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Home » IDENTIFICATION OF POTENT VIRTUAL LEADS AS TOPOISOMERASE-II INHIBITORS USING PHARMACOPHORE MODELLING, MOLECULAR DOCKING AND ADME STUDIES
IDENTIFICATION OF POTENT VIRTUAL LEADS AS TOPOISOMERASE-II INHIBITORS USING PHARMACOPHORE MODELLING, MOLECULAR DOCKING AND ADME STUDIES
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Sanal Dev 1 and Sunil R. Dhaneshwar*2
Department of Pharmaceutical Chemistry, B.V.D.U Poona College of Pharmacy, Pune, Maharashtra, India
Department of Pharmaceutical Chemistry, Ras Al Khaimah Medical and HealthSciences University, College of Pharmaceutical Sciences, Ras Al Khaimah- 11172, UAE
ABSTRACT: Topoisomerase enzymes are highly expressed in cells which undergo rapid multiplication. Inhibition of this enzyme represents a potential therapeutic approach for diseases such as cancer. In order to understand the structure activity correlation of 2, 4, 6 pyridine based topoisomerase inhibitor, we have carried out a combined pharmacophore modelling, 3D-QSAR studies, molecular docking and virtual screening studies. A five point pharmacophore with hydrogen bond acceptor (A), hydrogen bond donor (D) and three aromatic rings (R 5, R 6, R 7) was used to derive a predictive atom based 3d-qsar model. The generated model had showed good correlation coefficient for training set and test set (R2=0.91and Q2=0.827). It was also validated using enrichment factor (EF) and goodness of hit score (GH score) and was used for virtual screening of compounds from 'zinc drug like database'. Docking study of the hits retrieved from virtual screening revealed the binding affinity of these inhibitors at the active site of topoisomerase enzyme. In silico ADME predictions was also performed. These findings provide a set of guidelines for designing compounds with better topoisomerase inhibitory potential.
Pharmacophore, 3-D QSAR, Topoisomerase inhibitor, Pyridine, Docking
INTRODUCTION:DNA topoisomerases are ubiquitous enzymes playing a key role in solving topological problems associated with DNA molecule 1.
The extraordinary chain length of DNA and its double helical structure creates serious problems like twisting, knotting, tangling and super coiling during the process of replication, transcription and recombination.
DNA topoisomerases solves this problem by transiently breaking one or two strands through which the DNA strand can be passed in order to solve the topological problem and to relieve the torsion strain in DNA 2. There are two well-characterized classes of human topoisomerase. Topoisomerase I (Topo I) are monomeric which transiently break and relegate one strand of duplex DNA , while topoisomerase II (Topo II ) are homodimeric and will break both strands of a duplex DNA.Type I and type II enzymes are fundamentally different in both mechanism and cellular function. A common feature of topoisomerases is their catalytic mechanism, which in all cases consists in a nucleophilic attack of a DNA phosphodiester bond by a catalytic tyrosyl residue from the topoisomerase 3.
These enzymes are highly expressed in rapidly proliferating tumor cells and, the medical importance of these enzymes is highlighted by the fact that they are the specific targets for many promising anticancer agents. Topoisomerase inhibitors are found to be the most efficient inducers of apoptosis 4. Drugs that inhibit the topoisomerases include some of the most widely used anticancer drugs like irinotecan and topotecan which are known Topo I inhibitors and Topo II is the target for anticancer drugs like etoposide, teniposide, doxorubicin, idarubicin, epirubicin, and mitoxantrone.
Despite the wide use of Topo II targeted drugs as antitumour agents, several limitations hamper their benefits. Efforts for improving their clinical efficacy further by overcoming the drug resistance, myelosuppresion and poor bioavailability problems associated with them, were continued to be challenging 5.
Therefore, efforts have been made to develop novel topoisomerase inhibitors with a new scaffold distinct from existing drugs with improved bioactivity. The scaffold hopping strategy is a powerful approach for the discovery and modulation of medicinal ingredient by modifying the core structure of promising ligands 6. Furthermore, this strategy not only gives the opportunity to modulate both selectivity and affinity of a given ligand but also allows the development and the exploitation of innovative chemistry in order to provide eventually new drugs. With this in mind, we selected 2, 4, 6 trisubstituted pyridine derivatives which are reported to possess topoisomerase I and II inhibitory activity along with strong cytotoxicity against several cancer cell lines like breast cancer adenocarcinoma, cervix tumor, Human prostate tumor, colorectal adenocarcinoma and myeloid leukemia.
Since these are the most common types of cancer, a good understanding of their chemical properties at the molecular level such as their lipophilic, steric, and electronic characteristics may provide important information on the anticancer properties of these analogues for further development. The discovery of new types of Topoisomerase inhibitors that can be synthesized easily, increased sensitivity in drug resistant tumors and decreased dose-limiting toxicities would be a significant addition to the choices available in the treatment of cancer. Although a structure-based drug design approach is an attractive strategy, a ligand-based approach such as 3D-pharmacophore generation is useful for the identification of the pharmacophoric features which could help in designing new molecules 7. Pharmacophore is an important and unifying concept in rational drug design that embodies the notion that molecules are active at a particular enzyme or receptor because they possess both a number of chemical features that favour the target interaction and geometry complementary to it 8 .
In our efforts towards finding potent topoisomerase inhibitor, we undertook a comprehensive molecular modelling study to identify the pertinent features that could serve as a starting point for design of ligands with increased affinity and target selectivity.
In this study, we have developed a quantitative pharmacophore model based on topoisomerase inhibitors collected from the literature. The best quantitative model was used as a 3D search query for screening the Zinc "clean drug-like" database to identify new topoisomerase inhibitors. Once identified, the candidate compounds were subsequently subjected to filtration using molecular docking to get drug like molecules.
MATERIAL AND METHODS:
Hardware and Software Specifications: Molecular modeling studies were carried out on a personal computer running on windows 7. Pharmacophore modeling and virtual screening experiments were performed using Schrodinger suite 2009 and modules like Phase, Ligprep, Quick Prop, Glide, and Virtual Screening Workflow were used in the study.
Data Base preparation: Pharmacophore modelling correlates the spatial arrangement of various chemical features with their pharma-cological effectiveness. It requires structural and activity data for a range of active and inactive molecules for generation of the hypothesis. For the pharmacophore modelling studies, a total of 72 molecules belonging to 2, 4, 6 tri substituted pyridine analogue having topoisomerase inhibitory activity were selected from the literature 9–11.
The reported anticancer activity (IC50) against DU145, a human prostate tumor cell line was used and the IC50 value was converted into the corresponding PIC50 value.
IC50 is the dose in micromoles (µM) required to produce 50% inhibition. The structures of the 72 compounds used in this study, are given in Table 1.
TABLE 1: DATASET ANALYZED WITH EXPERIMENTAL AND PREDICTED ACTIVITIES
The structures of 72 molecules were sketched using maestro builder toolbar and all ligands were cleaned using the default Maestro settings. For each molecule, a set of conformers were generated through a mixed Monte-Carlo multiple minimum (MCMM)/Low mode (LMOD) with maximum number of 1000 conformers per structure using a process minimization of 100 steps and each minimized conformer was filtered through a relative energy difference 10 kcal/mol relative to the global energy minimum conformer with a maximum atom deviation of 2.00 Å. After the generation of conformers, only one conformer was displayed and carried for the further analysis. As active compounds are normally considered while developing common pharmacophore hypothesis, a "pharmaset" was defined by setting an active and inactive thresholds of PIC50 >5.5 for actives and PIC50 < 4.6 for inactives. Generation of the Common Pharmacophore Hypotheses (CPHs): Pharmacophore modelling provides a qualitative picture of the geometry of the active site by identifying the chemical features for binding of ligands and their spatial arrangements in 3D space 12. PHASE module of Schrodinger software 13 was used to generate 3D Pharmacophore models and QSAR studies for the present series of compounds.
PHASE provides a built-in set of six pharmacophore features such as hydrogen bond acceptor (A), hydrogen bond donor (D), hydrophobic group (H), negatively ionizable (N), positively ionizable (P) and aromatic ring (R). Common pharmacophores were identified using a tree-based partitioning technique that groups together similar pharmacophores according to their inter site distances, i.e., the distances between pairs of sites in the pharmacophore 14, 15. A five-point common pharmacophore hypothesis was identified from all the conformations of the active ligands having identical set of features with very similar spatial arrangement keeping minimum inter site distance 2.0Å in a final box size of 1.0Å with requirement that all actives should match.
Generated common pharmacophore hypotheses were examined by scoring alignment of actives against a reference ligand by using default settings.Scoring function such as score active, score inactive and rescore were applied for all five-featured common pharmacophore hypotheses. The scoring procedure provided a ranking of different hypotheses from which further investigation was carried out for appropriate hypothesis with rational choice.
Building the 3D-QSAR model: An atom-based 3D-QSAR model is more useful in explaining the structure activity relationship than pharmacophore based 3D-QSAR as the latter does not consider ligand features beyond the pharmacophore model 16. In atom-based 3D-QSAR, a molecule is treated as a set of overlapping van der Waals spheres. Each feature is categorized according to a simple set of rules:
(a) Hydrogens attached to polar atoms are classified as hydrogen-bond donors (D);
(b) Carbons, halogens, and C–H hydrogens are classified as hydrophobic/non-polar (H);
(c) Atoms with an explicit negative ionic charge are classified as negative ionic (N);
(d) Atoms with an explicit positive ionic charge are classified as positive ionic (P); and;
(e) N, O, and hydrogen-bond acceptors are classified as electron withdrawing (W) and all other types of atoms are classified as miscellaneous (X).
All the common pharmacophore hypotheses successfully generated and scored to generate atom-based 3D-QSAR models by correlating the observed and estimated activity for the set of 51 training molecules using PLS analysis. The PLS regression was carried out using PHASE with a maximum of PLS factor 5 and a grid spacing of 1.0Å. All models were validated by predicting activity of the set of 21 test molecules. The 3D-QSAR was evaluated by cross validated R2, Q2, SD, RMSE and Pearson-R. The predicted PIC50 at 5th PLS factor are tabulated in Table 2.
Evaluation of Pharmacophore model: The generated pharmacophore model should be statistically significant, predict activity of the molecules accurately, and it should also differentiate active and inactive compounds from a database. Therefore, the derived pharmacophore was further validated using the hit list (Ht), number of active percent of yields (%Y), percent ratio of actives in the hit list (%A), enrichment factor (EF), false negatives, false positives, and goodness of hit score (GH scoring method) 17.
A decoy set including 1001 molecules with unknown activity and 15 active compounds is prepared for this step. Enrichment Factor (EF) and Goodness of Hit Score (GH) were calculated to evaluate the hypothesis ADRRR.1. EF and GH were calculated using below equations:
GH Score = [(Ha/4HtA)(3A+ Ht)+(1- ((Ht -Ha)/(D-A))]
EF = (Ha/Ht)/(A/D)
Where Ht is the number of hits retrieved, Ha is the number of active molecules in the hit list; A represents the number of active molecules present in the database and D stands for the total number of molecules in the decoy set. The GH score ranges from 0, which indicates the null model, to 1, which indicates the ideal model.
Database search for new hits: The best-ranked five-point pharmacophore model, ADRRR.1, was used as a search query to retrieve compounds with novel and desired chemical features from Zinc "clean drug-like" database 18.
Protein preparation: The protein structure of human Topo II-α co-crystallised with DNA and etoposide (PDB code 3QX3, resolution 2.16 Å) complex was obtained from the RCSB PDB and was prepared using the Protein Preparation Wizard of the Schrödinger suite. H-atoms were added to the protein, including the protons necessary to define the correct ionization and tautomeric states of amino acid residues such as Asp, Ser, Glu, Arg, and His. The missing side chains of residues were corrected using prime interface incorporated in Maestro.
For each structure, minimization was carried out with the Impact refinement module, using the OPLS-2005 force field to alleviate steric clashes that may exist in the structures. The minimization was terminated when the energy converged or the Root Mean Square Deviation (RMSD) reached a maximum cut off of 0.30 Å 19. Topo-II complex, 3QX3 contains Mg2+ ions, and a co-crystallised DNA segment, which was retained during the docking study 20.
After protein preparation, receptor grid was set up, which was generated by employing the Receptor Grid Generation panel. All amino acids within 20 Å of the 3QX3 were included in the grid file generation. Since this protein was associated with ligand, the ligand was selected to define the position and size of the active site.
Docking study: Docking studies were performed by means of Glide v5.6 21. It performs grid-based ligand docking with energetics and searches for favourable interactions between one or more typically small ligand molecules and a typically larger receptor molecule, usually a protein 22. Glide provides three different levels of docking precision: HTVS, high-throughput virtual screening; SP, standard precision; and XP, extra precision. Docking calculations were first performed in HTVS mode and subsequently in SP and XP mode. All the molecules were built within Maestro using the Built module and an exhaustive conformational search was carried out for all molecules using OPLS-2005 force field, imposing a cut-off of allowed value of the total conformational energy compared to the lowest-energy state. A minimization cycle for conjugate gradient and steepest descent minimizations was used with default value 0.05Å for the initial step size and 1.00Å for the maximum step size. In convergence criteria for the minimization, both the energy change criteria and gradient criteria were used with default values 10−7 and 0.001 kcal/mol, respectively.
Virtual screening and drug-likeness prediction: For the exploration of novel scaffolds with Topoisomerase inhibitory activity, an in silico screening of 10,384,703 compounds from Zinc 'clean drug-like' database with derived pharmacophore model as a query was performed. The search criteria for compounds included in this database were: x log p ≤ 5.0, molecular weight in the range of 150–500, H-bond donor's ≤ 5, H-bond acceptor's ≤10.
Finally, compounds with the good estimated activity values were retrieved from databases and obtained molecules were chosen for subsequent molecular docking studies using virtual screening workflow by applying various filters in order to get more drugs like molecules.
RESULT AND DISCUSSION: The aim of this study was to elucidate the 3D structural features of 2,4,6 tri substituted pyridine derivatives crucial for binding, by generating 3D pharmacophore and to quantify the structural features of topoisomerase inhibitors essential for biological activity by generating atom-based 3D QSAR model.
