HTR 8 SVneo cells were seeded in 6 well plates just prior to transfection. For optimum transfection efficacy, cells were seeded to a final cell confluency of 30 50%. Cells were transfected with either STAT3 siRNA inhibitor Rucaparib or scrambled siRNA comple ed with G Fectin for 24 h. After treatment with OSM for 48 h, cells were dislodged from the surface of 6 well culture plate for western blotting. Indirect immunofluorescence Cells were cultured on microscope cover slips. Thereafter, the cells were stimulated with 20 ng mL OSM or left untreated for 48 h, with or without stattic pretreatment, and then fi ed with 4% paraformalde hyde in 0. 01 M phosphate buffered saline for 5 min at room temperature. Ne t, these cells were incubated in 2% BSA containing 0. 1% Triton 100 for 30 min at room temperature.

Triton was used for permeabilization. We tested several blocking methods and solutions and found that 2% BSA was ideal as a blocking solution. Cells were then incubated with a mouse anti human monoclonal antibody against E cadherin in blocking solu tion for 1 day at 4 C, to allow good penetration of the pri mary antibodies. The cells were washed in PBS and incubated in the presence of appropriate secondary anti bodies conjugated with Cy3 for 2 h at room temperature. The fluorescent specimens were mounted using Vectashield mounting media. Digital images were acquired using a Zeiss LSM 510 Meta confocal microscope and were imported into Photoshop. We used Photoshop software to de crease the background on confocal images with DAPI staining, and adjusted contrast of the DIC images to im prove visualization of the cell morphology.

Ne t, the cells were treated with OSM for 48 h with or with out pretreatment with stattic for indirect immunofluorescence staining. The ne t steps were the same as those described above. Migration assay Cell wounding assays were also conducted as described by Jones et al, with minor modifications. Briefly, 5 105 HTR8 SVneo cells were plated in 6 well plates in 2 mL medium. The cells were then incubated in a humidified chamber with 5% CO2 at 37 C until they reached conflu ence, and were then wounded using a sterile pipette tip, leaving a denuded area and a sharp demarcation line. Total STAT3 protein e pression did not change sig nificantly at any time point. Stattic, a STAT3 inhibitor, suppressed OSM induced STAT3 phos phorylation in HTR8 SVneo cells.

Monolayers were then rinsed 4 times with s PBS to remove the scraped cells. The cells were incubated for 12 h at 37 C in 5% CO2 with or without OSM or function blocking anti gp130 antibodies, and then photographed. Wound closure was assessed using a LEICA DM IRB DC 300 microscope Cilengitide at 100�� magnification. Cell migration distance was measured using Olympus 6. 51 software and compared with baseline mea surements. To evaluate the effects of stattic on OSM induced cell migrations, cells were incubated for 12 h at 37 C in 5% CO2 with or without OSM or stattic and then photographed.

Notably, e pression levels of Mcl 1 in the three cell lines was high compared to that found in the non transformed mammary epithelial contain cell line MCF10A, indicating that signaling pathways leading to enhanced e pression of Mcl 1 are active in transformed mammary epithelial cells, and in HER2 overe pressing ones in particular. Transformed mammary epithelial cells, including established breast cancer cell lines such as BT474 cells, e hibit an inherent phenotypic plasticity and har bor a subpopulation of cells with features of cancer initiating cells. The latter cells, which are charac terized by numerous parameters, including their ability to form spherical colonies in non adherent culture con ditions, were frequently described as being resistant to cell death induction by numerous sti muli.

This suggests that they may rely on survival signals distinct from these that are critical for the rest of the population. We thus investigated whether the Mcl 1 dependence of BT474 cells revealed above applies to the subpo pulation of CICs. To test this, we reasoned that, if BT474 CICs are Mcl 1 dependent, then a diminished ability to form mammospheres should be observed in a population of BT474 that has been depleted in Mcl 1. The ability of BT474 cells to form mammospheres after transfection with siRNAs was thus evaluated. As shown in Figure 2, the ability of Mcl 1 depleted BT474 cells to form mammospheres was significantly decreased compared to that of the same cells treated with a con trol siRNA. In contrast, Bcl L or Bcl 2 knock down was insufficient by itself to affect mammosphere for mation by BT474 cells.

