy
The U-T welcomes and encourages community dialogue on important public matters. Please visit this page for more details on our letters and commentaries policy.
- E-mail email@example.com
- Mail: Andrew Kleske, Reader Outreach Editor
San Diego Union-Tribune
P.O. Box 120191
San Diego, CA 92112-0191.
A question to weigh
"Our meat, on drugs" (Business, May 9) left me wondering whether the antibiotics that fatten our livestock, in less time and using less feed, pass through the food chain to similarly fatten our own population. Has there been research to check on this possible connection? – Nancy Eastman, Rancho Peñasquitos
Debate on Prop. 29 lights up
Congratulations on your endorsement of Prop. 29 ("A vote for public health: Yes on Prop. 29," Editorial, May 9). I agree wholeheartedly with your assessment that prevention and research is a cost-efficient way to reduce health costs. You also take issue with the deceptive ads paid for by Big Tobacco that wrongly suggest that the physicians are against the proposition.
Following the same logic, I urge you to decry the Republican bill in the House that would eliminate $63 billion from the prevention fund in health care reform in order to pay for keeping the student loan rates low. – Ricardo A. Jimenez, Chula Vista
Sixty million Americans (20 percent of our population) enjoy smoking. It is our choice, we enjoy it, and it is our own business. Why are WE being criminalized when more dangerous choices such as alcoholism, drug use and obesity are ignored? When has "smoking" ever caused automobile accidents, family disunity or criminal activity?
Now we are being intimidated with a proposed $1 hike in a package of cigarettes (with a proportional increase in other tobacco products) "supposedly" to fight cancer. Is it not really another attack on our freedoms by those who want to control our lives? Why not add an increase on alcohol to fight rampant alcoholism, or an increase on sugar to fight the growing obesity epidemic? And why not a SERIOUS attack on the ravages of drug use?
I hope that every person who enjoys his personal freedom will vote "No" on Proposition 29! An attack on YOUR lifestyle could be next! – James D. Lemon, San Diego
Keep term limits as they are
You say that we should adopt Prop. 28 and allow legislators to have longer terms in each house than they have now. [Tuesday] on "PBS NewsHour," Sen. Tom Coburn discussed his new book, "The Debt Bomb." Sen. Coburn is more Republican than a cloth coat, wise about the ways of legislators, and he knows how we got our massive Federal debt.
He says it was generated by legislators he calls "careerists" whose only goal in life is re-election. Sen. Coburn recommends term limits as the way to reduce careerism, and he says reducing careerism is the royal road to the federal government's fiscal health. He's right, and his term-limit recommendation applies to state legislatures and the Congress equally. We should adopt term limits for both.
In a choice between you recommending continued legislative careerism with its fiscal recklessness, and the senator recommending term limits, I choose him. He's got it right. You've got it wrong.
Yes on fiscal sanity. No on Prop. 28. – Jim Varnadore, City Heights
Al-Qaeda still a threat
In reference to [Wednesday's] paper, I am surprised that "American Idol" took up about half of the first page of the front section. Meanwhile, the story on the double agent and the possible future bomb threats to the U.S. by al-Qaeda was on Page 4. I would bet that a survey of highly regarded newspapers would show that the double-agent article is on the first page of their papers today.
The news that al-Qaeda is seriously still attempting to bring down airliners headed toward the U.S. is much more important for the first page than a local "American Idol" contestant. Wouldn't the "American Idol" article regarding a local resident be more fitting in the Local section? – Debbie Jezior, Scripps Ranch
And the Obama administration tells us the "war on terror" is over. Is this another way of voting "present" by our "leader"? – Julianna Faulkner, Mission Beach
Sweetwater cuts start at the bottom
In response to "Sweetwater schools OK about 200 layoffs" (Local reports, May 9): Looking at the cuts, I noticed there were none at the administration level. As always, cuts never start at the top. – Greg Pharis, Spring Valley
San Diego mayoral race
In response to "Carl DeMaio for mayor" (Editorial, May 6): The problem with DeMaio is not that he's a contractor who wants to privatize as many services as possible, nor that he refuses to let evidence cloud his judgment, it's that he would seemingly be satisfied taking his crusade anywhere.