For the pharmacophore modelling and QSAR studies, we have used Phase module of Schrodinger suite. This hypothesis generated by Phase will also convey the relative binding of the ligands inside the active site of the receptor.
Hence, we have used conformation suggested by the hypothesis for generating 3D-QSAR model to identify overall aspects of molecular structure that govern the activity.
Generation of the common pharmacophore hypotheses (CPHs): For the generation of pharmacophore model, compounds in the active "pharmaset" are normally considered. We included nine compounds having PIC50 >5.5 as active and PIC50 < 4.6 as inactives and used four minimum sites and six maximum sites to have optimum combination of sites or features common to the most active compounds. Sixteen common pharmacophore models were generated with different combination of variants in which top three ranking models were considered based on survial-inactive score for further QSAR generation. The top model was found to be associated with the five-point hypotheses was having hydrogen bond acceptor (A), hydrogen bond donor (D) and three aromatic rings (R 5,R 6,R 7) and was denoted as ADRRR1 (Fig. 1a). The special arrangement of features along with their distance aligned on most active compound 44 are depicted in Fig. 1b.
FIGURE 1A: GENERATED PHARMACOPHORE MODEL ADRRR.1, FIGURE 1.B: PHARMACOPHORE HYPOTHESIS AND DISTANCE BETWEEN PHARMACOPHORIC SITES. ALL DISTANCES ARE IN Å UNIT.
The 72 molecules were divided into a training set (54 compounds) and a test set (18 compounds) for the purpose of atom-based 3D-QSAR. The training set molecules were selected in such a way that they contained information in terms of both structural features and biological activity ranges. The most active, several moderately active, and some inactive compounds were included in the training set in order to obtain critical information on pharmacophoric requirements 22. In order to assess the predictive accuracy of the model, a set of 18 compounds was arbitrarily set aside as the test set. Training set compounds were aligned on the common pharmacophore hypotheses and analyzed by PLS with five factors. The predictivity of the generated 3D-QSAR model was analyzed by test set prediction.
Considering the flexibility of all the molecules, the predictive qualities of the QSAR model was satisfactory, based on R2, Q2, SD, and RMSE, as well as on the high value on the Pearson-R. The predicted and observed activities of the training and test set compounds are shown in Figure 2.
The training set correlation is characterized by PLS factors (R2 = 0.9101, SD=0.1168, F =91.1, P =2.175e-015).
The test set correlation is characterized by PLS factors (Q2 =0.8276, RMSE=0.1633, Pearson-R =0.9159).
FIGURE 2: GRAPH OF OBSERVED VERSUS PREDICTED BIOLOGICAL ACTIVITY OF TRAINING SET AND TEST, RESPECTIVELY
The large value of F (91.1) indicates a statistically significant regression model, which is also supported by the small value of the variance ratio (P), an indication of a high degree of confidence. Further, small values of standard deviation (0.1168) of the regression and RMSE (RMSE = 0.1633) makes an obvious implication that the data used for model generation are best for the QSAR analysis. Validity of the model can be expressed by cross-validated correlation coefficient (Q2 = 0.8276). The Q2 value is more reliable and robust statistical parameter than R2 because it is obtained by external validation method by dividing the dataset into training and test set.
The QSAR model displays 3D characteristics as cubes and the blue cubes indicate positive coefficients which are favourable while red cubes indicate negative coefficients which are unfavourable regions for activity. This might give a clue to what functional group are desirable or undesirable at certain positions in a molecule. The blue cubes in 3D plots of the 3D pharmacophore regions refer to ligand regions in which the specific feature is important for better activity, whereas the red cubes demonstrates that particular structural feature or functional group, which is not essential for the activity or likely the reason for decreased binding potency.
The basic pyridine nucleus was found to be essential for the activity. The volume occlusion maps generated from 3D-QSAR studies highlight the structural features required for topoisomerases inhibition. when the QSAR model is applied to the most active compound 44 and least active compound 27 is shown in figure 3(a-b).
3a 3b
FIGURE 3: A. ATOM BASED 3D QSAR MODEL VISUALIZED IN THE CONTEXT OF MOST ACTIVE COMPOUND 44; B. LEAST ACTIVE COMPOUND 27 (BLUE CUBES INDICATE FAVOURABLE REGIONS WHILE RED CUBES INDICATE UNFAVOURABLE REGION FOR THE ACTIVITY
Blue cubes were observed near the OH group of phenyl ring in ligand 44, suggesting that the presence such group at this position, capable of acting as hydrogen-bond donor, seemed to have a favourable effect on biological activity. Owing to the presence of this H-bond donor, compounds 11 and 39 are active. In the case of least active compound, 27 which was having a five member ring and also lacking the OH group.
It was reconfirmed by docking study, in the binding mode analysis of 44 with PDB 3QX3, H-bond contacts between Thiamine fragment (DT.D9) of DNA and the OH group of ligand 44 was observed. Most active compound 44 showed good binding affinity with the protein and the docking score was found to be -7.29 and for the least active compound it was - 0.116.
Visual analysis demonstrates that the presence of the blue cubes at the phenyl ring, attached to 4th position of pyridine ring is pointing out the positive potential of hydrophobic group and is requisite for the activity at this particular place.
It can be suggested that addition of appropriate hydrophobic group at these positions, will append the topoisomerase inhibition, whereas the addition of hydrophobic groups on ring attached to 6th position of pyridine ring will contribute to decreased receptor binding.
The effect of H-bond donor, Hydrophobic, Electron-withdrawing substituents are given in Figure 4.
FIGURE 4A-F: EFFECT OF H-BOND DONOR, HYDROPHOBIC, ELECTRON-WITHDRAWING SUBSTITUENTS FOR COMPOUND 27; 4(A-C) AND FOR COMPOUND 44; 4(D-F). (BLUE CUBES: FAVOURABLE INFLUENCE ON ACTIVITY; RED CUBES: UNFAVOURABLE INFLUENCE ON ACTIVITY).
The predicted and actual activity was good for the developed model for both test and training set. The predictions of different activities have been classified according to the following residual scale (i.e. residual is computed as the difference between the experimental activity and the estimated activity): residuals less than 0.8 are considered as good predictions; residuals between 0.8 and 1.6 are considered weak predictions and residuals higher than 1.6 are considered poor predictions.
In this study, residual scale (figure 5) was in the rage of ±0.3 which is an indication of a good model.
FIGURE 5: RESIDUAL SCALE
Assessment of Pharmacophore quality: The quality of pharmacophore was analyzed using a set of parameters such as hit list (Ht), number of active percent of yields (%Y), percent ratio of actives in the hit list (%A), enrichment factor (E), false negatives, false positives, and goodness of hit score (GH) (Table 4). The false positives and true negatives are 3 and 8 respectively. EF and GF are 48.38 and 0.78 respectively, When GH score is higher than 0.7, the model is very good indicated the quality of the model and high efficiency of the screening test.
TABLE 2: STATISTICAL PARAMETER FROM SCREENING DECOY SET
S. No. Parameter
1 Total number of molecules in database (D) 1016
2 Total number of actives in database (A) 15
3 Total number of hit molecules from the database (Ht) 21
4 Total number of actives molecules in the hit list (Ha) 15
5 % Yield of actives [(Ha/Ht) ×100] (%Y) 71.4
6 % Ratio of actives [(Ha/A) ×100] (%A) 100
7 Enrichment Factor (EF) 48.38
8 False negatives [A-Ha] 0
9 False Positives [Ht -Ha] 6
10 Goodness of Hit score (GH) 0.7809
Virtual screening: A pharmacophore-based virtual screening has been carried to find out potential topoisomerase inhibitors. Hypothesis ADRRR.1 was used to carry out a query of ZINC "drug-like" database of 10,384,703 molecules, which includes a wide variety of chemical scaffolds. The sequential virtual screening performed in this study is schematically represented in a flowchart in Figure 6, from which we can witness the number of hits reduced after each screening step.
FIGURE 6: SCHEMATIZATION OF THE VS WORKFLOW
The initial filter using database query returned over 12009 hits. Next, various conformers of these hits were searched thoroughly for matches to pharmacophore model. While retrieving hits, matching of all five pharmacophoric features was made mandatory. A hit list of 1099 compounds matching the pharmacophore model was obtained. The virtual screening workflow (VSW) in Maestro was used to dock and to score the lead-like compounds. In the first step, Glide was run in high-throughput virtual screen mode. A total of 127 hits obtained after drug-likeness screening by the application of Lipinski's 'rule of five' and kept to go onto the next, Glide Single Precision (SP), stage.
The search retrieved 13 molecules were retained and docked using Glide Extra Precision (XP) mode. Among these, the molecules after visual inspection and estimated XP GlideScore ≥ −9.6, were considered as potential topoisomerase inhibitors and are shown in Figure 7.
FIG. 7: POTENTIAL TOPOISOMERASE INHIBITORS
Docking study of hits: The ligand interaction diagram of most active hit compound (ZINC09009213) is shown in Figure 8. It is observed that both the lead molecules were able to occupy the same sites occupied by the co-crystallized ligand (etoposide) molecule, indicating that this site was the most probable interaction site for these compounds. As presented in Figure 9, compound ZINC09009213 and ZINC09357466areinserted between the base pairs of DNA and may effectively blocking religation of the cleaved phosphodiester bond and hence as etoposide, therefore, the docking results suggest that the DNA site in Topo II-α is the more probable binding site for this type of molecule. So a mechanism of action similar to that of etoposide can be proposed for these lead compounds.
TABLE 3: DOCKING SCORE OF SELECTED HITS:
Sl. No. Entry ID XP G Score Docking score
1 ZINC09009213 -10.299378 -9.756578
2 ZINC09357466 -9.637271 -9.171671
5 Etoposide -8.298543 -8.298543
FIGURE 8: CRYSTAL STRUCTURE OF ZINC09009213 BOUND TO TOPOISOMERASE II (PDB: 3QX3)
FIGURE 9: PICTORIAL REPRESENTATION OF THE GLIDE XP DOCKING OF COMPOUND ZINC09009213 (GREEN) ZINC09357466 (YELLOW) ALONG WITH ETOPOSIDE (ORANGE) INTO ACTIVE SITE OF TOPO-II
To validate the procedure, and adequate scoring function selection, the co-crystallised ligand etoposide was removed and redocked it into the receptor (PDB-3QX3). It was found that this structure, when superimposed on the crystal structure, has a root mean square deviation (RMSD) of only 0.065673Å. Docking score of all lead molecules and etoposide are shown in Table 3. It can be seen that two of the lead molecules were having docking score higher than that of etoposide.
Predicted ADME Properties: The ZINC database was evaluated for drug-likeness of the lead molecules by assessing their physicochemical properties and by applying Lipinski's rule of five. Their molecular weights were <500 Daltons with <5H-bond donors, <10H-bond acceptors and a log p of <5; these properties were well within the acceptable range of the Lipinski rule for the final lead molecules.
Drug-like behaviour of the potential leads was assessed by analysing pharmacokinetic parameters required for ADME using QikProp 3.2 24. QikProp is frequently used in in-silico screening studies for ADME analysis of potential leads 25. QikProp properties for the final hits were calculated.
The molecular weights of all the lead molecules were in the range of 424–461. QP log Po/w shows the partition coefficient, which is important for the estimation of absorption and distribution of drugs within the body. For the lead compounds, the partition coefficient (QPlogP o/w) and water solubility (QPlogS), critical for estimation of absorption and distribution of drugs within the body ranged between 3.264 to 4.527 and −5.258 to −6.286.
Cell permeability (QPPCaco), a key factor governing drug metabolism and its access to biological membranes, ranged from 29.7 to 1207.5. QPPMDCK value which are considered to be a good mimic for the blood brain barrier ranges from 77.853 to 310.456. Overall, the percentage human oral absorption for the compounds ranged from 86.47 to 100 %. The standard drug etoposide the predicted percentage human oral absorption was only 41.52% all the virtual lead molecules retrieved from virtual screening has showed betted oral absorption.
All lead molecules shown pharmacokinetic parameters within the acceptable range defined for human use (see Table 4 footnote), thereby indicating their potential as drug-like molecules.
TABLE 4: ADME PROPERTIES OF SELECTED HITS
Zinc ID QPlogPo/w a QPPCacob QPlogS c QPlogHERG d QPPMDCK e % Absorptionf
09009213 3.268 180.725 -5.304 -6.668 77.853 86.47
Etoposide -0.806 335.935 -2.375 -4.241 152.155 41.52
aMolecular weight. aPredicted octanol/water partition co-efficient log p (acceptable range: −2.0 to 6.5). bPredicted Caco-2 cell permeability in nm/s (acceptable range , <25 is poor and >500 is great). cPredicted aqueous solubility; S in mol/L (acceptable range: −6.5 to 0.5).
CONCLUSION: In conclusion, A ligand-based pharmacophore model was generated for the series of 2, 4, 6 pyridine derivatives with topoisomerase inhibitory activity to reveal the structural features responsible for biological activity. A five-point pharmacophoric feature with three aromatic rings (R) and hydrogen bond acceptor (A), hydrogen bond donor (D) was developed and evaluated by 3D-QSAR model. It resulted in good statistical significance and predictive ability with R2 = 0.9101 and Q2 =0.8276. The developed pharmacophore was also validated using EF and GH score. Furthermore, visualization of the 3D-QSAR model in the context of the molecules under study provided details of the relationship between structure and activity and thus provides explicit indications for the design of better analogues. We have also shown how straightforward virtual screening can be used to rapidly retrieve compounds with a desired activity from a large compound pool with limited experimental effort.
QikProp pharmacokinetic prediction provided physicochemical properties along with BBB permeability and percentage oral absorption. These parameters are helpful to find out bioavailability and toxicity prediction in human body. Compared with currently known active compounds, these newly identified inhibitors have significant potential for further development.
ACKNOWLEDGEMENT: The authors are thankful to University Grants Commission (UGC), New Delhi, for providing financial support through UGC Major Project.
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Teli MK, Rajanikant GK, Identification of novel potential HIF-prolyl hydroxylase inhibitors by in silico screening. Molecular Diversity 2012; 16:193–202.
Esteves-Souza A, Claudio E. Santos R, Cistia CN, Silva DR, Carlos M R. Sant Anna: Solvent-Free Synthesis, DNA-Topoisomerase II Activity and Molecular Docking Study of New Asymmetrically N,N'-Substituted Ureas. Molecules2012; 17:12882-12894.
Glide, version 5.5 (2009) Schrödinger, LLC, New York.
Golbraikh A, Shen M, Xiao Z, Xiao YD, Lee KH, Tropsha A. Rational selection of training and test sets for the development of validated QSAR models. Journal of Computer Aided Molecular Design 2003; 17:241-53.
QikProp, version 3.2, Schrödinger, LLC, New York, NY, 2009.
Bandgar B, Hote B, Gangwal R, Sangamwar A: Synthesis, biological evaluation, and pharmacokinetic profiling of benzophenone derivatives as tumor necrosis factor-alpha and interleukin-6 inhibitors. Medicinal Chemistry Research 2012; 21:1-5.
Dev S and Dhaneshwar SR: Identification of potent virtual leads as Topoisomerase-II inhibitors using Pharmacophore Modelling, Molecular Docking and ADME studies. Int J Pharm Sci Res 2013: 4(8); 2939-2954. doi: 10.13040/IJPSR. 0975-8232.4(8).2939-54
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Page No: 2939-2954
Authors: Sanal Dev and Sunil R. Dhaneshwar*
Authors Address: Faculty of pharmacy- university of Aleppo. Department of analytical and food chemistry, Syria
Email: sunil.dhaneshwar@gmail.com
Received: 09 March, 2013
Revised: 12 June, 2013
Accepted: 21 July, 2013
DOI: http://dx.doi.org/10.13040/IJPSR.0975-8232.4(8).2939-54


