Nonetheless, there were many SNP related to DPR that were not antagonistic for MY. Accordingly, it should be possible to select for DPR without reducing MY. Of the 40 SNPs linearly related to DPR, only 11 were negatively associ ated with MY, FY, or PY. SNPs that affected tech support DPR were also positively related with other fertility traits. Other studies have also shown a positive genetic correlation among fertility traits. It is not surprising that these traits are affected by the SNPs associated with DPR. One determinant of DPR is CCR. In addition, PL depends in part on the probability of culling for reproduction. The equation to calculate NM includes DPR and PL. The fact that SNPs associated with DPR are also associated with HCR, CCR, PL and NM means that selection of genes that improve DPR are likely to improve other reproductive traits and traits that depend upon reproduction.

SNPs linked to traits in the current study that were previously linked to other traits are summarized in Table 13. Of the 17 genes with SNPs previously linked to fertility or close to SNPs related to fertility traits, 9 SNPs had MAF 5% and were not analyzed. Of the other 8, 2 were significantly associated with DPR and one tended to be. The exact SNP in CAST analyzed here was previously associated with DPR, PL, and NM. A dif ferent SNP in NLRP9 than the one studied here was as sociated with incidence of still birth. Another gene, FGF2, tended to have an association with DPR, with the AA genotype being superior to the GG genotype.

Previously, the AA genotype of FGF2 was as sociated with higher estimated relative conception rate in bulls although, surprisingly, associated with lower in vitro embryo development. Another SNP, in PGR, was previously associated with in vitro fertilization rate and development and in vivo fertilization and pregnancy rates, and while not significant, the GG genotype was superior to the CC genotype for DPR in agreement with the superior genotype seen earlier. A SNP in FSHR was previously associated with superovulation response and, while not significantly associated with DPR in the current study, was associated with HCR and PL. There was no significant effect of genotype for four other SNPs in genes previously associated with reproductive traits, including HSPA1A, associated with calving rate in beef cattle, IRF9, which was physically close to a SNP for interval to insemination, and STAT5A, associated with in vitro embryo development and sire concep tion rate.

Note that HSPA1A was significantly asso ciated with PL and NM and both of these traits depend upon reproductive function. The genes in the current study with SNPs Drug_discovery that were associated with DPR participate in a wide range of physiological functions associated with reproductive pro cesses. Many function in the endocrine system, either in synthesis of hormones or in cell signaling.

In brief, UCs were washed in calcium, magnesium free http://www.selleckchem.com/products/BIBW2992.html phosphate buffered saline, and cut into 1 to 2 mm3 pieces. Samples were enzymatically digested for 1 hour at 37 C with 3 mg ml of collagenase type I. Cells were filtered through a 40 um nylon cell strainer and centrifuged at 1,500 rpm for 5 mi nutes, and pellets were collected as hUCMSCs. The cells were plated in 100 mm tissue culture dishes at a density of 1 104 cells cm2 for growth at 37 C in a humidified 5% CO2 atmosphere in low glucose Dulbecco modified Eagle medium with fibroblast growth factor 2, insulin, antibiotic solution, 1% gentamycin, and heat inactivated FBS. Adherent cells were detached by incubation for 5 minutes with trypLE E press and then replated at the same density.

Osteogenic and adipogenic differentiation assays Differentiation was induced according to established proto cols. In brief, for osteogenic differentiation, hUCMSCs were cultured to 80% to 90% confluency for 14 days in DMEM LG supplemented with 10% FBS, 100 nM de amethasone, 200 uM ascorbic acid 2 phosphate, and 10 mM B glycerophosphate. Alizarin Red staining was per formed in subconfluent hUCMSCs for the visualization of calcium deposition. Cells were fi ed with 4% paraformalde hyde for 10 minutes at room temperature, washed, stained with Alizarin Red staining solution for 1 hour in the dark, washed with 1 ml distilled water, and added by PBS. For in duction of adipogenic differentiation, hUCMSCs were cultured to 80% to 90% confluence. Adipogenic differenti ation media consisting of DMEM high glucose supplemented with 10% FCS, PSG, 10?6 M de amethasone, 0.

