Not surprisingly, numerous transcripts coding for reactive oxygen scavengers were found to be strongly induced, many of them by selleckchem multiple stresses, e. g. superoxide dismutase, glutathione S transferase Z1, ger min like oxidase and several catalases, peroxidases and ascorbate peroxidases. Also, the strong and multiple stress induction of aspartyl protease, various cysteine proteases, a subtilisin like protease and a vacuolar processing enzyme supports a role for protein recycling processes in response to stress, similarly to what was found during the salinity stress adaptation competence process in the extremophile T. halophila, whereas the expression of expansins, xyloglucan endotransglycosylases, several cellulose synthase subu nits, glycine, proline and hydroxyproline rich proteins is supported by the observed capacity to adjust cell wall properties in many plants undergoing stress.

Many of these carbohydrate active genes were also highly expressed in stems. Of particular importance were genes highly expressed by several stress treatments, not previously reported in amaranth or related halophyles extremophyles. These have obvious potential biotechnological applications and could also contribute to the elucidation of molecular mechanisms leading to resistance to multiple stress con ditions.

A selection includes the following, Drm3, required for de novo DNA methylation in Arabidopsis thaliana where it is proposed to regulate gene silencing processes, Enhancer of SOS 3 1 which encodes a chloroplast localized protein that interacts with the criti cal SOS3 and SOS2 regulators of salt Brefeldin_A stress tolerance in Arabidopsis, YCF3 and HCF101 proteins deemed to be essential for assembly and accumulation of the photosystem I complex and prevention of photo oxidative damage, translational initiation factor eIF1, found to be a determinant of sodium tolerance in yeast and plants, implying that translation is a salt toxicity target and that its recovery might be a crucial mechan ism for cell survival under NaCl stress conditions in addition to its proposed regulation of ion accumula tion and the intracellular redox status, ATP dependent FtsH protease 9, involved in the degradation of the D1 protein of photo damaged, a step which is needed to avoid the accumulation of excessive levels of reactive oxygen species, the ACD1 LIKE elec tron carrier, resembling the Arabidopsis accelerated cell death gene product, involved in the oxygenation of pheophorbide a that is required to prevent photooxida tive destruction of the cell and also found to be up regulated during salt stress adaptation process in T.

cDNA quantity and quality were evaluated using ND 1000 spectrophotometer measurement. Microarray assay The Affymetrix normally Wheat Genome GeneChipW Array was used to measure the gene expres sion changes within the bulked RNA samples of cv. Dream and cv. Lynx. RNA labelling and microarray hy bridisation were performed according to the Affymetrix technical manual at the Max Planck Institute for Terrestrial Microbiology, Marburg, Germany. The fol lowing wheat samples were analysed cv. Dream, F. graminearum inoculated, 32 hai, cv. Dream, mock inoculated, 32 hai, cv. Dream, F. graminearum inoculated, 72 hai, cv. Dream, mock inoculated, 72 hai, cv. Lynx, F. graminearum inoculated, 32 hai, cv. Lynx, mock inoculated, 32 hai, cv. Lynx, F. grami nearum inoculated, 72 hai, and cv. Lynx, mock inoculated, 72 hai.

Three biological replications per genotype treatment timepoint were performed. Gene ex pression intensities were extracted from the scanned GeneChip images, data analysis was performed using the Bioconductor packages affy, gcRMA and limma within the R environment. Data were preprocessed using the affy package and normalised by the gcRMA method. The limma package was used for the analysis of differentially expressed genes. Genes with an absolute t value 1. 96 that were at least two fold regulated were selected as differentially expressed genes. Such genes were assigned as induced or repressed. To identify enriched gene ontology terms, a gene set enrichment analysis was carried out using the GSEA platform. The gene ontology annotations were received by using Blast2GO.