This page presents the Google satellite map (zoomable and browsable) of Garlefan Farm in Canterbury district in New Zealand.
Geographical coordinates are -43.9865 and 171.7952 and its feature type is Hstd. More information below.


Borders joined the organization in October after leaving her position as the president of the WNBA.
The resignation of Lisa Borders as the CEO and president of Time's Up followed sexual misconduct allegations against her son, according to a statement from the gender equality initiative.
"Lisa Borders informed members of TIME'S UP leadership that sexual assault allegations had been made against her son in a private forum," according to a statement from the organization released early Friday.
Borders made the decision to resign within 24 hours and "we agreed that it was the right decision for all parties involved," the group said.
"All of our actions were fully guided by our support for survivors," the statement said, which did not elaborate on the nature of the allegations.
She did not detail the nature of the family concerns.
Borders did not immediately respond to request for comment.
She joined the organization in October after leaving her position as the president of the WNBA.
Time's Up was formed last year following the #MeToo movement and the wave of allegations against powerful men in Hollywood. The group has a legal fund which aids workers seeking to fight workplace harassment.
Time's Up said in its statement Friday that it strongly encourages anyone who has experienced sexual harassment, assault or retaliation in the workplace to contact the legal defense fund for assistance.
The group said its COO, Rebecca Goldman, would serve as interim CEO following Borders' departure.