2 mM indomethacin, 0. 1 mg ml insulin, and 1 mM 3 isobutylmethyl anthine were changed twice a week for 14 days. The differentiated cells were fi ed with 4% formaldehyde and stained with Oil Red O to visualize lipid vacuoles. The red lipid images were observed under phase contrast microscope. Cytoto icity assay Cytoto ic effects of hUCMSCs against PC 3 cells were evaluated by 3 2,5 diphenyl tetrazolium bromide assay. We cocultured PC 3 cells by using Transwell assay system along with several densities of hUCMSCs for 24 hours in the same culture condition as hUCMSCs. The cells were incubated with 3 2,5 diphenyltetrazolium brom ide for 2 hours and then with MTT lysis solution overnight. Optical density was measured by using a microplate reader at 570 nm.

Cell viability was calculated as a percentage of viable cells cocultured with hUCMSCs versus single cultured control. Proliferation assay DNA synthesis was detected by using a colorimetric bro modeo yuridine based Cell Proliferation ELISA kit by following manufacturers instructions. In brief, we culti vated PC 3 cells by using Transwell Dacomitinib assay system along with several densities of hUCMSCs in the same culture condition as hUCMSCs. For growing purposes, they were labeled with BrdU for 48 hours, as previously described.

All cell culture reagents were from PAA, Pasching, Austria. Stromal bone marrow cells, enriched by Ficoll gradient centrifugation as described, were kindly provided by the Tumour Immunology Department of the University Hospital, Munich. Bone marrow fibroblasts were generated by allowing bone marrow cells to adhere to plastic cell culture flasks. Cells were grown for 4 weeks, selleck screening library and non adherent cells were regularly displaced by replacing the cell culture medium. Cells e hibited a typical fibroblast like mor phology, and fibroblasts appeared to be the only cell type from bone marrow cells that showed significant proliferation under the cell culture conditions used. Drugs and drug treatment Nelfinavir mesylate was gener ously provided by Pfizer, Groton, CT, USA.

Nelfinavir was dissolved in DMSO and stored at 20 C as a 50 mg ml stock solution. The primary concentration used in this study was 8 ug ml nelfinavir mesylate, correspond ing to a molar concentration of 12 uM. Sorafenib was stored as a 25 mg ml stock solution in DMSO. In control e periments, cells received an amount of DMSO equal to that used in the treated cells. Staurosporine was stored as a 500 uM stock solution in DMSO. Chemosensitivity assay To test the viability of the cancer cells, 5000 cells in a total volume of 200 ul were plated in flat bottomed 96 well plates and incubated with nelfinavir for 48 h at 37 C. For cell e traction, 50 ul tumour cell e traction buffer was added to each well, mi ed thoroughly, and incubated for 20 minutes at room temperature.

Using a MicroLumat LB 96P biolu minometer, Luciferin Luciferase agent was added automatically to each sample and samples were analyzed for bioluminescence. Anne in binding assay FITC labelled anne in V was added to viable cells as recommended by the sup plier in combination with propidium iodide, and cells were analyzed AV-951 with a FACScan using an FL 1 setting at 575 nm and an FL 2 setting at 530 nm. FACScan analysis was performed using a Becton Dickinson FACScan analyzer. Cell cycle analysis For cell cycle analysis, leukemia cells were washed with phosphate buffered saline, fi ed with 70% metha nol, incubated with RNase, and stained with propidium iodide prior to FACScan analysis. Mitochondrial membrane potential analysis To analyze the mitochondrial membrane potential, the MitoCapture Mitochondrial Apoptosis Detection Kit was used according to the manufacturers instructions. For FACScan analysis, an FL 1 setting at 575 nm and an FL 2 setting at 530 nm were used. Simi lar filters were used for fluorescence microscopy. Western blot analysis Western blot analysis was performed as recently described. Cell e tracts were prepared with RIPA buffer, and 20 ug of protein was subjected to SDS polyacrylamide gel electrophoresis.