Throughout the campaign, he has failed to show that he appreciates any sense of place or pride in San Diego. He would measure his performance based on numbers of pensions reduced and positions eliminated rather than quality of life for San Diego residents and businesses. The other candidates can articulate why they want to be mayor of San Diego. Mr. DeMaio can only describe why he wants to a mayor. – Jeremy Haas, Point Loma
You got it exactly right this time! I don't always agree with your recommendations ("Carl DeMaio for mayor," Editorial, May 6), but after considering the candidates, I agree that Carl DeMaio is the one to get our city back on track.
I've been following the mayoral campaign closely, reading many articles and attending debates and forums. Nathan Fletcher claims to be "tested." However, he has no experience in city government. DeMaio has been on City Council, practicing what he preaches, to reform San Diego government. He said he'd clean up the mess in City Hall and return the money back projects that help San Diego. That's what he's done on the City Council and what he'll continue to do as mayor. He understands how the city works – and doesn't work – and won't need a learning-curve period to become effective.
I attended DeMaio's La Jolla Town Hall on pension reform last week to further educate myself. I never thought I was part of the "99 percent," but maybe I am. When over 500 city retirees earn $100,000-plus in pension and benefits, it is time for taxpayers to "Occupy" City Hall. Boy, would I like a pension of that size – or any size, in fact! In hindsight, I chose the wrong career path. As a freelance writer, I have only my SEP-IRA plus Social Security (and expensive health insurance, as well).
The pensions for just those 500 retired city employees total at least $50 million, and is probably closer to $100 million, in city obligations each year. Apparently Marion the Librarian got more than just the books; she also got a $250,000 annual pension, too! I think education and libraries are important, but $250,000? How many teachers, textbooks, and extra branch library hours can that buy? Carl DeMaio has been a leader in reforming these runaway pensions and is the one we can trust to get them under control.
Already there are letters saying that we don't need the U-T San Diego to tell us who to vote for. Fine – no one has to be a blind follower. The U-T has provided extensive coverage of all candidates, with detailed articles about their plans for San Diego. Not to undercut the importance of the U-T's endorsement of DeMaio, but sometimes it really isn't rocket science – DeMaio's campaign and record of public service focuses on two common sense ideas that will produce uncommon results: fix City Hall and fix San Diego.
It's time to say good night ladies to the big spenders at City Hall and hello DeMaio for mayor. The U-T's endorsement was right on in identifying the problems in our city and the right candidate to fix them. – Marlene Bellamy, La Jolla
In response to "Fletcher: principled or panderer? (Local, May 7): It is unconscionable that Nathan [Fletcher] has missed 70 percent of his official duties yet continues to take state-tax-funded salary, plus all those other expensive goodies he and his fat-cat Sacramento pals spend our hard-earned cash on.
Let's see, could he be trusted there? No.
So, can we trust him here? You know the answer. – Claudia Strasbaugh, Scripps Ranch
Nathan Fletcher continues to show that he is incapable of making any decisions. Nothing says bold solutions like skipping the days, and votes, that really make a difference. Perhaps Fletcher's group should consider changing their name to something more appropriate like, "Movement to Irrelevant." – William Mitchell, San Diego
Risks not worth reward
In response to "More steam tubes plugged at San Onofre" (Business: May 9): Southern California Edison officials are delusional if they think the San Onofre generating station is ready to be put back online. On the contrary, a growing number of people believe it should be permanently decommissioned for the safety of the 8.4 million Southern Californians who live within a 50 mile radius of the facility.
Recently the entire City Council of the city of Irvine endorsed the permanent shutdown of San Onofre. And with good reason. Complaints by employees at San Onofre about safety violations have been as high as 10 times the average for a nuclear generating plant. Whistle-blowers have allegedly been disciplined or fired.