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NGC 669 (другие обозначения — UGC 1248, MCG 6-5-4, ZWG 522.4, IRAS01443+3519, PGC 6560) — галактика в созвездии Треугольник. Открыта Эдуаром Стефаном в 1883 году. Описание Дрейера: «довольно тусклый, довольно крупный и сильно вытянутый объект».

Этот объект входит в число перечисленных в оригинальной редакции «Нового общего каталога».

Вместе с NGC 662, NGC 668 и несколькими другими галактиками входит в скопление галактик , которое является частью сверхскопления Персея-Рыб.

Галактика NGC 669 входит в состав группы галактик . Помимо NGC 669 в группу также входят ещё 34 галактик.

Примечания

Спиральные галактики
0669
Треугольник (созвездие)

redpajama_set_name: RedPajamaWikipedia


This piece is from our HUGE selection of unstretched Original Oils and Print on Canvas. All from different artist all over the world, some well known, some not. If we had to list everything we have, you would see over 10,000 pieces of art from different Artist and Subjects. Thank you for taking time to look at our art. For more great pieces, please visit our Ebay store. Have a great day!


Lindsey A. Dentino received her Bachelor of Arts in Psychology from Arizona State University. After graduation Lindsey worked as an adoption specialist. As an adoption specialist, Lindsey facilitated the adoption special needs children who were in the state foster care system. After working as an adoption specialist for three years, Lindsey moved to San Diego to attend California Western School of Law where she earned her Juris Doctor in 2017. While obtaining her J.D. at California Western, Lindsey volunteered with The Community Law Project, which provides free legal information to low income and indigent communities throughout San Diego County.
Lindsey began working at Moore, Schulman & Moore, APC, through her participation in California Western's clinical internship program as a law student. After graduation, Lindsey returned to the firm as a law clerk and was later hired as an associate attorney.
Lindsey has assisted the attorneys at Moore, Schulman & Moore, APC in a variety of recent cases and has experience with family law issues including dissolution of marriage, child custody and visitation, move-away requests, child support, spousal support, and pre and postnuptial agreements.
In her free time Lindsey enjoys hiking, horseback riding and cheering on the Arizona State Sun Devils.


I'm purchasing this item, But there is no yarn or any supplies list for this item. I would like to know the color of the Cross. It looks like it could be a goldish yellow color, but I'm not sure. I wished you could find the yarn color list for this product so that I can get the right colors for it, or any type of substitute color in yellows, or gold for the cross. Thank you for your input.
This is all that is listed on the chart itself and the supply list doesn't give a brand of yarn to use; just states 4 ply yarn. Hope this helps! Thanks.


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A San Antonio man spoke out against Texas ending mask mandate. Then his noodle shop was vandalized with 'ugly display of hate.'
John Bacon
A San Antonio man whose restaurant was vandalized with racist graffiti days after he criticized Texas Gov. Greg Abbott's decision to rescind a statewide mask mandate says he has no regrets and will continue to speak out.
Mike Nguyen appeared on CNN last week, saying his stance was based on the safety of his staff, himself and the community. Since that interview, he said he has faced death threats on social media. On Sunday, Nguyen arrived at his Noodle Tree restaurant to find "no mask," "go back 2 China" and other hateful messages scrawled in red paint on his storefront.
"I was born here and I'm not Chinese," Nguyen, whose background is half Vietnamese and half French, told USA TODAY on Monday. "It's not right that as an American I can't state my opinion without being attacked."
A string of attacks against Asian Americans has rattled California's Bay Area: This group of 700 volunteers is taking action
More than 535,000 Americans have died from the virus, including more than 46,000 Texans. Abbott, however, cited declining numbers of deaths, hospitalizations and infections in announcing earlier this month he was going to "open Texas 100%." No masks required, no restrictions on businesses.
Nguyen said he believes more confrontations are ahead in Texas as shop owners try to protect employees by requiring customers to wear masks without a mask mandate from Abbott.
"Instead of being a leader, he wants us to deal with it," Nguyen said. "The blood is on his hands if anything happens."
Neighbors came to Nguyen's aid, helping him clean up the mess. Mayor Ron Nirenberg thanked them, saying the effort "proved that we're better than this one hateful act."
"We must work together to eradicate racism from our city," Nirenberg said.
'It's wrong, it's un-American and it must stop': President Biden denounces attacks
The Chinese American Citizens Alliance and the Asian American Alliance of San Antonio released a joint statement denouncing the "ugly display of hate."
"This ignorant act had not only damaged a business, but it also dishonored the distinguished reputation of San Antonio," the groups said.
Incidents of hate against Asian Americans have risen sharply during the pandemic, sparking outrage and activism in the Asian American community and spurring some lawmakers and organizers to respond to the threat.
"Racially motivated violence and other incidents against Asian Americans have reached an alarming level across the United States since the outbreak of COVID-19," a United Nations report released last year found. It cited increases in vandalism, physical assaults and robberies against people, businesses and community centers.
Last week, President Joe Biden condemned the violence Asian Americans have endured throughout the pandemic, saying "it's wrong, it's un-American and it must stop."
Nguyen said he is encouraged by the support he received. He had to open late Sunday due to the cleanup, but said he was overwhelmed with customers. When he sold out, some just left a donation.
"The day started with negativity but ended positive," he said. "People here, we are not going to tolerate racism and hate."

redpajama_set_name: RedPajamaCommonCrawl


Taiwan's PRESOTEA: Now Serving Brew-To-Order Tea in Robinsons Magnolia!
What is PRESOTEA and how is it different from the hundreds of milk tea shops in the metro?
Presotea is a well-known bubble tea chain in Taiwan with almost 400 shops internationally. What makes Presotea different from other brands is their unique brew-to-order style! They use this cutting edge tea-brewing machine known as the Teapresso-- each cup is pressed with high quality tea leaves using espresso machine technology to maximize flavor and freshness-- to concoct the freshest and healthiest tea drinks. Compared to other tea shops that brew tea in bulk, Presotea uses one tea bag per order. Every cup is freshly made in front of the customer so they'd be sure to enjoy only the freshest flavors from the tea leaves. WOW!
It's located on the 2nd level of Robinsons's Magnolia! It's near the escalators.
Smelled all of these fresh tea leaves. So fresh!
They made us try their 10 best-sellers-- they have milk tea, fruit tea and coffee! So good!


5 Reasons LEGO Shakes Up Imagination!
It's been a serious love affair in our house with LEGO going on for 3 + years now. I mean when I say we're dedicated lovers of the brand I mean it. We've shed blood, sweat and tears over lego creations… that's just what happens when you mix a passionate soul (Waylon our 5 year old) with the ingenuity and creative expression produced by a LEGO brick.
Waylon loves to follow the directions on the box when he firsts receives them but once his masterpiece is crafted he usually adds his own artistic expression to it by building outside the boxed instructions adding bricks to different parts that all make up something amazing in his mind and frankly mine too since I'm always blown away at what he creates. Zeke our 2 year old is definitely not left out of this picture as he wants to do everything his big brother does, he's taken up to building his own special creations and playing with his "guys" for hours. It was so sweet to see Waylon helping him build his new train while teaching him to count the numbers on the side of the bricks. Precious brother bonding right there.
So since my kids have had a fascination with Legos for a while now I developed a list of 5 reason how I've seen LEGO shake up their imagination and why it benefits their brains and lives!
Building with Legos has provided hours of playtime in our house. Boom that's a gift in itself.
Fine Motor Skills: I've seen our 5 year old's fine motor skills take off in Kindergarten evidenced by insanely good drawings, coloring in the lines, and penmanship.
Community: Playing with LEGO has helped our kids connect to other kids that play with Legos. They bond over creations and even trade certain guys and legos.
LEGO have taught our kids how to follow instruction but also use their imagination to create beyond the box of instructions in turn giving them greater satisfaction and joy in what they create.
Pride: The look of pride on Waylon's face after he finishes building a creation is priceless. He has labeled himself a master builder…I love seeing the confidence it builds in him.
I'm sure the love affair with theses bricks will continue for a long time. If you're looking for a gift that keeps on giving this year we highly recommend LEGO!
*This was a sponsored post for LEGO but as always all words, thoughts and experiences are my own.