Taken together, these data indicate that the HER2 overe pressing BT474 cells require Mcl 1 to survive in vitro, and that this Mcl 1 dependence e tends to their subpopulation of CICs. To investigate whether pathways driving Mcl 1 e pres sion are specifically active in HER2 overe pressing can cers, compared to other breast cancers, we analyzed the e pression of 20 pro and anti apoptotic Bcl 2 family members from published gene e pression profiles of breast cancer patients. We based this analysis on studies in which the HER2 status of each tumor was available and had been evaluated by immunohistochemistry, and that were performed using Affymetri microar rays. Two studies corresponded to these criteria, allowing to investigate e pression profiles of 41 HER2 overe pres sing tumors and 170 HER2 ones.

Our evalua tion was performed in a probe matching way, using the 2 pooled aforementioned Carfilzomib cohorts. Regarding the e pres sion of anti apoptotic genes, this evaluation revealed a statistically sig nificant enrichment, in HER2 overe pressing breast tumors compared to other breast tumors, in one MCL1 specific probe and also in one BCL2L1 one. In contrast, other breast tumors appeared sta tistically enriched for three BCL2 specific probes.

It included a num ber of glycosyl transferases and several glycosyl hydro lases representing families having cellulase, b 1,3 glucanase, xylanase, xyloglucan endotransglucosylase hydrolase, glucan endo 1,3 beta D glucosidase, invertase and b D Sutent galactosidase activity. These enzymes are var iously required for cell wall loosening and elongation, formation of the secondary cell walls of vascular tissues, hydrolysis of the xylan backbone, post translational modifications of proteins and mobili zation of energy in form of sucrose. Also detected were pectin methylesterases involved in the modifica tion of the physical, chemical, and biological properties of pectins. The concomitant expression of a PME inhibi tor probably represented a need to regulate PME in young amaranth stems in order to avoid the wall rigidi fication associated with PME activity.

In addition, a putative b expansin protein was detected, these proteins modulate the interaction between hemi celluloses and cellulose presumably via a disruption of their shared hydrogen bonds. Within the extracellular oxido reductases group were found two peroxidases, belonging to the peroxidase 25 and 64 families, respectively. Peroxidases have been found to be expressed at moderate to high levels in developing stems, where they are believed to reduce cell wall extensibility due to their role in the formation of covalent links between pectin residues, hydroxyproline rich proteins like extensins, and lignin precursors. One gene encoding a multicopper oxidase of the SKS family was identified.

The function of these proteins in stem development is not well known, although the expression of SKS5 was latterly found to be up regulated in metal hyper accumulating ecotypes of Thlaspi caeru lescens. Another oxido reductase identified in amaranth stems was an 2 OG Fe oxygenase protein of unknown function that was recently found to be asso ciated with defense mechanisms against fungal infection in Arabidopsis. Several genes encoding proteins with putative interac tion domains with polysaccharides and or other proteins were identified. Many of the genes classified within this category are kinases, peptide receptors and receptor like kinases that regulate developmental processes in plants such as the CLAVATA1 like receptor, CLA VATA3 ESR related receptor, Abnormal Leaf Shape 2 receptor like kinase, leucine rich repeat receptor like kinase RLK7 and LRR XI 23 kinase. A number of hydroxiproline rich proteins, most probably representing arabinogalactan proteins, structural proteins and a related prolyl 4 hydroxylase needed for the hydroxylation Cilengitide of proline residues, were also highly expressed in stems.

Root to shoot ratios of the Katherine and Mt. Isa populations increased while in the Petford population selleck chemical Abiraterone they decreased under stress treat ment however, these differences were not significant. RNA sequencing and differential gene expression In total, 52 million reads were generated from 12 sam ples. Reads per sample ranged from two to nine million with an average of 4 million reads per sample. Reads from high throughput sequencing were analysed with TopHat package to develop gene models. Reference guided mapping was used to predict gene models by mapping the reads against the E. grandis reference gen ome sequence without using E. grandis annotations. By using the coordinates from the predicted gene models we identified the E. grandis genes mapping to the pre dicted gene regions E. Camaldulensis.