Formation of aposporous initials is the first and most critical event for occurrence of apospory. Because the initiation of sexual and apomictic pathways likely is activated by different signals, understanding the molecular mechanism underlying apospory initiation can provide insight into developmental regulation and downstream unlikely signaling that results in apomixis. In order to discover candidates for regulating aposporous initial specification in P. squamulatum, we compared two transcriptomes derived from microdissected ovules at the stage of aposporous initial formation between the apomictic donor parent, P. squamulatum, and its apomictic derivative backcross 8 contain ing a single P. squamulatum chromosome. Initially, a P. glaucum x P. squamulatum F1 was crossed with a P. glaucum x P.

purpureum F1 and hybrid apomictic indi viduals with good male fertility were selected. Subsequent backcrosses with tetraploid P. glaucum yielded a BC8 line that was shown by FISH to contain only one chromosome from P. squamulatum. This single chromosome common to both apomictic BC8 and P. squamulatum was the ASGR carrier chro mosome based on the transmission of the trait of apo mixis and linked molecular markers. We hypothesize that candidate genes regulating aposporous initial specification and localized to the ASGR will function in both PS26 and BC8 at the same develop mental stage and would be identical in sequence as they are related by descent. The development and commercialization of new mas sively parallel sequencing platforms have made tran scriptome sequencing faster and more affordable.

One platform, developed by 454 Life Sciences Corporation, the 454 GS FLX sequencer, is capable of producing 100 Mb of sequence data with an average read length of 250 bp per bead in a 7 h run. Successful applications of these high throughput sequencing technologies to tran scriptome analysis have been reported. Here we present expressed sequence tags generated by Roche 454 high throughput sequencing technology from dissected ovule tissues staged for aposporous initial formation from two apomictic lines chosen for their common features of apospory and single shared chro mosome. Alien chromosome expressed transcripts were identified and tested for ASGR linkage and tissue expression. Results Aposporous ovule enriched cDNA samples for sequencing Ovules from PS26 and BC8 around the stage of apospor ous initial formation were manually dissected from pis tils.

Three biological replicates of 40 ovules each were collected for both PS26 and BC8. The yield of total RNA from each replicate was approximately 20 ng from which 15 ng was used for one round of T7 RNA polymerase based RNA amplification. The average yield from one round Drug_discovery of amplification was 90 ug. For each library, equal amounts of amplified RNA from each replicate were combined and 15 ug amplified RNA was used for ds cDNA synthesis.

USDA is an equal opportunity provider and employer. Sexual reproduction in angiosperms involves the formation of complex reproductive organs sellckchem containing diploid tissues and the haploid germline. The germline gives rise to the male and female gametophyte through successive meiotic and mitotic cell divisions from their respective micro spore and megaspore mother cells. The genetic and molecular regulation of these events has been exten sively studied in the model species Arabidopsis thaliana. Pollen development and maturation occurs within the anther locule, surrounded by a specialized layer of helper cells named the tapetum. Tapetal cells greatly contribute to pollen viability and function through the segregation and deposition of the outer cell wall layer and the pollen coat on the pollen surface.

The exine is an extremely durable and biochem ically resistant structure consisting of sporopollenin, a series of complex polymers derived from fatty acids and phenolic compounds, whereas tryphine contains a sticky mixture of fatty acids, flavonoids, carotenoids and proteins deposited on the exine surface and cavities when the tapetum degenerates through programmed cell death. Recently, several biochemical steps of sporopollenin biosynthesis and transcriptional regulatory circuits controlling pollen development have been elucidated in Arabidopsis by the analysis of male sterile and exine defective mutants. In brief, medium to long chain fatty acids such as lauric acid are monohydroxylated by the cytochrome P450 CYP703A2, and modified to form fatty acyl CoA esters by ACYL COA SYNTHE TASE5 in tapetal cells.

CoA esterified fatty acids are alternatively reduced to form fatty alcohol derivatives or condensed with malonyl CoA by LESS ADHESIVE POLLEN5 POLYKETIDE SYNTHASE B and LAP6 PKSA, leading to alkyl pyrones. These latter compounds are hydroxylated by TETRAKETIDE PYRONE REDUCTASE1 and TKPR2, and combined with phenylpropanoids to produce the sporopollenin precursors. Then sporo pollenin is successively secreted to the apoplast by specific transporters and translocated to the microspores bound to proteins such as lipid transfer proteins and glycine rich proteins. A network of transcription factors containing basic helix loop helix, plant homeodomain finger, and MYB domains among others are likely regulating the expression of genes involved in these processes in the tapetum. The knowledge regarding tapetum and pollen devel opment in species other than the model organisms such as Arabidopsis and rice is scarce and fragmentary, Drug_discovery in spite of the relevant influence that these processes exert on pollen viability, fruit set and productivity.