In a time when online chat support is a key part of customer service offerings, ensure your employees have the reading and writing skills needed to have positive interactions with your customers with our 35-minute written English test.
Using a computer in either online or offline mode, candidates respond to workplace-related situations that require written English, including taking notes, summarizing, and responding to emails.
With test results available in minutes, corporations and call centers trust the Versant Writing Test's advanced auto-marking to make an unbiased evaluation of candidates' writing abilities.
Test takers respond to questions and provide writing samples via computer allowing us to evaluate each candidate's grammar, vocabulary, organization, voice & tone, and reading comprehension. Millions of data points are collected throughout the Versant Writing Test that feed into our patented scoring algorithm.
In this task, test takers read a sentence that has a word missing, and they supply an appropriate word to complete the sentence.
Performance on this task feeds into the vocabulary score.
Performance on this task feeds into the grammar and reading comprehension scores.
In the Email Writing task, test takers will read a description of a situation and write an email addressing the issues described in the situation.
Performance on this task feeds into the grammar, vocabulary, voice & tone, organization, and reading comprehension scores.
Other question types in the Versant Writing Test include Typing and Dictation.
The Versant Writing Tests are very useful in developing email skills. We use the tests to create content for our custom email 2.0 classes. The most useful feature for me? The assessment of email "Voice and Tone" - such email skills are the ones that make all the difference for employees working across cultures and teams. I see the Versant Writing Test as a key tool to help us maximize the return on our time and training dollars.


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Egypt: Mubarak sentenced to life in prison
Jun 2, 2012 | Politics, Uncategorized | 0 |
Hosni Mubarak
2 June 2012. Hosni Mubarak was sentenced to life in prison Saturday for failing to stop the killing of protesters during the uprising that forced him from power last year. The ousted president and his sons were acquitted, however, of corruption charges in a mixed verdict that swiftly provoked a new wave of anger on Egypt's streets. They were acquitted on corruption charges because of a 10-year statute of limitations that had lapsed since the alleged crimes were committed.
Revolutionary groups and the powerful Muslim Brotherhood have called for a massive protest at Tahrir Square, the heart of the uprising, at 5 p.m. local time.
After the sentencing, the 84-year old Mubarak suffered a "health crisis" while on a helicopter flight to a Cairo prison hospital, according to security officials who spoke on condition of anonymity because they were not authorized to speak to the media. One state media report said it was a heart attack, but that could not immediately be confirmed.
Mubarak finally moved to the Torah prison hospital
Watch this video: Hosni Mubarak, Egypt's Ousted President, Sentenced To Life In Prison
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and this: Egyptians react to Mubarak verdict
PreviousUSA: Assessor John Noguez steps aside amid corruption probe
NextAfghanistan: EU blocks aid to Police
Canada: Construction corruption charged in Suburban Montreal
Pakistan PM indicted for contempt
Brazil: Ex-president Lula guilty in 2nd corruption case


This cabochon has been cut and polished by hand in natural Peacock Turquoise/Chalcosiderite from Nevada. The bottom is flat and unpolished.
The coin in the photo is a US dime (18 mm) for size reference.
Join us on Instagram @stonephase every Tuesday at 8:00 pm Central time for our live sales. This is where we sell the majority of our work.


Electricity generated by the use of the thermal energy released from the fission of nuclear fuel in a reactor. Data are reported as net consumption as opposed to gross consumption. Net consumption excludes the energy consumed by the generating units. .


Alan F. Rozich explains in his new book, Other Inconvenient Truths Beyond Global Warming, that, although global warming is important as "An Inconvenient Truth," there are "Other Inconvenient Truths" affecting the functionality of society. These other truths are much more likely to adversely impact human civilization far sooner than global warming.
Global warming is a symptom with CO2 as its poster child. Cognizance of global warming is important as it rightfully earned the categorization, "An Inconvenient Truth." However, there are a number of "Other Inconvenient Truths" that comprise a "Super Nexus". The Super Nexus is an emerging economic and environmental reality requiring both cognitive understanding and action.
What does this all mean? What is the answer? It is that resolving the super nexus needs to be the focus and not CO2. Resource security is pivotal for societal functionality. If left unchecked, one possible consequence is that resource costs will skyrocket, threatening the very fabric of societal functionality. Although the challenges are daunting, viable solutions are within our grasp.
Dr. Alan F. Rozich has been working in the environmental and renewable space for over 30 years. Rozich is a world-renowned expert on biological processes with vast experience in biological conversion systems. Rozich obtained degrees at The Ohio State University and University of Delaware. He is an accomplished author with previous books and numerous articles and presentations to his credit. Rozich is currently Chief Science Officer of In-Pipe Technology of Chicago, Illinois (In-Pipe) and resides in West Chester, Pennsylvania with his wife, Gemma.
Other Inconvenient Truths Beyond Global Warming is available in softcover (ISBN 978-1- 935355-15-1) and Ebook (ISBN 978-1-935355-420-6) from New Shelves (http://www.newshelves.com), Amazon, and Barnesandnoble. This book can also be ordered from most bookstores around the United States and United Kingdom.


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Category Archives: Secretin Receptors
The M-protein is the major reference measure for response in multiple
The M-protein is the major reference measure for response in multiple myeloma (MM) and its correct interpretation is key to clinical management. 23%). ASIPs were not associated with new clonal plasma cells or other lymphoproliferative processes, and molecular remissions were documented. This is the first time this phenomenon has been seen with regularity in non-myeloablative therapy for MM. Analogous to the ASCT experience, ASIPs do not transmission incipient disease progression, but rather herald strong response. hybridization (FISH) testing were performed centrally at New York Presbyterian Hospital laboratories SB939 before study enrollment and to confirm CR, when appropriate, as explained previously (Niesvizky = 72) The baseline monoclonal Ig, the altered protein banding pattern witnessed, the period of treatment until the time of the first appearance of an ASIP, and the treatment response for each patient with one or more ASIPs was recorded. (Observe Supplementary table for any case-by-case compilation of all observed ASIPs.) The ASIPs that were seen were diverse SB939 in their type, number, and period of monoclonal Ig(s) detected. The median duration of ASIP appearance was relatively prolonged at 236 days, yet varied widely per individual individual, with a range from as short as 27 days to as long as 758 days. A total of 7 patients (29%) exhibited ASIPs with monoclonal Ig bands that frequently disappeared and reappeared periodically throughout the course of treatment, indicating that the protein level fluctuated near the lower limits of detection by immunofixation. An example ASIP time course is shown in Fig 1B which shows alternating M-proteins appearing and disappearing over time in a reciprocal fashion; comparable patterns was also seen in other patients with an ASIP during the course of treatment. The appearance of oligoclonal banding over time was also a common phenomenon, occurring in 10 patients (42%). ASIPs tended to appear after a continuous course of therapy, with the median first appearance after 6 months of treatment. At the last follow-up, 10 patients (42%) still experienced evidence of an ASIP. The isotype distribution of initial and newly discovered monoclonal Ig for sufferers who continued to build up ASIPs is normally depicted in Fig 2A,B. The brand new Ig distribution was approximately equivalent to the initial study population using a preponderance of IgG-. Nevertheless, the introduction of a fresh IgM band within the ASIPs was observed, with 12 sufferers (50%) developing either IgM-, IgM-, or both, on do it again serum electrophoretic examining. In summary, ASIPs had been noticed after extended classes of Parrot therapy typically, they fluctuated and persisted in level for a long time, and had been protean within their Ig make-up. Fig 2 Evaluation of monoclonal proteins creation and response for sufferers with atypical serum immunofixation patterns (ASIPs). SB939 The distribution of M-protein for sufferers on the initiation of lenalidomide and dexamethasone with clarithromycin (Parrot) therapy … Relationship of ASIPs with scientific response rate Sufferers who created ASIPs acquired a considerably better reaction to Parrot versus non-ASIP sufferers (= 00001), (Amount 2). Every affected individual with an ASIP attained a minimum of a Rabbit polyclonal to Wee1. incomplete response (PR) weighed against an 85% response price within the non-ASIP group. Additional analysis demonstrated that the great PR or better price (96% vs. 60%; = 00017) and CR price (71% vs. 23%; = 00002) had been also better for the ASIP versus non-ASIP sufferers, respectively. Many sufferers with an ASIP continuing to get tumor-burden decrease with additional cycles of Parrot treatment even following the ASIP initial appeared. The level of Parrot therapy ahead of advancement of first ASIP didn't correlate with response price (= 050). To verify the reaction to treatment, bone tissue marrow aspirates had been frequently examined for residual disease by karyotype and Seafood evaluation when feasible. Overall, 18 pre- and post-treatment samples were evaluable out of the 24 individuals with an ASIP (Table III C should be in product). In all cases, there was resolution of prior karyotypic abnormalities, with the exception of a prolonged pericentric inversion of chromosome 9 in a patient. All FISH recognized abnormalities in the.
June 7, 2017 Ian Oliver	Rabbit polyclonal to Wee1., SB939
Previously, we have demonstrated the current presence of anti-calcium-sensing receptor (CaSR)
Previously, we have demonstrated the current presence of anti-calcium-sensing receptor (CaSR) antibodies in patients with autoimmune polyglandular syndrome type 1 (APS1), an illness that's characterized partly simply by hypoparathyroidism involving hypocalcemia, hyperphosphatemia, and low serum degrees of parathyroid hormone. these peptides was verified in ELISA. Anti-CaSR antibody binding sites had been mapped to amino acidity residues 41C69, 114C126, and 171C195 on the N-terminal from the extracellular area from the receptor. The main autoepitope was localized in the 41C69 amino acidity sequence from the CaSR with antibody reactivity confirmed in 12 of 12 (100%) APS1 sufferers with anti-CaSR antibodies and in 1 AHH individual with anti-CaSR antibodies. Small epitopes were situated in the 114C126 and 171C195 amino acidity domains, with antibody reactivity proven in 5 of 12 (42%) and 4 of 12 (33%) APS1 sufferers, respectively. The outcomes indicate that epitopes for anti-CaSR antibodies in the AHH affected individual and in the APS1 sufferers who were examined are localized in the N-terminal FST from the extracellular area from the receptor. Today's work provides confirmed the successful usage of phage-display technology in the breakthrough of CaSR-specific epitopes targeted by human anti-CaSR antibodies. ? 2010 American Society for Bone and Mineral Research. gene.(5) In our previous study, anti-CaSR antibodies were detected using immunoprecipitation assays in 12 of these patients.(16) One AHH patient (female, age 73 years) with a positive antinuclear antibody titer of 1 1:5120 and anti-ribonuclear protein antibodies developed hypercalcemia, an elevated level of intact PTH and marked hypocalciuria (10 to 40 mg/24 h), prompting investigation of possible AHH. Three individual serum samples were available, and anti-CaSR antibodies were detected in each of them (unpublished data) using immunoprecipitation assays.(16) Twenty healthy individuals (9 male, 11 female; mean age 32 years, with range 24 to 48 years) who experienced no present or past history of autoimmune disorders were included as controls. No individual experienced anti-CaSR antibodies when tested in immunoprecipitation assays.(16) Specific anti-CaSR antibodies Anti-CaSR rabbit polyclonal antibody against a synthetic peptide corresponding to amino acids 12C27 of the rat CaSR was purchased from Alexis Biochemicals (Nottingham, UK). The antibody has cross-reactivity with the human CaSR. Anti-CaSR mouse monoclonal antibody against a synthetic peptide corresponding to amino acids 214C235 of the human CaSR was obtained from Acris Antibodies (Herford, Germany). Phage-display library construction Vector pComb3(22) was used to construct a phage-display library of CaSR peptides. The vector is designed to allow the expression of cloned DNA fragments and the subsequent surface exposure of the peptides encoded therein on phage particles. For surface expression, DNA fragments are required to be cloned in frame with the PelB leader peptide PD153035 and the gene III phage coat protein present in pComb3 at PD153035 the N- and C-terminal, respectively. To construct a CaSR cDNA fragment library in pComb3, full-length CaSR cDNA was prepared from pcCaSR-FLAG(16) by restriction of the plasmid with XL1-Blue cells (Stratagene, La Jolla, CA, USA), as explained by the manufacturer. The library size was estimated by plating out samples of electroporated cells onto Luria-Bertani (LB) agar(23) made up of 100 g/mL ampicillin and 10 g/mL tetracycline. To prepare the CaSR peptide phage-display library, the electroporated cells were PD153035 incubated for 1 hour at 37C before superinfection with 1??1012 plaque-forming units of VCMS13 helper phage (Stratagene) at 37C for 15 minutes. The culture subsequently was transferred to 100 mL of LB medium(23) supplemented with 100 g/mL ampicillin, 10 g/mL tetracycline, and 10 g/mL kanamycin. After overnight incubation at 37C, the lifestyle was centrifuged and phage precipitated in the supernatant with 0.2 amounts of 20% (w/v) polyethylene glycol 4000/2.5 M NaCl. The phage had been resuspended in 2-3 3 mL phosphate-buffered saline (pH 7.4; PBS, Sigma, Poole, UK) and kept at ?20C. The phage titer was dependant on infecting log-phase XL1-Blue with an aliquot from the phage-display collection and plating out examples onto selective LB agar. Biopanning tests For biopanning tests, individual sera or pet anti-CaSR antibodies (10-L aliquots) had been put on the wells of Corning polystyrene 96-well microtiter plates (Bibby Sterilin, Ltd., Mid Glamorgan, UK) in 50 L of buffer filled with 1.5 mM Na2CO3, 3.5 mM NaHCO3, and 3.0 mM NaN3 (pH 9.2). Plates had been incubated at area heat range for 2 hours to permit antibody PD153035 binding before cleaning with PBS/0.05% (w/v) Tween 20 (PBS/Tween). To stop any nonspecific phage binding in the task afterwards, 400 L 2% (w/v) bovine serum albumin (BSA) in PBS was put into the wells, and incubation at area temperature continuing for 2 hours. The wells had been rinsed once again with PBS/Tween prior to the addition of the 100-L test of phage-display collection filled with 1??1010 colony-forming units (cfus). Plates had been PD153035 incubated right away at 4C to permit the connections of anti-CaSR antibodies with peptides shown on the areas from the phage contaminants. The wells were washed with PBS/Tween to eliminate unbound phage extensively. Bound phage after that had been eluted with 150 L 100 mM HCl (altered to pH 2.2 with great glycine) and neutralized with 9 L 2 M Tris-HCl (pH 7.6). The phage suspension system was utilized to infect 2 mL of exponentially subsequently.
June 3, 2017 Ian Oliver	FST, PD153035