Significant enriched gene sets were selected based on a FDR 25% and a gene set size 15. The following publicly available databases were consid ered for functional annotations, PLEXdb, NCBI, RGAP 6. 1, TAIR, the Gene Ontol ogy Database, the Fusarium Comparative Database and the MIPS Fusarium graminearum Genome Data base. Generally, a homology was considered as a significant hit according to a threshold at an e value of 1e 20 and a sequence identity of 70% in a sequence seg ment of at least 100 nucleotides for all BLAST analyses. Quantitative real time PCR assay The qPCR expression analyses for selected genes were realised using the 7500 Fast Real Time System with its corresponding software 7500 v2. 0. 4. Each reaction contained 5 ul Power SYBRW Green Master Mix, 4 ng cDNA, 1 uM of both for ward and reverse primer in a final volume of 10 ul.

The following thermal profile was used, 2 min at 50 C, 10 min at 94 C, 45 cycles of 45 s at 94 C, 45 s at anneal ing temperature 60 to 62 C, and 45 s at 72 C. All cDNA samples of each treatment were amplified simultan eously in one PCR plate. After the final PCR cycle, a melting curve Anacetrapib analysis was conducted to determine the specificity of the reaction. Target gene expression was quantified using the com parative 2 Ct method.

Yap1p activates transcription by binding to specific DNA sequences located in the promoter of its target genes. Currently, four predicted Yap1p binding sites selleck inhibitor have been identified in hundreds of genes. Obvi ously, the Yap1 regulated adaptation to various stimuli strongly depends on the expression of these target genes. To gain insights into how Yap1p regulates the protective response and how the yeast cell adapts to a changing en vironment, it is very important to get a global overview of changes in expression of these target genes. DNA microarrays provide a practical and economical tool for studying expression of nearly every gene in yeast. This approach can, in principle, be used to identify all the transcription targets of regulatory pro teins like Yap1p.

However, accumulating evidence indi cates that mRNA abundance does not always correlate well with protein expression levels. The present study was conducted to explore the changes in expres sion of Yap1p targeted proteins at the proteome level. For this purpose, we utilized an S. cerevisiae transfor mant overexpressing Yap1p and performed triplicate analyses of the proteome by two dimensional gel elec trophoresis. Proteins of interest were identified using mass spectrometry. This study provides the mapping of the Yap1p targeted proteins in S. cerevisiae and offers a global overview of the ubiquitous cellular changes elicited by overexpression of this important yeast transcription factor. To our knowledge this is the first report on the effect of Yap1 overexpression on the yeast proteome. Results Overexpression of Yap1p in S.

cerevisiae To obtain a global overview of the in vivo Yap1p targets at the proteome level of S. cerevisiae, a comparative ana lysis was performed using a yeast transformant harbor ing a control plasmid and a transformant with a plasmid carrying the YAP1 gene. Considering the possibility to control pH and maintain anaerobic conditions, yeast transformants were cultivated in a multi bioreactor and the fermentation was discontinued when the cells were still in the exponential growth phase. Before 2 DE ana lysis, overexpression of Yap1p was validated by western blot analysis. As expected, the Yap1 protein was present at elevated levels in the Yap1p overexpressing transfor mant. The level was estimated to be approx. four fold higher than in the control transformant.

2 DE analyses of protein extracts from S. cerevisiae Yeast Entinostat cells from both cultures were harvested and pro teins were extracted using the extraction protocol devel oped by Kolkman et al. which we further optimized for our yeast samples. In the protocol developed by Kolkman et al. the cells are lyophilized and subse quently vortexed with glass beads prior to boiling with SDS. In order to improve cell disruption, we introduced an additional step. Before the SDS boiling, the yeast cells were disrupted in extraction buffer containing thiourea.

Rat recombinant globular adiponectin was purchased from Biovision. Antibodies for the phosphorylated Akt at Ser473, total Akt, cleaved caspase 3, poly poly merase, inhibitor of PI3K LY29002, inhibitor of p ERK1/2 U0126, HRP conjugated anti rabbit or anti mouse secondary Paclitaxel human endothelial cells antibodies were obtained from Cell Signaling Technology. Antibodies for the phosphorylated at Thr202/Tyr204 extracellular regulated kinase, total ERK1/2, B actin, and Enhanced chemiluminescence reagent were purchased from Millipore. Palmitate and Bovine Serum Albumin were obtained from Sigma Aldrich. The stock solutions 5 mM PA/10% BSA that can be stored at ?20 C was prepared reference from. The 5 mM PA/10% BSA stock solutions are heated for 15 min at 55 C, and then cooled to room temperature before use.