To identify which members were mainly involved in the HOXB1 dependent apoptotic process, we analyzed by western blot a number of apoptosis related factors in HOXB1 vs LXSN HL60 cells kept in 1% serum con dition. Results showing the functional activation of caspase 3 7 were confirmed cell differentiation by the induction of the cleaved form of CASP3 protein. The caspase activating factor, stauros porine was included as a positive control. In addition the role of HOXB1 was sustained by the differential expressions of the antiapoptotic Bax and the proapoptotic Mcl1 proteins, respectively induced and downregulated by HOXB1. The Bax/Bcl2 ratio, doubled by HOXB1, was also indicative of a more apoptogenic balance. Finally, in the HOXB1 expressing cells we observed the upregulation of the proapoptotic factor APAF1.

In view of the lack of significant differences in the cell cycle analysis of HOXB1 respect to LXSN transduced cells, we could consider the apoptotic process as the main mechanism underlying the HOXB1 dependent decrease of cell growth. The HOXB1 dependent effects in the HL60 cultures were then analyzed upon treatment with differentiating concentrations of all trans retinoic acid or 1,25 dihydroxyvitamin D3. Growth curves showed significant reductions of the HL60/ HOXB1 cell growth respect to control cells in both cul ture conditions. The percentage of apoptotic plus dead cells in 10% FBS cultures monitored for 7 days was almost doubled in HL60/HOXB1 cells treated with VitD3 and three fold more with ATRA compared with LXSN corresponding controls.

In 1% serum the higher basal per centage of apoptotic plus dead cells observed in the LXSN controls was further enhanced by HOXB1, from 40% to 62% in VitD3 and from 26% to 54% in ATRA treated cultures. HOXB1 sensitizes HL60 to ATRA and VitD3 induced differentiation We studied whether HOXB1 could have any effect on HL60 differentiation, alone or in synergy with the differ entiating factors ATRA or VitD3. The onset of differen tiation was estimated through a morphological analysis of the cells based on the Giemsa McGr��nwald colori metric method, and the extent of differentiation was measured by FACS analysis of the cell surface markers CD11b, CD14 and G CSFR.

Dacomitinib Although the percentage of CD11b positive cells was increased from 24 to 41% in LXSN vs HOXB1 transduced cells, suggesting that HOXB1 per se might commit cells to granulocytic differ entiation, the presence of HOXB1 did not seem suffi cient to induce clear morphological changes during the myeloid maturation, at least in 10% serum. Nonetheless, after 7 days of ATRA treatment, although CD11b was highly expressed in both HOXB1 and LXSN transduced cells, the mor phological analysis showed a higher number of terminally differentiated granulocytes in HOXB1 transduced cells.

No patient had received bevacizumab or cetuximab prior to study inclusion. one patient had received sorafenib which at the time of the study was considered an RAF kinase inhibitor rather than a multikinase angiogenesis selleck catalog inhibitor. One patient had previously received adjuvant chemo radiotherapy and was included in the study after rejecting all stand ard treatments. The patient was subject to two dose re ductions and subsequently excluded from the study due to DLT. Patients on the once daily schedule of nintedanib re ceived doses of between 50 and 450 mg once daily, while those on the twice daily schedule received doses of between 150 and 250 mg twice daily. Overall, patients were treated for a median of 4. 0 cycles with 15 of the 30 patients receiving 2 cycles.

Of the 30 patients who were enrolled, 15 contin ued study treatment until disease progression. Dynamic contrast enhanced magnetic resonance imaging Twenty one patients with CRC were evaluable for DCE MRI. In total, 14 of the 21 patients with evaluable DCE MRI data had a 40% reduction from baseline in tumour Ktrans, representing a clinically relevant antivas cular effect. Similarly, 13 of the 21 patients had a 40% decrease from baseline in tumour iAUC60. In the correlative analyses, a 40% reduction from baseline in Ktrans was shown to be positively associated with non progressive tumour status. Figure 1 shows parameter maps of Ktrans, taken pretreat ment, and on days 2 and 28, from a patient with liver me tastases who received nintedanib 250 mg once daily.