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Goniothalamus kostermansii este o specie de plante din genul Goniothalamus, familia Annonaceae, descrisă de Mat-salleh. Conform Catalogue of Life specia Goniothalamus kostermansii nu are subspecii cunoscute.

Referințe

Goniothalamus


Earth has finally accomplished FTL! While preparing to launch our first global space program we were horrifically attacked by the Erkelians, a brutal warlord race bent on enslaving our people and depleting the Earth of its resources. By pure luck we were able to defeat our attackers, but we know it won't be long before they are back and with a bigger fleet. Our training facility gone, hundreds of trained explorers dead or wounded, and all but one of our FTL ships salvable, our hope lays in the hands of one woman...the Atomic Starlet, Galactic Ambassador to the stars! From the wreckage our engineers were able to recover parts to restore one of three tools. Knowing that she only has herself to rely on in a pinch out there she stretches out her hand to the Redshirts toolkit. It's ability to repair complicated machinery (or disrepair it if needed), open locked doors, and generally wreak havoc with technology might be just what's called for. Do you choose to grab the Redshirts toolkit?


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Aid groups: demolishing Syrian refugee settlements in NE Lebanon could affect thousands of children
July 9, 2019 /0 Comments/in All News, Featured 2, Middle East, Other News /by asadian
SHAFAQNA- Press TV: A group of international aid agencies said that Lebanese military units have begun demolishing Syrian refugee settlements in northeast Lebanon following the expiry of a government ultimatum, warning that the move could affect thousands of children.
"At 4:30 a.m. [local time] on July 1, military units moved into several camps in Arsal and demolished at least 20 homes," said a joint statement by seven aid groups, including Save the Children, the Norwegian Refugee Council, and Oxfam, on Monday.
The aid groups also expressed their deep concern about the demolitions, saying they "fear that this is the start and more demolitions will take place tomorrow."
According to the statement, an estimated 15,000 Syrian refugees, including at least 7,500 children, would be affected by demolitions of some 3,000 units.
Separately, Bassel al-Hujeiri, the head of the Arsal municipality, said that Monday's demolitions had been very "limited" and impacted only huts made almost completely out of cement.
Beirut's ultimatum of July 1 had prompted refugees to demolish their own shelters in the weeks leading up to the deadline, the statement said, adding that since June 27, less than half of the hard huts in Arsal had been demolished by their dwellers.
"Depriving refugees of their already very basic shelter and leaving them out on the streets is not a solution. The demolitions in Arsal come in the context of deteriorating conditions for Syrian refugees, who in the past months have faced an increasingly coercive environment making their lives even harder," the statement added.
Lebanon, with a population of around 4 million, hosts some 1.5 million Syrian refugees. Facing a crisis-hit economy, Lebanon is toughening its enforcement of rules governing the refugees.
Beirut only allows building informal camps in a bid to prevent refugees from settling permanently.
Some Lebanese politicians have already called for mass returns of Syrian refugees to their homes after fighting and violence ended in many parts of the war-torn country over the past three years.
While some refugees have gone back to Syria, many others appear to be reluctant to return, with some citing fears of retribution or a fresh upsurge in violence and fighting.
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Daesh militants, family members of Kosovo brought back from Syria+ Video
Tags: Bassel al-Hujeiri, children, Lebanese military, Oxfam, Syria, Syrian refugee, the Norwegian Refugee Council
https://en.shafaqna.com/wp-content/uploads/2019/07/68590ea5-e28d-4d30-996e-fc1ef7fa05f1.jpg 365 650 asadian https://en.shafaqna.com/wp-content/uploads/2018/10/new-logo-s-en-fine.png asadian2019-07-09 12:49:012019-07-09 12:49:23Aid groups: demolishing Syrian refugee settlements in NE Lebanon could affect thousands of children
Video: Emotional return to Shrine of Lady Zainab (S.A)
Suspected US-led airstrikes claim 23 civilian lives in North Syria
Developing countries host 85prc of the world's refugees +Video
Syrian MP: The Balance of Power will Change after Receiving S-300
At least 29 civilians killed in US airstrikes on Syria's Raqqah
US, UK, France targeted Syria with 110 missiles: Syrian Comdr


The Washington is an acrylic fleece version of our traditional Old Friend Loafer moccasin. Available in men's sizes 8-14. The Washington is hand stitched with cinch adjustable tie and cushioned midsole for a snug, comfortable fit. The durable rubber outsole ensures long wear.