Cell culture H9c2 cells obtained from Chinese Collection of Cell Cultures, were grown in Dul beccos Modified Eagles Medium supplemen ted with 10% fetal bovine serum and 1% penicillin streptomycin in a humidified atmosphere of 95% air 5% CO2 at 37 C. In addition, the various treatments for cells were carried out only when cells reach about 80% of confluence in appropriate culture dish. Nuclear staining with Hoechst 33342 Cell were plated in 6 well chamber slides and allowed to adhere. Following 12 h different treatment, cells from each group were washed with phosphate buffered saline and fixed with 4% formalin for 10 min. Hoechst 33342 was applied for 30 min under dark condition to stain the nuclei of fixed cells. Slides were then washed with phosphate buffered saline and mounted in a mount ing medium, and observed under a fluorescence microscope.

Apoptotic cells were identified as those with a nucleus exhibiting brightly stained condensed chromatin or unclear frag ments. For each experimental condition, four separate cell populations were prepared. Apoptotic indices were deter mined by direct visualization and counting of a minimum of 500 cells per population. The apoptotic index was calcu lated as the ratio of number of apoptotic cells to total cells counted 100. Cell viability assay Cell viability was measured using the MTT dimethylthiahiazo 3,5 diphenytetrazoliumromid assay, based on the MTT conversion into formazan crys tals using mitochondrial dehydrogenases. Briefly, H9c2 cells were plated at a density of 1 104 cells/well in 96 well plates. After different treatment for 12 h, the culture medium was replaced with 200 uL MTT solution. After 4 h incubation at 37 C, this solution was removed and the produced formazan was solubilized in 150 uL dimethyl sulfoxide. The absorbance was measured at 550 nm using an automated microplate reader. Immunoblot Cells were lysed Entinostat in ice cold RIPA buffer and the protease of inhibitor phenylmethanesulfonyl fluoride.

The protein expression ratio was calculated using MS Excel. Western blot analysis was performed as previously described. DNA microarray and microarray selleck chem data analysis DNA microarray analysis was performed as previously described. In brief, K562 cells were treated with 1 uM tozasertib for 16 h. Following incubation at 37 C, the cells were washed twice with ice cold phosphate buffered saline and collected immediately for RNA isolation. In this study, we used the Human Genome U133A Genechip, which contains more than 47,000 transcripts. Target prepar ation was carried out following the manufacturers ex pression analysis manual. All arrays were screened for quality by standard methods, and the mean fluorescent intensity for each probe set was determined.

Primary samples This study was approved by the Institutional Review Board of Tokyo Medical University, and informed con sent was provided by all patients in accordance with the Declaration of Helsinki. Primary samples were obtained from the peripheral blood of CML patients. Mono nuclear cells were isolated from blood samples and separated by Lymphosepar. The cells were cultured in RPMI1640 medium containing 10% fetal calf serum and analyzed as described. Flow cytometory analysis Cells were treated with the indicated concentrations of tozasertib for 48 h. Annexin V/propidium iodide apop tosis assays were performed according to the manufac turers instructions. The cells were gently mixed and immediately analyzed by flow cytometry. Statistical analysis Differences between treatment groups, in terms of dose response and apoptosis, were determined using Students t test.

P values of less than 0. 05 were considered significant. Background Endometrial cancers are one of the most common gynecological cancers in the United States, with over 35,000 women diagnosed each year. Endometrial endometrioid carcinomas represent 80 85% of all endometrial cancers. When diagnosed at an early stage, the prognosis for EC has improved over recent years. However, for patients diagnosed Anacetrapib with late stage disease they have an overall poor prognosis. There fore, there is urgent need to further understand the molecular mechanism underlying the development and progression of EEC. Recent evidence has suggested that epigenetic mecha nisms contribute to the development, progression and metastasis of cancer including endometrial cancer. These epigenetic changes occur apart from primary gen omic sequences and include DNA methylation, histone modifications, and miRNA expression. In human neo plasias, CpG island hypermethylation is associated with transcriptional silencing of tumor suppressor genes in cluding genes that encode miRNAs, which are produced by DICER1, a cytoplasmic RNase III enzyme.