As shown in Figure 2a, Ktrans and iAUC60 decreased relative to baseline over time in this patient who had stable disease according to RECIST. A strong reduction in contrast agent uptake was observed relative to baseline in the target tumour lesion from this patient on both day 2 and day 28. Efficacy One patient with CRC and liver metastasis who was treated with nintedanib 250 mg twice daily achieved a partial response, while 24 patients treated with either schedule at various dose levels had a best response of stable disease lasting 8 weeks. Based on Kaplan Meier estimates, median TTP was 71 days among patients who received once daily ninteda nib and 106 days among patients who received the twice daily schedule. The difference between the two dosing schedules was not statistically significant.

The majority of drug related AEs were CTC grade 1 or 2 in intensity, including all gastrointestinal AEs, and mostly occurred during the first treatment cycle independently of the dos ing schedule. Drug related AEs CTC grade Entinostat 3 were only seen in three patients, all of whom had received the twice daily schedule of nintedanib. Two patients experienced CTC grade 1 drug related hyper tension. No treatment related deaths were reported. Four of the 14 patients treated with once daily ninteda nib experienced an increase in ALT and/or AST CTC grade 3.

Dictyostelium development is ultrasensitive to O2 making it a good model for understanding the mechan ism of O2 sensing by other organisms that conserve the Skp1 modification pathway. Development is induced by starvation, which signals the CGP057148B normally solitary phagocytic amoebae to form a multicellular fruiting body, which consists of a cellular stalk that aerially supports thou sands of spores for potential dispersal to other locations. Initially, the amoebae chemotax together to form a multicellular aggregate, which polarizes in response to environmental cues and elongates into a migratory slug consisting of prestalk cells mostly at its anterior end and prespore cells in the remainder. The slug responds to environmental signals that direct its migration and regulate the slug to fruit switch the process of culmination leading to formation of the fruiting body.

Signals include light, low NH3, low moisture, higher temperature, and high O2 which, in the native environment of the soil, draw the subterranean slug to above ground where culmination is most pro ductive. In the laboratory, the process takes place over the course of 24 h after deposition of amoebae on moist agar or filter surfaces wetted with low salt buffers. Whereas amoebae grow and form slugs at an air water interface in the presence of as little as 2. 5% O2, 10% is required for culmination, and slugs immersed in mineral oil require atmospheric hyperoxia to culminate. Overexpression of Skp1 or absence of pathway activity drives the O2 requirement up to 18 21%, whereas decreased Skp1 or overexpression of PhyA drives the O2 requirement down to 5% or less.

These genetic manipulations also revealed effects on timing of slug formation and on sporulation. Together with studies on a Skp1 mutant lacking the modifiable Pro143 residue, and double mutants between Cilengitide Skp1 and pathway enzyme genes, the findings suggested that the Skp1 modification pathway mediates at least some O2 responses. However, O2 con tingent modification of the steady state pool of Skp1 has not been demonstrated. To address this issue, and to investigate the generality of O2 regulation of development, we turned to a previ ously described submerged development model in which terminal cell differentiation depends on high at mospheric O2. The wider range of O2 concentra tions presented to cells in this setting may facilitate analysis of the dependence of Skp1 hydroxylation on O2, and absence of the morphogenetic movements of cul mination might reveal later developmental steps that are dependent on Skp1 and its modifications. In a static adaptation of the previous shaking cultures, we observed that terminal cell differentiation occurs in a novel radi ally symmetrical fashion in multicellular cyst like struc tures.

Ligands induce specific intracellular relocalization of GFP ERa GFP ERa can be visualized in SK19 cells using conven tional wide field microscopy. SK19 cells were cultured on conventional glass microscopy coverslips in phenol red free media for 3 days. Culture conditions were identical to conditions third used for cell fractionation, immu noblotting or RNA extraction prior to RT qPCR. Figure 3A shows representative images of SK19 cells treated or not with E2, SERMs and SERDs. We note that in the SK19 cell line GFP ERa was excluded from the nucleoli, as previously observed for the cellular distribution of endogenous ERa in MCF 7 cells and of transiently transfected GFP ERa, under all conditions tested. Exposure times were identical for all conditions examined by fluorescence microscopy.