While several of the predicted gene models map to E. grandis gene mod els there were however several predicted gene models E. Camaldulensis that did not map to E. grandis gene mod els. We used E. grandis gene names wherever the pre dicted models mapped to the E. grandis models. Where there are no E. grandis annotations mapping to the pre dicted gene models we used the gene names with a CUFF prefix. The coordinates of these genes are pre sented in Additional file 2. Reference guided transcriptome mapping Reads from all the 12 libraries were mapped against the Eucalyptus reference genome sequence to generate gene annotations using the TopHat and Cufflinks packages. A total of 32,474 transcripts were predicted including a large number of alternatively spliced transcripts.

The identity of the transcripts was investigated by BLAST searches against the Arabidopsis protein database. This analysis revealed 15,538 unique genes from the total transcripts. Read counts mapping to the gene annota tions generated by reference guided transcriptome map ping were used for testing differential expression of the genes between control and stress treatments using the edgeR package. Before testing for differential expression, diagnostic tests were performed to test the consistency of the data between the populations. A high correlation was observed in gene expression between the three populations from a given treatment as measured by the read counts. The Pearsons correlation coefficient between the read counts of the three populations before stress treatment ranged from 0.

94 to 0. 99 and the correlation coefficient between the three populations of control plants at the end of the experiment ranged from 0. 93 to 0. 95. Similarly in the stress treatment Entinostat the correlation coefficients between the populations ranged from 0. 94 to 0. 97. This is further reflected in clustering analysis. Multi dimensional scal ing plot of the count data clearly separated the 12 libraries into three groups. The six libraries from the three populations before treatment were clustered together.

In particu lar, the relative abundance of Hxk2p and Cdc19p increased more than two fold. This is most likely due to promotion info the fact that they are rate limiting enzymes in glycolysis. In S. cerevisae, the first irreversible step of glycoly sis can be catalyzed by three enzymes, namely the hexokinases Hxk1p and Hxk2p and the glucokinase Glk1p. However, Hxk2p appears to play the main role since it is the predominant isoenzyme during growth on glucose. Moreover, Hxk2p has been identified in the nucleus of the cell and is required for glucose induced repression of several genes including HXK1 and GLK1. Our results are consistent with these findings, since Hxk1p and Glk1p were not detected on the 2 D gels. Cdc19p, which catalyzes the final step of gly colysis, namely the conversion of phosphoenolpyruvate to pyruvate, is the main pyruvate kinase in the glycolysis pathway.

In the present study, the relative abundance of Cdc19p increased more than two fold in the Yap1p tion of fatty acids, amino acids, and sugar alcohols. Importantly, the pathway is also necessary to protect yeast cells against oxidative stress, since NADPH is an essential cofactor for anti oxidative enzymes. In the present study, two proteins involved in this pathway were identified on the 2 D gels as occur ring at higher levels in Yap1p overexpressing yeast. Overexpression of Yap1p in S. cerevisae resulted in up regulation of a number of proteins involved in stress response, including seven heat shock and chaperone proteins, and one peroxiredoxin. The ex pression of Hsps is one of the conserved mechanisms of cellular protection.

Expression of Hsps was first observed when fruit flies were exposed to high tempera tures. However, an elevation of temperature is not the only way to induce the expression of Hsps. Heavy metals, ethanol, oxygen radicals and peroxides are among a large group of agents that can induce Hsps. Since stress response also induce the activity of Yap1p, our re sult suggests that Yap1p may be an important activator for Hsps when yeast cells are exposed to stress conditions. The peroxiredoxin Tsa1p was 1. 4 fold up regulated upon overexpression of Yap1p. Tsa1p belongs to a family Drug_discovery of thiol specific peroxidases that catalyze the reduction of peroxides through oxidation of Cys. It has also been identified as the key peroxidase suppressing genome in stability and protecting against cell death in yeast. However, the up regulation of Tsa1p was relatively modest, and the role of Tsa1p in Yap1p mediated stress response remains elusive. The number of identified anti oxidant proteins was rather less than expected, since Yap1p has been described primarily as a central regulator of the response to oxidative stress in S. cerevisiae.

The results demonstrated that ERK1/2 signaling pathway was involved in palmitate http://www.selleckchem.com/products/MLN8237.html induced apoptosis in H9c2 cells and adiponectin partially inhibited palmitate induced apoptosis through decreasing the level of phosphorylated ERK1/2. PI3K/Akt and ERK1/2 signaling pathway crosstalk plays a role in regulating adiponectin attenuated palmitate induced apoptosis in H9c2 cells Previous results indicated that 2. 5 ug/mL globular adi ponectin can attenuate palmitate induced apoptosis in H9c2 cells through decreasing the level of p ERK1/2, and simultaneously increasing the level of p Akt. An inter esting question was whether partial recovery of the activity of PI3K/Akt signaling pathway could lead to a decreased activity of ERK1/2 signaling pathway.