The steam generators are failing only a couple of years after they were installed and have the potential to release deadly radiation into the air. And Southern California Edison officials want captive ratepayers to pay $64 million for a seismic study to determine if there are undiscovered faults capable of causing an earthquake that could cause a catastrophic meltdown similar to the one that occured in Fukishima, Japan. The risks San Onofre poses far outweigh the advantages. – Elliot Becker, Escondido
Marketplace determines value
I find the headline to Eric Taylor's letter (May 9), "Pay doesn't match level of achievement," to be somewhat disingenuous at best. What we expect from the future might not be realized based on the times and the economy. The marketplace will determine what value it places on credentials.
Taylor states that Kilpatrick's full-time salary of a "mere" $62,964 "is quite small, given the cost of living in San Diego County." Taylor simply doubled the figure that I calculated and then neglected to state that it was a NET salary, not gross.
Quoting from simplyhired.com, "According to government data, the average salary for jobs in San Diego, California, is $43,478, and the median income of households in San Diego was $55,637." Those are GROSS salaries and incomes!
I believe a great supermajority of San Diegans would be extremely happy with a NET income of a "mere" $62,964 for a full-time job! Maybe U-T San Diego could do one of their polls.
Eric challenges me to "attempt the same position at the same pay" re Kilpatrick's lecturer position at CSU San Marcos. I would be happy to. CSU San Marcos? I await your call. – Russel Ray, La Mesa
Junior Seau
Why does Ruben Navarrette Jr. ("What we can learn from Junior Seau," Opinion, May 9) inject ethnicity into his column? In the second paragraph he states, "... hard-hitting linebacker of Samoan descent." This adds nothing to the piece. Later in the column he mentions another player, Gary Plummer, but doesn't refer to him as white.
Mr. Navarrette makes a living by finding racial and ethnic injustices wherever he looks. In my opinion, the media makes more of this stuff than is warranted. Can't we leave this alone just for a little while? Maybe it would just go away. – Bill DiMasi, Jamul
Ranks of school nurses dwindle
[May 9] is National School Nurse Day, thanks goes out to all of the San Diego Unified School District nurses for their dedication and hard work! Throughout the year the nurses advance the well-being, academic success and lifelong achievements of all students by providing a critical safety net for our children's health.
The San Diego school board members have let us down by proposing to cut/layoff nurses to a dangerously unsafe level. If SDUSD goes through with these cuts there will only be 84 nurses to care for over 130,000 next year.
The cuts also include the many valuable teachers, counselors and maintenance personnel within our district. Board President John Lee Evans stated in the May 5 U-T San Diego that he "was hopeful that students would consider the teachers role models and consider following in their footsteps."
A student following a teacher's footsteps can expect to receive layoff notices for years.
The district board needs to stop this practice for the sake of our children and our future. – Dean Williams, Carlsbad
Rise above self-interest
In response to "Bishop Brom's message of service (May 8): I was moved by the U-T San Diego's recent interview with San Diego Roman Catholic Bishop Robert Brom. I am not a Catholic, yet I resonate to his universal appeal, to "get out of the cocoon of self-absorption and embrace a life of selfless service." Brom pointed out that "leadership at every level has to get beyond self-interest in order to be of service to others." This echoes John Kennedy's message of 50 years ago: "Ask not what your country can do for you; ask what you can do for your country."
Our society has digressed from that message and that mission in the past several decades. As a liberal Democrat, I am dismayed by the Supreme Court's affirmation of the citizenship of corporations; by the national tax structure which so favors the wealthy; by San Diego's continued push for downtown redevelopment at the expense to our school budgets and our children for 40 years into the future; and by deep potholes in our streets while publicly funded stadiums owned by the wealthy are still being promoted. Although these are all Republican causes, my fellow Democrats are culpable as well. They are unwilling to stand up to public employee unions who act like greedy corporations, caring little for the common citizen and focusing only on feathering their own retirement nests at the expense of future generations who must support them. We need only look to Southern Europe to see the selfishness and long-term folly of such policies. Nor do I favor tax-averse libertarians, because I believe that those of us with financial means have a responsibility to pay taxes for the common good.