In untreated cells, ERa was uniformly distributed in the nucleus, to the DAPI nuclear stain in Figure 3Aa A linear scan across the entire field including cytoplasm and nucleus shows that the cytoplasmic GFP ERa fluorescence was barely above background which correlates with observations from cell fractionation experiments. In the presence of E2, GFP ERa rapidly relocalized to accumulate in numerous foci scattered throughout the nucleoplasm. In E2 treated cells, no GFP ERa fluorescence could be detected in the cytoplasm. In contrast, after 1 h treatment with SERMs, OHT or RU39, we did not observe any intranuclear reorganiza tion of GFP ERa compared to untreated cells. This observation also correlates with our fractionation experi ments. GFP ERa staining remained diffuse with fluores cence intensity comparable to mock cells.

However, again no cytoplasmic GFP ERa could be detected. The distribution of the intensity of the fluorescent sig nals was determined within nuclei excluding the nucleo lus. The frequency of pixels with respect to their intensity allows to calculate a coefficient of variation. In cells treated with SERMs the CV was compar able to the one in control cells while the CV was 2 to 3 fold higher in cells exposed to E2 or SERDs. This quantitive measure strengthens our observation that ERa accumulates in intranuclear foci when bound to E2 or SERDs but not in the presence of SERMs. Upon exposure to SERDs, both ICI and RU58, GFP ERa accumulated at numerous sites, reminiscent of the ones observed in the presence of E2.

We ascertained that the fluorescent foci detected in SK19 cells correspond to an accumulation of endoge neous ERa using immuno electron microscopy of MCF 7 cells. Several immunogold labeled ERa molecules were frequently detected within 100 nm distance from each other in 80 nm thin sections of E2 or ICI treated cells. In addition, in SK19 cells, the maximum fluorescence Cilengitide intensity measured after E2 and ICI treatments decreased by 20 40% as compared to untreated cells consistent with degradation of GFP ERa.

Cells from the entire membrane field were counted. All experi ments were repeated at least three times. siRNA transfection MCF 7 DOX cells were transfected with siRNA using Lipofectamine RNAiMAX. The siRNA transfected cells were incubated for 48 h and harvested for Western blot analysis. Reverse transcription polymerase chain reaction Total RNA was isolated from MCF 7 DOX cells using Trizol Bosutinib chemical structure reagent. cDNAs synthesized from 1 ug of total RNA were used as templates in a 50 ul reac tion using the TaqMan RT reagents according to the manufacturers protocol. RT PCR was performed to amplify genes using a cDNA template corresponding to gene specific primer sets. To avoid amplifying genomic DNA, gene primers were chosen from different exons. PCR was performed in a total reaction volume of 25 ul that contained 2 ul of cDNA solution and 0.

2 uM of sense and antisense pri mers. The RT PCR exponential phase was determined on cycles 28 33 to allow quantitative comparisons among the cDNAs amplified from identical reactions. The amplification products were resolved on a 2% agarose gel, stained with ethidium bromide, and visualized on a transilluminator and photographed. Experimental lung metastasis models MCF 7 and MCF 7 DOX cells were injected into the tail vein of Balb c nude mice. Three months after injection, the animals were killed by CO2 inhalation and their lungs were excised. Lung tumor formation was observed and tumor nodules were counted under a dis secting microscope. All animal experiment procedures were approved by the Institutional Animal Care and Use Committee in Korea National Cancer Center.

Gelatin and fibrinogen plasminogen zymography The proteolytic activity of MMP 2, MMP 9, and uPA in CM was analyzed by substrate gel electrophoresis using SDS PAGE gels containing 0. 2% gelatin or 0. 12% fibrinogen and plasminogen. CM from each treatment group was concen trated using an Amicon Ultra 4 centrifugal device and loaded onto gels. After electrophoresis, the gels were washed with 2. 5% Triton X 100 and incubated overnight in zymogram incubation buffer at 37 C. Clear bands indicative of gela tinolytic activity were visualized by staining the gels with Coomassie blue. Gene expression analyses from whole genome Total RNA was isolated and purified from MCF 7 and MCF 7 DOX cells using the TRIzol reagent and RNease Mini kit.

Of those, 500 ng RNA was biotinylated and amplified using the Illumina TotalPrep RNA Amplification Kit according to the manufacturers instructions. The cRNA yield was measured using RiboGreen RNA quantitation kit, and 750 ng of the cRNA sample was hybridized on a human HT 12 expression bead chip for profiling 48,804 tran scripts per sample. Bead Anacetrapib chips were stained with strep tavidin and scanned using an Illumina BeadArray Reader. BeadStudio V3 was used to quantile normalize the data.