Moritz might after induction cookware to Schoneck I have heard nothing from him but I believe upper hand the enjoy the induction cookware nerve strengthening air to me to likes she will is said to be induction cookware beneficial effect on the millie corretjer organs.At the present time, the corporate office of the company is based in Austin, TX.Now, in an ironic circle, applications once more have peoplehidden inside them.This general disposition is seen most distinctly in eastern Morocco and Algeria.Possibly because they never went.
He is very committed to doing everythinghe can to better his people's situations while also defending the Jews andtheir claim to the land as well.
Include hills to increase your strength and prepare for the Gateway Bridge climb.Low serum cholesterol concentration and short term mortality from injuries in men and women.
I-take note of the fact that you consider Spiegel the greatest and most wonderful news magazine of all times and lands.These features resonate among users in human resources, sales and marketing, and training departments, CEO Adam Slutsky says.Since her retirement she has become interested in the tedmiques and design motifs of the people of prehistory and is concentrating on relief tiles using the stamping techniques found in cultures around the world.The point is still relevant.
The Administrator would be appointed by the City Council taking the place of the elected City Clerk and City Treasurer positions.The reason troops can pull out is because of the surge that Republicans supported.
This follows because John baptized many people before receivingofficial confirmation from God that Jesus was indeed the Christ.This unlikely couple's homicidal urges are then further complicated when god knows how, but they fall in love.Places likesome of the satellites of Jupiter,like Europa and Ganymede where the surfaceof the planet is covered with a mixture of ice and rocky debris,and certainlyon Europa we think that that crust of ice might be lying on top of perhapseven fluid water.Since he was unable to read maps with any accuracy he was unable to pinpoint on a map of the mountain where it was that he had seen and climbed on the Ark.Pour the yeast mixture into the center and the egg yolks, and with a large spoon, gradually stir in the flour into the liquid ingredients.Macrina in Uniontown, Pa.Truth be told, it would fare better in the market as RWD.This proposed rule is astonishing.It's entirely possible that Buckeye fans in the area may already know that there are much more fun places to see Ohio State games.
Check out the line of Sony Vaio CR Laptops or the NR Series for the price savvy shopper.


Quasi-periodic features of the inter-annual and inter-decadal spatial-temporal variability of the wind speed absolute value (W) in the Black Sea area are assessed based on a series of 60–100-year-long direct observations performed at eight coastal stations, and also on the reanalysis data obtained from the NDP-048, WDC, NCDC, NCEP/NCAR (1948–2011) and MERRA (1980–2015) arrays. Three groups of oscillations with the periods T about 60–90, 20–40 and 3–15 years that present in the W direct data are shown by means of the wavelet analysis. As for long-period oscillations (Т ~ 60–90 years), possible relations of such quasi-periodicity with the phases of the North Atlantic Oscillation index characterizing the atmospheric circulation features are investigated. Calculated according to the MERRA array reanalysis, the mean anomalies of January – February average W series separately for groups of years with the values of the NAO index > +1 and ←1 demonstrated in their dynamics the values of different signs for the same Black Sea areas. It is revealed that during the periods when the index positive phase is mainly predominant, the W positive anomaly is formed over the south-western, south-eastern and eastern parts of the sea adjacent to the coast at a distance 20–100 km from it, and the W negative anomaly – over the rest of the sea. During the years when the index phase is mainly negative, the signs of the W anomalies over almost the same regions change to the opposite ones.
the Black Sea, wind speed module, inter-annual variability, quasi-periodicity, North Atlantic Oscillation.


You might think your hiding spot is safe, but burglars know your tricks. Be sure you're not hiding your valuables in such obvious places.
You might think these things aren't a big deal, but they can take a toll on your body over time. Do you have any of these bad habits?
On busy days, it's so tempting to hit up a drive-through so you can skip cooking and cleanup time. But you can also skip that stuff with a slow cooker. With these tips on how to master that handy kitchen appliance, dinner will be way more satisfying, too.
Paging Dr. Google. According to Medicare Health Plans, these are the most Googled conditions in each state, and that can sometimes predict where outbreaks are going to happen next.
Frequent fliers share their favorite tips to ensure a smooth travel day.
Belly fat affects your risk for heart disease, diabetes, and other serious health issues. These bad habits can increase your chances of developing belly fat or prevent you from losing it.
From the high prices at duty-free to the free unadvertised water and WiFi, here are the secrets airports won't tell you.
We know a LOT about science, but there's still so much that we don't know. These mysteries have stumped scientists throughout the ages—and will leave you scratching your head.
You can't open your kitchen cabinet door and have a good mood jump out at you, but a few items on those shelves can help you feel more upbeat.
If you've ever second-guessed yourself while trying to spell words like "receipt" and "license" and turned to Google for help, you're far from the only one.
We found out how to get a six pack without doing endless crunches. With these expert-back tricks, you'll have killer abs in no time.
These history questions are harder than you'd think, and you probably won't find the answers in your old textbooks. If you can get 12/16 correct, you're an official history buff.
You may know not to wave around a firearm or joke about having a bomb in your carry-on, but it's still surprisingly easy to mess up at the airport.
Get the best results by avoiding these common errors with your dishwasher, microwave, and other items in your kitchen and beyond.
Everything from your diet to your friends to your very attitude has major effects on your longevity. Find out which habits and traits will give you a better shot at hitting that 100 mark.
Don't bother looking for an expiration date on these foods. They can stay in your pantry and kitchen cabinets and never expire.
Swiffer products may be convenient and easy to use, but cleaning experts say they're not safe for all surfaces. Here's what to use instead.
Everyone has the occassional sleepless night, but if disrupted sleep persists it could be a symptom of a more serious health condition.
Even though the answers were very wrong, all of these students should at least get some points for creativity.
Don't commit to staying in a hotel until you've scoured the room for the conveniences and health risks that travel experts always look for.
If you find yourself leaving the warehouse store with more than you originally bargained for, you're not alone. Costco employs retail tricks to encourage customers to spend more.
Concerned about your parents' health? Learn how to approach your aging parents with these key steps.
If you try to avoid germs by using your foot to flush public toilets or your knuckle to press elevator buttons, prepare to be disappointed.
You're already recycling paper, bottles, and cans, but there's so much more that you can keep out of landfills.
You may be increasing the risk of someone robbing your house without even realizing it. These not-so-obvious indicators can make your home a target for thieves.
It may come as a surprise, but all of these things are legal in the U.S., at least in some parts of the country.
Panera Bread is more than just bread bowls and free WiFi! Here's everything you need to know about the eatery.
The most important documents of your life aren't always easy to retrieve from their original source or even online. It's up to you to stash your documents for safe keeping.
Antibacterial wipes are a time-crunched cleaner's best friend—but here are ten things you should never use them on.
The rules of etiquette change with the times. Here's what you need to know about the proper way to act right now.
For better or for worse, texting is almost as prevalent as talking, so it pays to stay on top of the constantly evolving rules of text etiquette.
Which president enjoyed skinny-dipping? Which one is Presidents' Day really named after? And who befriended his burglar while in office? Presidents are people too, and they have been wild, wonderful, and just plain weird.
Once you freeze to death, that's it, right? Not always. With the right techniques, doctors may be able to bring a frozen person back to life.


text:
Will Kevin Smith Still Visit Tromaville After All? We Hope So
Lloyd Kaufman's appearance on Kevin Smith's Comic Book Men show may be a hint.
March 21, 2018, 10:00 AM EDT
Troma
Image via AMC / CC BY
I'm sure most of our readers here are familiar with the TV show Comic Book Men, if only because it immediately follows The Talking Dead every Sunday. Comic Book Men focuses on the day-to-day adventures of the staff of Kevin Smith's personal comic book store in Red Bank, New Jersey. This most recent episode from Sunday (dubbed "Troma-tized") was particularly interesting in that it featured a visit from "Uncle" Lloyd Kaufman, who is a director, producer, and co-founder of the legendary B-movie studio Troma Entertainment.
He is most famous for The Toxic Avenger series of films, although my personal favorite is Surf Nazis Must Die! The premise of the episode was putting the guys at Jay and Silent Bob's Secret Stash through a series of auditions to see who will star in his next film. In typical Troma fashion a series of hilarious, yet strange hijinks ensued with each Secret Stash staffer acting out scenes inspired by the films such as screaming, running from an axe-wielding Toxie, and even directly recreating the iconic toxic waste-spewing "Troma meltdown."
Besides being wildy entertaining (I'll watch Kaufman any time doing anything, the guy is crazy!), perhaps this was a sublte hint that Kevin Smith may return to the directing chair for a future Toxie or Nuke 'Em High film? We reported back in December that Smith was once tapped to direct the remake of The Toxic Avenger but ultimately turned it down due to a difference in opinion regarding how it should be filmed.
A Toxie remake was officially confirmed back in 2010 and, as of 2016, was set to be helmed by Conrad Vernon, one of co-directors of Sausage Party. However, there has been almost no news about the film since the announcment of Vernon as the director. As we all know, projects like these can easily get stuck in development hell and actors, producers, and directors come and go throughout the development process. Therefore it's not so outside of the realm of possibility that the door may have opened for Kevin Smith to take over a film project from a franchise that inspired him to try his own independent film.
So sure, Kaufman and Troma's appearance on Smith's Comic Book Men simply could have been a fellow New Jersey native helping a local legend get some exposure and do a little cross-promotion, or it could have been a sublte hint that Kevin Smith is still very invested in trying his hand at making a Troma-piece. Likewise, while I'm sure this episode was filmed quite some time ago, in light of Smith's recent massive heart attack, maybe he realized taking on The Toxic Avenger is a previously-missed opportunity that he wants to have another shot at.
This is all speculation, but we here at Dead Entertainment really hope there's a grain of truth to it. Would you like to see Kevin Smith direct a piece set in Tromavilla? Let us know in the comments and stay tuned for updates on the remake, Kevin Smith, and all things horror.
Kevin Smith Almost Visits Tromaville
The New Jersey native was once tapped to direct the remake of The Toxic Avenger.
L.B. Lubomski
Lawrence "L.B." Lubomski is an avid horror movie fan, gamer, musician, historian, and aspiring author. Born and raised in Pittsburgh, Pennsylvania, L.B. was exposed to the works of local filmmaker and godfather of zombie cinema George A. Romero early on. He has since developed a particular fondness for Italian zombie/cannibal and slasher films. This passion for horror extends into other media, from survival horror video games such as Resident Evil to horror-inspired musical artists. In his spare time, L.B. pursues many interests including building his collection of vinyl records, action figures, and vintage video games as well as drumming in various local bands.
Legendary Taps Macon Blair to Direct The Toxic Avenger Reboot
The studio acquired the feature film rights for the Troma classic back in December.
Surf Nazis Must Die Review: Too Good to Be Troma
Who rules the beaches? Surfers. And who rules the surfers? Surf Nazis!