Although many politicians may privately disagree with all of these messages, they have to submit to one of them in order to procure campaign funding to win their elections. One solution? National and state legislation to limit campaign contributions and spending. Otherwise, politicians will have no choice but to support greed and self interest on one side or another other, and not be able to serve those who most need their support, our future generations. – Dr. Janet Farrell, San Diego
Privatized profits, socialized costs
The latest Fortune Magazine list of 500 richest companies is out and look who is almost right in the middle: Sempra Energy!
Yes, the parent company of Southern California's public utilities is number 266 in the listing of the 500 most successful capitalist companies.
Yes, the same parent of SDG&E, that wants its customers – we, the ratepayers – to be liable for the additional costs to settle the valid and lawful claims of those who were hurt and wronged by SDG&E in the SDG&E equipment-caused the 2007 wildfires.
Yes Sempra Energy, the company that wants to make the profit, but wants the customer to pay the loss!
I guess that's one way to stay on the Fortune 500 listing.
The root of this problem is the fact that SDG&E is an investor-owned public utility.
This is, in itself, an obvious conflict. When the company makes a profit, that money goes to the investor-owners. But, when there is an operating financial loss, the ratepayers are expected to make up the difference – usually by rate increases. In other words, profits are privatized while costs are socialized. This is what we are seeing much of in the U.S. today where the profits are concentrated among the few, the shareholders, the politically connected; and the liabilities or losses are spread out among the many, the taxpayers, the politically disassociated. This is what I call Republican socialism.
If the PUC allows these to be approved, it will set a dangerous – and likely illegal – precedent. For what we have here is truly a wolf in sheep's clothing: SDG&E is couching the concept of WEBAs [Wildfire Expense Balancing Account] as a device for tracking the amount of money that "may be" needed above the amount of insurance settlements. But, if that is truly all that is needed, internal accounting methods can be used to track these costs even more easily. No, what we have here is a public monopoly trying to pull the wool over our eyes and allow the precursor to a rate increase.
I urge the PUC to reject the WEBAs as a gross abrogation of responsibility and the transparent money grab that it is. – Michael-Leonard Creditor, La Jolla
Homeowners need protection
The Homeowner's Bill of Rights is anything but consumer protection run amok, as Norm Miller, real estate professor at the University of San Diego, would suggest ("Why the Homeowner Bill of Rights is 'consumer protection run amok,' " Business/Real Estate, utsandiego.com, May 8). In my opinion it is needed homeowner protection from lenders who ran amok and helped bring about the real estate market collapse.
I am a North County real estate agent, and dues-paying Realtor, but I don't buy the industry line that consumer protection hurts business. Consumers of all sorts need protection, be it for clean air, healthy food or predatory lending.
Legislation that would require banks to assign a single point of contact for troubled-loan cases, fine banks for robo-signing and fight neighborhood blight, is long overdue. I believe that what's good for troubled homeowners and neighborhoods is good for the real estate industry and by extension is good for the economy. And I don't believe the legislation will stall the foreclosure process, as the California Mortgage Association claims, because the free market process and profit motive will compel smart lenders to adjust their business practices. – Charles Dodson, Carlsbad
Indiana moderate ousted
So defeated Sen. Richard Lugar is "too moderate," according to his tea party opponent ("Indiana's Lugar loses; N.C. passes ban on gay marriage," May 9). I decided to check out Lugar's crime by consulting my dictionary. Webster defines "moderate" as "avoiding extremes of behavior or expression – observing reasonable limits – calm, temperate – avoiding extreme political or social measures."
I realize that Webster and politicians have differing definitions for many terms, but isn't it a shame that some choose to denigrate such qualities in a man? – James Ransom, San Diego


text:
GAINESVILLE, Fla. - A woman was killed when a tree fell on her north Florida home in the middle of the night, according to officials.