Therefore, we fur ther investigated the relationship between ERK1/2 and PI3K/Akt signaling pathway in adiponectin mediated anti apoptosis in H9c2 cells. Cells were exposed to 2. 5 ug/mL globular adiponectin plus palmitate in the absence or the presence of PI3K inhibitor, 10 uM LY294002. Data showed that the level of p ERK1/2 was increased dramat ically. Meanwhile, the level of p Akt was also increased after cells were exposed to palmitate combined with ERK1/2 inhibitor 10 uM U0126. These results indicated that partial inhibition of PI3K/Akt signal ing pathway resulted in activation of ERK1/2 signaling pathway, which attenuated effects of globular adiponectin on anti apoptosis. and partial inhibition of ERK1/2 signal ing pathway resulted in activation of PI3K/Akt signaling pathway and also attenuated palmitate induced apoptosis in H9c2 cells.

Discussion Free fatty acids, such as saturated fatty acids are now recognized as significant contributors to lipotoxicity pathology including insulin resistance, type 2 diabetes, and cardiomyopathy. Palmitate as a kind of satu rated fatty acids can induce apoptosis in diverse cell types, such as cardiomyocytes. Apoptosis or pro grammed cell death is basically cellular suicide which occurs after sufficient cellular damage. Caspase 3, a mem ber of cysteine aspartic proteases that play a central role in the execution of the apoptotic program, exists as an in active 32 kDa proenzyme in normally. The cleavage GSK-3 within the 19 kDa fragment generates a p17 kDa subunit as an activity form. During apoptosis, caspase 3 cleaved the 116 kDa PARP protein to yield a 24 kDa DNA binding fragment and an 89 kDa catalytic fragment. In this study, our results showed that palmitate induced apoptosis through increasing the activ ity of caspase 3 and PARP in H9c2 cells. Adiponectin, an abundant circulating adipokine, is al most exclusively secreted from adipose tissue and exists in the range of 3 30 ug/mL in plasma.

Alternatively, ribosome associated www.selleckchem.com/products/INCB18424.html factors could act extraribosomally to influence the sub cellular localization or fate of Ty1 RNA and associated proteins, thereby interfering with nuclear import or integration of Ty1 cDNA. The majority of RHF genes, when deleted, result in 2 fold change in the level of Ty1 cDNA, suggesting that they exert their effects on retrotransposition at steps sub sequent to the synthesis or accumulation of Ty1 cDNA. This set of RHF genes includes several chromatin organization genes that have a potential role in the inte gration of Ty1 cDNA into the host genome. Ty1 inte grates into nucleosomes upstream of RNA polymerase III genes, but the chromatin determinants of this integration pattern are not known.

A recent genome wide analysis of Ty1 integration sites revealed a significant correlation be tween Ty1 integration hotspots and nucleosomes enriched for H3K14 acetylation and histone variant H2A. Z substi tution. RHF genes that act after cDNA synthesis and are known to influence chromatin organization include Snf1, Gal83, and Sip4 . Caf40 and Ccr4 . Hda1 and Hda3 . Ume1 and Ume6 . Ino2 and Ino4 . Swr1 and Vps72 . and Nat4, an N acetyltransferase involved in the N terminal acetylation of histone H4 and H2A. These chromatin modifiers could enhance inte gration of Ty1 cDNA by modifying the accessibility of the target DNA. Our data indicate that Nat4 is a potent co factor for chromosomal Ty1his3AI retrotransposition even though Ty1 cDNA levels are not decreased in a nat4 mutant. Thus, Nat4 may modulate Ty1 retrotransposition through its effects on the chromatin structure of the target DNA.