text:
MultiTouch delivers LCD display for digital signage
MultiTouch, a provider of modular, multitouch LCD screens for large-scale displays, is now shipping its MT469 MultiTouch Cell 46in HD LCD, which offers more than 500lm of brightness.
The MultiTouch Cell Advanced product line, originally introduced in September 2010, has already been installed in such venues as the North American International Auto Show (NAIAS) in Detroit and at CES 2011, where six MultiTouch Cell Advanced units were used in the official 2011 International CES Twitter Wall.
The MT469 leverages the company's MultiTouch Cornerstone software to enable touch and object recognition capabilities. MultiTouch's Computer Vision Through Screen (CVTS) technology allows an unlimited numbers of users and the stacking of displays into any table or wall configuration. The MT469 improves upon the previously introduced MT467, which was one of the industry's first large-scale LCD multitouch displays for retail and digital signage applications.
Vizio Updates Features to Help Viewers Discover and Manage Streaming Content
Interoperability: No Media Solution is an Island


text:
import parcs.*;
import java.math.*;
import java.io.*;

public class SeriesS implements AM{

        public static void main(String[] args) {
                Const.parseArgs(args);
                task curtask = new task(new TieRecoveries(
                                          new SafeRun("test"), new EventTrace(), true));
                curtask.addJarFile("Series.jar");
                (new SeriesS()).run(new AMInfo(curtask, (channel)null));
                curtask.end();
        }

        public void run(AMInfo info) {
                try {
                DataInputStream in = new DataInputStream(System.in);
                String func = in.readLine();
                String Lnumb = in.readLine();
                int numb = (int)(new Integer(Lnumb));

                String Lscale = in.readLine();
                int scale = (int)(new Integer(Lscale));

                String Llim = in.readLine();
                long lim = (long)(new Long(Llim));

                String dt = in.readLine();
                BigDecimal AR = new BigDecimal(dt);

                AR.setScale(scale);
                point[] p = new point[numb];
                channel[] c = new channel[numb];
                for (int i=0; i<numb; i++) {
                        p[i] = info.createPoint();
                        c[i] = p[i].createChannel();
                        p[i].execute(func);
                }
                for (int i=0; i<numb; i++) {
                        c[i].write(numb);
                        c[i].write(i);
                        c[i].write(scale);
                        c[i].write(lim);
                        c[i].write(AR);
                }
                BigDecimal res = new BigDecimal(0);
                res.setScale(scale);
                for (int i=0; i<numb; i++) {
                        res = res.add((BigDecimal)c[i].readObject());

                }
                System.out.println(res.toString());
                } catch (Exception e){System.out.println(e);}

        }
}

redpajama_set_name: RedPajamaGithub


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Supervision is £45.00 per hour for individuals.
Counselling sessions are held at the same time every week that is suitable to both the client and the counsellor. However, I can accommodate shift work.
The rate is negotiable for students and those on benefits.


text:
Locked out NHLers complain of boredom, having to watch baseball
By Mike HalfordOct 12, 2012, 3:30 PM EDT
It appears the players are getting stir crazy.
With the lockout nearing the 30-day mark, some NHLers are becoming a little too comfortable — and therefore, uncomfortable — with the prospect of having nothing to do.
For example, consider what Winnipeg Jets forward Bryan Little told the Free Press:
"It feels like we should have been playing a long time ago. Everything is going by slow. It's hard to believe it's the middle of October. It's strange. I've found myself watching lots of baseball, which I don't normally do. That's all there is to do right now, watch baseball playoffs.
"That's pretty upsetting when you're supposed to be playing and you're sitting at home."
Or, what Patrick Kane told the Chicago Tribune:
"We just want to be playing hockey. As fun as it is to sit on the couch all day at home and do nothing, it starts getting boring after a while."
Or, what Evander Kane told the New York Times:
"It's definitely weird to not be in training camp right now. For me, it's been a little bit too long for my liking not to be playing in the N.H.L.
"You get a little bored sometimes."
Of the three, only Evander Kane has found work, signing with Dinamo Minsk of the KHL. (Where he reportedly showed up out of shape, drew the ire of his head coach, then watched his head coach get fired today — not so boring now!)
For Little and Patrick Kane, the goal will be to stave off further boredom and avoid the "nothin' else to do, might as well eat" trap that ensnared Keith Tkachuk during the last lockout.


Make elegant evening wear jewellery with these lovely Preciosa Twin Hole Round Beads in Black/Blue Iris. These twin holed beads are domed on both sides and so will sit beautifully around your neck or wrist – why not try working them into intricate 3-D shapes? The beads are a stunning blue/green colour on one side and black on the other - they are ideal for pairing with seed beads in all sizes.
Each bead measures approx. 6mm.


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We are also open whenever there are classes scheduled, so please stop by and join us!


As election officials tallied up votes for the two open Maplewood City Council seats, Tou Xiong, 25, and his supports tracked the results at Stargate Nightclub, off Rice Street near Larpenteur Avenue. Across town, Bryan Smith, 40, gathered with friends, family and his campaign team at Jake's City Grille.
The two new candidates struck up a partnership months ago, even pausing to lend each other support during the final hours on Nov. 3 when Smith dropped by Xiong's get-together.
"We were certainly running our own campaigns, but I think we both believed in each other early on and thought we could help bring some fresh ideas to Maplewood," Smith says.
It's a synergy they'll soon bring to council chambers.
Xiong and Smith won the election with 3,153 (30 percent) and 2,729 votes (26 percent), respectively. Incumbent council member and former mayor Bob Cardinal trailed closely behind Smith with 2,584 votes (24 percent) and former mayor Diana Longrie came in last with 2,053 votes (19 percent).
It's a historic election for Maplewood, as Xiong will serve as the city's first Hmong council member — an accomplishment that he largely attributes to having run a positive campaign.
"I think the community saw the energy that our campaign had; the effort that we put in," Xiong says. "We tried to make the campaign fun, make it a positive campaign. I think the community saw that; and we're very excited."
"I think the combination of hard work and a positive campaign helped the voters see that Tou and Bryan are the best candidates to serve the city," she says. "I think that the voters of Maplewood were ready for new, fresh voices."
The two ran active campaigns — knocking on doors, mailing out letters and seeking endorsements.
Campaign finance records show Xiong raised $24,300 for the general election, nearly double the amount he raised for the primary. He spent $17,607, largely on print materials, postage, food for volunteers, and events.
Smith raised $3,121 for the general election, citing multiple endorsements from labor and business organizations representing a wide range of supporters. He spent $3,956 on advertising, postage, print materials and other campaign resources.
Longrie and Cardinal, combined, raised roughly $3,000 for the general election.
Xiong, an urban planning organizer for Harrison Neighborhood Association and a Maplewood resident of 12 years, says he only got four hours of sleep the two days leading up to Election Day because he and his volunteers were toiling over last-minute literature drops and additional door-to-door visits.
With a council seat now secured — for a four-year term that begins in January — he's already turning his attention toward three key issues: encouraging more civic engagement, building the local economy and addressing infrastructure needs "all the way from the north end to the south end" of the city.
While he's grateful for the support of Hmong voters, he's focused on serving all taxpayers.
"I'm not the Hmong candidate," says Xiong. "I'm the Maplewood candidate. I'm here to support the entire city."
Likewise, Smith, a marketing manager at Tennant Company who has lived in Maplewood for five years with his wife and 7-year-old son, says he sought a wide range of supporters with the promise of focusing on marketing the city to business owners and developers, improving code enforcement and maintaining a path of reasonable government.
"I think our method of wanting to continue the trajectory of reasonable government — creating a functional environment for citizens to work with the city — that resonates with people," he says.
The issue of home ownership stood out as a hot topic during the campaign season. Xiong shares a rental home with his parents, and Smith rents with his family.
Longrie and Cardinal distributed separate campaign materials that brought their opponents' credentials into questions, based on their lack of home ownership.
The move didn't sit well with state Rep. Peter Fischer, DFL-Maplewood, who posted a comment to his Facebook page Oct. 27 calling such tactics "bigoted, elitist and racist" because they "imply that if you don't own property and/or pay property taxes, you don't belong on the Maplewood City Council."
In a follow up phone interview, Fischer told the Review "on the surface, those comments don't appear racist, but it leads to classism and institutionalized racism" because it discredits many minority and elderly citizens who rent.
"We're trying to improve race relations in our community," Fischer adds. "These are not the types of statements we should be making. They did more harm than good."
Xiong says a number of his supporters took offense to the remarks attacking his status as a renter.
"Many Hmong families came here poor. They were trying to work their way up. They do want to be homeowners, but they just can't [afford it]," he says, noting the remarks made his supporters even more determined to get him in office.
"It's a cultural thing too," he explains. "If you're a mother or daughter and you're not married yet, it looks bad if you move out to live on your own and don't come back to take care of your parents."
Both Longrie and Cardinal deny Fischer's allegations, claiming there were no racial undertones in any of the campaign materials they sent out.
Longrie, who points out some of her closest friends are racial minorities, says the growing financial demands of running for city office is more concerning. For the primary and general elections combined, Xiong raised a total of $37,594; Smith raised $9,234.
"It's unfortunate that candidates have to raise anywhere from $9,000 to $38,000 in a municipal election to win a campaign," she says.
The spike in campaign finances also caught the attention of citizen services director Karen Haag during the primary election.
In the general election, she was most impressed with the 27 percent voter turnout, up from just 8 percent during the primary in August. Typically, she says, the city would see a turnout at that rate when the mayor's seat is on the ballot as well.
With election season in the rearview mirror, everyone will begin to prepare for the upcoming transition.
"We will do everything we can on the staffing level to bring Tou and Bryan up to speed so we can get on with the business of the city," Haag says.