The Alachua County Sheriff's Office said 42-year-old Dawn M. Ewing and her husband, 40-year-old Michael L. Braton, were in bed early Wednesday morning when an enormous oak tree crashed through their roof and crushed them. Ewing died, but rescuers managed to get Braton to a Gainesville hospital for emergency surgery.
The couple's three children were in the home, but they were not injured.
Officials believe strong winds caused the tree -- estimated to stand at least 50 feet and roughly 4 feet around -- to fall on the single-wide mobile home.
A neighbor told The Gainesville Sun that the family had only moved in a few weeks ago.
Copyright 2013 by ClickOrlando.com. The Associated Press contributed to this report. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.


text:
On Nov. 15, the Coventry High School Early College Experience (ECE) French class, under the direction of teacher William Scheideman, placed third in the Annual Quiz Bowl and High School Day at the University of Connecticut.
Students competed against schools from across the state that offer ECE French. Students earn college credit for the course while developing fluency, reading, and writing skills to compete in a global marketplace. On Nov. 15, prior to the culminating Quiz Bowl competition covering French and Francophone language, culture, and history, students participated in immersion activities led by UCONN graduate students. All of the students agree that the camaraderie, cultural immersion, and interaction with students from other Connecticut high schools outweighed the ultimate result of the competition.


text:
: Research:
Battle Tower Pokémon Offsets (Pokémon Ruby)
View Single Post
July 16th, 2010 (6:30 PM). Edited July 17th, 2010 by Chaos Rush.
Chaos Rush
Join Date:
May 2007
After some researching, I managed to find out how to edit the Battle Tower Pokemon. The offset of the Pokemon Data base starts at
Pokemon Ruby
The offset in Emerald is somewhere around 0x5D97CC. I tried porting Emerald's list into Ruby, but I hit a brick wall when I couldn't find the offset. I
the pointer in Ruby was at 0x134F54, but when I changed the bytes there to repoint to where I pasted the Emerald Pokemon list, the Battle Tower Pokemon were exactly the same as Pokemon Ruby & Sapphire. So then I tried simply pasting Emerald's list directly over Ruby's (even though Emerald has a much larger list than Ruby's, lol), all that did was crash the game when I tried to make a Battle Room challenge.
Anyways, I don't think I need to explain how to change it, but at 0x4038E0 you see the bytes
19 00
, right? Yeah, and if you use GoldFinger, if you look up at the top while 19 is highlighted, you should see, "19 (25)". And Pikachu's hex number is also 25, meaning that the first byte is the species of the Pokemon. According to
this page
, there are only two Pikachu you will encounter in the Battle Tower, so at 0x4038E0, change 19 to 18, and congratulations, 50% of all Pikachus will now turn into Arboks.
I'm too lazy to figure out how to edit their moveset, but it's pretty easy, since the move bytes are very close to the species byte(s).
So here's some screenshots to prove that I'm not making up stuff:
I didn't edit the movesets, I just changed random 01s into 00s to change some of their species numbers, which is why you see non Hoenn-Pokemon using illegal moves (their movesets originally belonged to Hoenn Pokemon).
Obviously, I'm no advanced hacker, but the reason why I'm posting this is because hopefully some of you advanced hackers can figure out how to port Emerald's Pokemon list over Ruby's.
EDIT: I did a little more experimenting, I pasted part of Emerald's list over Ruby's, and that doesn't exactly work correctly. Usually it freezes right before you battle a trainer, but if you manage to make it into battle, the Pokemon use glitchy moves. One time where I made it into a Battle, when my foe Dustox attacked, it said, "Dustox used EXIT" and then it froze.
*Serebii.net - for posting the Battle Tower list that helped me find Ruby's offset
*Peterko (a member at Smogon) - for making
this thread
that helped me find Emerald's offset
*HackMew - for making A-Trainer, which I used to mimic some Battle Tower Pokemon to help me find the offsets for both Ruby and Emerald
*Whoever made GoldFinger, the hex editor I used to find everything.