This finding may be useful in understanding the role of Nat4 in chromatin dynamics, which is poorly understood. Deletion of 43 RHF genes resulted in 2 fold decrease in endogenous Ty1 cDNA levels. A retrotran sposition defect has previously been reported for eight of the 43 corresponding rhf mutants, and we verified the retrotransposition defect in seven additional rhf single mutants. Thus, the reduced cDNA levels in these mutants provide independent verification that these 43 RHFs affect Ty1 elements globally, rather than having specific effects on the marked Ty1his3AI element. This class includes three genes of unknown function DGR2, HIT1, and OCA4. A forth gene, YDL124W, encodes an evolutionarily conserved NADPH dependent alpha ketoamide reductase, but its cellular function has not been elucidated. However, most of these RHF AV-951 genes en code proteins that are involved in RNA metabolism, raising the possibility that they affect the metabolism of Ty1 RNA or its tRNAiMet primer or trafficking of Ty1 RNA between different functions in the mobility cycle.

The irreversible loss of E cadherin expression emerges as a critical step driving epithelial mesenchymal transition in various human cancers. HTS The loss of E cadherin expression increases tumor invasiveness in vitro and in vivo and also increases the resistance of cancer cells to chemotherapeutic agents. Recent reports have implicated a crucial role for the miR 200 family in the regulation of E cadherin transcriptional repressors zinc finger E box binding homeobox 1 and zinc finger E box binding homeobox 2. In addition, the downregulation of DICER1 has been associated with the miR 200 family EMT pathway and tumor metasta sis, which indicates poorer prognosis. Here we presented for the first time a comprehensive analysis of miR 130 family and DICER1 expression in endometrial cancer tissues, compared with normal endo metrium.

In addition, with EC cells as experimental model we explored the mechanism and functional con sequences of dysregulation of some miRNAs, whose ex pression was linked to aberrant DNA methylation and histone modification and regulated the growth and inva sion of EC cells. Materials and Methods Cell culture and treatment The human endometrial cell lines Ishikawa and AN3CA were obtained from the Chinese Academy of Sciences Committee Type Culture Collection cell bank. The cells were grown in Dulbeccos modified Eagles medium /F12 supplemented with 10% fetal bovine serum, 100 u/mL penicillin, and 100 ug/mL streptomycin in a humidified atmos phere of 5% CO2/95% air at 37 C. The cells were treated with 10 uM 5 Aza 2 deoxycytidine or 10 uM HDAC inhibitor Trichostatin A.

Cell transfection Cells were washed with PBS and transiently transfected with 100 nM pre miR 130b or anti miR 130b with their corresponding negative controls in Opti MEM using siPORT NeoFX transfection agent following the manufacturers protocol. Medium was replaced 8 h later. small interfering RNA expression vectors targeting DICER1 were transiently transfected into AN3CA and Ishikawa cells using lipofectamine 2000 following the manufacturers instructions. Quantitative real time PCR Fresh frozen EEC tissue samples and normal endometrial samples were obtained from patients at the Obstetrics and Gynecology Department of Shanghai First Peoples Hos pital, affiliated to Shanghai Jiao Tong University School of Medicine.

Following excision, tissue samples were imme diately snap frozen in liquid nitrogen and stored at ?80 C until RNA extraction. Total RNA was extracted from the tissues or cells using TRIzol RNA Isolation Reagents. The cDNA was generated using Prime Script RT reagent Kit Real time quantitative PCR of miRNAs was performed using TaqMan assay. The relative fold change was calculated based on the differences in Ct values between fold change 2 Ct. Three biological and technical replicates were done for each sample. All values were expressed Entinostat as mean standard deviation.

We also analyzed Pgp activity determining the accumulation of daunomycin, a fluorescent sub strate of Pgp, this research in cells treated or untreated with iHDACs, using as a positive control L1210R cells. The presence of an active Pgp in these cell lines was demonstrated by the increased accumulation of DNM observed after addition of the Pgp inhibitor verapamil. Our results in Figure 1C show that no functional Pgp was present in IMIM PC 1, IMIM PC 2 and RWP 1 cell lines, either in the presence or in the absence of TSA. I P D whether the MDR1 mRNA present in pancreatic carcin oma cell lines included the longer or the shorter 50 UTR form, and whether the translational control was a general phenomenon in pancreatic cancer cells. In order to do that, we extended this study to other pancre atic cell lines.