Also, this might help with research, but here's a patch for Ruby that makes some Kanto Pokemon appear in the Battle Tower with unfitting movesets:
Attached Files
(1.8 KB, 10 views)
(Save to Dropbox)
My works:
Final Fantasy II NES translation
Final Fantasy III NES translation
Mother 1/EarthBound Beginnings Color Restoration for GBA
Non-Pokémon hacks I support:
Sonic Classic Heroes
Sonic 3 Complete
Chaos Rush
Joined May 2007
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text:
Thu 7th Feb at 7.30pm - 9pm
Choir of the Royal Holloway
@ St Martin In The Fields, London
Featuring: Choir of the Royal Holloway, The Brandenburg Sinfonia
This event has been and gone!
Catch the next one:
Check out the info below for future tour dates, and track them to receive alerts when they tour near you
See all dates for Brandenburg Choral Festival
Brandenburg Choral Festival: Mozart Requiem: Choir of the Royal Holloway, The Brandenburg Sinfonia
"Rupert Gough conducts the acclaimed choir of Royal Holloway and the Brandenburg Sinfonia in three pieces by Mozart, including his Salzburg Symphony No. 3 and his Requiem.
Combined tickets are available for this concert and the later event. Contact the box office for more details on 020 7766 1100."
Info provided by the Event Organiser
Choir Of Royal Holloway
The Choir of Royal Holloway is considered one of the finest mixed-voice collegiate choirs in the country.
The 24 choral scholars that constitute the College's Chapel Choir undertake a busy schedule of services, concerts and tours and record exclusively for Hyperion Records.
The choir is available for hire to sing concerts, commercial and corporate events, as well as weddings and funerals etc.
Choir Of Royal Holloway tour dates
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Venue Facts
Venue: St Martin In The Fields
Website: stmartin-in-the-fields.org
Phone: 020 7766 1100
→ Charing Cross 6 min walk
→ Charing Cross 3 min walk
→ Leicester Square 3 min walk


text:
Legal Question in Family Law in Georgia
I have a child support case pending for 1 child, but i have 3. Do i have to go to court to get credit for my other two children
Asked on 3/13/13, 4:02 pm
2 Answers from Attorneys
Tahira Piraino Tahira P. Piraino
If all three children have the same father all three should have been included under one order. If the two children not included in the pending child support case have a different father or fathers, you will have to set up a case for each father.
Answered on 3/14/13, 4:18 am
Harold Holcombe Harold D. Holcombe, P.C.
Need more info. What stage is this case in? If it has already been decided you may need a modification. Whether you get credit depends on a variety of legal factors.
Answered on 3/14/13, 7:31 am


text:
Alternative Title: PF
Learn about this topic in these articles:
armed forces of Panama
The national police organizations are now under civilian control and include the Public Force (PF) and the Technical Judicial Police, a special investigative unit. National defense is also entrusted to the PF, which has limited combat capabilities but some military components, including air and naval units. In the late 1990s concern was raised that Panama needed greater resources to secure its...
...was defeated in a referendum, as unemployment soared and corruption remained widespread. The administration succeeded in abolishing the PDF and replacing it with a new national police known as the Public Force, and it amended the constitution to prohibit the creation of a regular military; the amendment was ratified in October 1994.


text: Jack the Great Dane has had quite the journey from being dumped at a high kill shelter to a battle for his life with pneumonia, but he has now fully recovered both emotionally and physically from his adventures! He won the heart of new mom, Vickie, in St Louis, Missouri, which is where he now calls "home". She is a nurse, so she was the perfect one for the job of taking care of him while he recovered. His new 4-legged brother, Petie the Lab/Pyrenees Mix), was also a gracious host along with Izzie the cat. After owning a Dane previously, Vickie really missed having a gentle giant in her life and he was the perfect match! Here's to a brighter future, Jack, and the life you deserve!