Results in Figure 2B show that neither the pancreatic carcinoma cell lines, IMIM PC 1, IMIM PC 2 and RWP 1 nor the new added cell lines Hs 766 T, BxPC 3 and PANC 1 express the longer 50UTR MDR1 mRNA, suggesting a generalized translational control of Pgp expression in human pancreatic carcinoma cell lines, since the new added cell lines were also negative for Pgp expression in Western blot analysis. We have used K 562 ADR and HTC 15 as positive controls, since both of them express a functional P glycoprotein. Furthermore, Figure 3 shows the effects of TSA on MDR1 mRNA levels, as determined by real time RT PCR in IMIM PC 1, IMIM PC 2, RWP 1, PANC 1, Hs 766 T and BxPC 3 cells. All the cell lines except BxPC 3 show an increase in MDR1 mRNA levels after TSA treatment.

TSA regulates differentially the ABCB1 promoters The longer and the shorter 50 UTR forms of MDR1 mRNA have been related to the existence of two ABCB1 alternative promoters. To determine Drug_discovery the effects of TSA on both ABCB1 gene promoters, we analyzed the levels of MDR1 mRNA after 24 h treatment with TSA in a panel of cell lines of different origin that have been divided in two groups. Group A includes cell lines Translational control of Pgp mRNA in pancreatic carcinoma cell lines After demonstrating that the increase in MDR1 mRNA levels due to iDHACs treatment did not correspond with an increase in Pgp protein expression or activity, and since these results resembled our previous findings in colon carcinoma cell lines, we decided to determine that we have analyzed previously and use the down stream promoter which transcribes the short 50UTR MDR1mRNA. Group B includes cell lines which use the upstream pro moter that transcribes the long 50UTR MDR1 mRNA. Results in Figure 4 clearly demonstrate differential effects of TSA on DSP and USP promoters and suggest that TSA is able to downregulate the USP promoter and upregulate the DSP promoter in the ABCB1 gene.

We did not find a significant correlation between MYC, FBXW7, and TP53 mRNA expression. SB1518 Thus, only a tendency toward correlation between an increase in MYC mRNA ex pression and a decrease in FBXW7 mRNA expression was detected. Table 2 summarizes the associations between various clinicopathological features and the RQ of MYC, FBXW7, and TP53 mRNA expression in tumor and paired non neoplastic specimens. An increase in MYC mRNA level was associated with the presence of lymph node metasta sis and GC tumor stage III IV. A significant reduction in FBXW7 mRNA level was also associated with the presence lymph node metastasis and tumor stage III IV. Nuclear MYC protein staining is associated with intestinal type GC Positive staining for nuclear MYC and p53 was found in 64. 5% and 19.

4% of GC samples, respectively. No positivity was found for FBXW7. Table 1 summarizes the clinicopathological features and MYC and p53 immunostaining results. Expression of MYC was more frequent in intestinal type than diffuse type GC. Furthermore, MYC immunostaining was associated with increased MYC mRNA level. No association was found between p53 immunostaining and clinicopathological characteristics, TP53 copy number, or TP53 mRNA expression. Comparison of ACP02 and ACP03 cell lines Both ACP02 and ACP03 cells contained three MYC copies and only one FBXW7 copy. The number of TP53 copies was undetermined in both cell lines. Compared with mRNA expression in ACP03 cells, ACP02 cells expressed a higher level of MYC and lower levels of FBXW7 and TP53 mRNA.

Western blot analyses revealed that MYC expression was significantly higher in ACP02 cells than ACP03 cells. Moreover, FBXW7 expression was significantly lower in ACP02 cells than ACP03 cells. How ever, there was no significant difference in p53 expression between the cell lines. Immunofluorescence analysis of both proteins showed a punctiform pattern of labeling, supporting the Western blot results showing an increase in MYC and reduction in FBXW7 expression in ACP02 cells compared with ACP03. Matrigel invasion assay results showed that ACP02 cells were more invasive than ACP03 cells. Migration assay results showed that fewer ACP02 cells migrated compared with ACP03 cells. Both ACP02 and ACP03 cells presented four gelatinase activity bands, MMP 9 latent, MMP 9 active, MMP 2 latent, and MMP 2 active. We found no significant differences in MMP 9 latent, MMP 2 active, and MMP 2 latent between ACP02 and ACP03 cells. However, significant differences were found between ACP02 and ACP03 cells with respect to MMP 9 active. Discussion In the current study, we observed AV-951 that MYC mRNA ex pression was increased in GC samples compared with corresponding non neoplastic samples.