Materials and methods Cultured cells, and SPRR2A stable transfectants useful site The human intrahepatic cholangiocarcinoma cell line HuCCT 1 was maintained as reported. Methods to obtain stable transfectants with a SPRR2A expressing vector were previously published. Plasmids We used the C terminal His V5 tagged human SPRR2A expression vector previously described. A human Halo tagged p300 vector was purchased from Promega. Other plasmids, including luciferase re porter plasmids, were purchased from Addgene luc p21 promoter constructs. luc p53 wt. luc p53 mut. Ha p300. and Ha p300 CH3 deletion. Florescence imaging The SPRR2A sequence was cloned into a DsRed mam malian expression vector and transfected into HuCCT 1 cells grown on glass coverslips. 48 hours after transfection, the coverslips were fixed for 1 hour in 1% paraformaldehyde.

Nuclear staining was done with Hoechst dye. DsRed SPRR2A and Hoechst florescence was captured using an AxioImager M1 microscope with a 40X objective lens, NA 0. 95. Biotinylated oligonucleotide precipitation assays The probes for DNA pull down assays are shown in Figure 2A. The assays were carried out as described. Briefly, twenty four hours after transfection, cells were lysed with HKMG buffer containing protease and phosphatase inhibitors. Extracted proteins were pre cleared with ImmunoPure streptavidin agarose beads. Pre cleared lysates were then incubated 12 hours with 1 ug of the 5 biotinylated double stranded oligonucleotides and 10 ug of competitor DNA poly to eliminate non specific pro tein/DNA interactions.

Oligo specific bound proteins were collected with streptavidin agarose beads, separated Anacetrapib by SDS PAGE, and protein identification done by West ern blotting. Transfections and luciferase reporter assay Transfections with DNA plasmids or empty vector were done with Lipofectamine 2000 using the manufacturers recommended protocol for adherent cells. p300 and HDAC1 knock down transfections were done with target specific or negative control SilencerW Select siRNA using RNAiMAX. Luciferase assays were carried out with a Promega assay kit system 24 hours post transfection and mea sured on a luminometer. Western blotting Cell lysates were obtained using TNE buffer con taining protease inhibitors 48 hours after treatments. Cytosolic and nuclear proteins were separated using an NE PER extraction kit. Proteins were separated by SDS PAGE and visualized using enhanced chemiluminescence reagents. Antibodies used are the following p53, GAPDH, p300, and Ha HDAC1 . V5 . Halo . PCAF, acetylated lysine, and Ac K382 p53. Western blots were measured using imageJ software. Immunoprecipitation Cell lysates were obtained 48 hours post treatment using TNE buffer containing protease inhibitors.

Thus, high temperature causes an increase in the expression and chaperone activ ity of PfHsps causing increased trafficking AMN-107 of KAHRP and ETRAMP5, leading to increased PfEMP1 presentation on the pRBC membrane. Figure 2a shows PPI associated with PfEMP1 presentation and sequestration. Platelet activation Glycoprotein integrin gpIIIa is seen to interact with the merozoite surface protein MSP 1. MSP 1 is initially expressed as a protein precursor, which undergoes pri mary proteolytic processing within the pRBC during late trophozoite and schizont stages resulting in four frag ments. All the remaining fragments, except for the p19 Glycosylphosphatidylinositol anchor present on the pRBC, are shed during schizogony to form a complex in association with MSP 6 and MSP 7.

Platelets are known to be activated via membrane glycoprotein integ rins such as gpIIb IIIa. It is known that parasite derived products released at schizogony can act as trig gers for platelet activation via platelet membrane glyco proteins followed by TGF B release. It can be hypothesized that the MSP 1 complex interacts with gpIIIa in vivo resulting in platelet activation. On the other hand, platelets can also get activated on contact with pRBCs. It is possible that the MSP 1 GPI anchor on the pRBC surface interacts with gpIIIa during platelet pRBC contact resulting in platelet activation. Thus, there are two possible in vivo scenarios for the MSP 1 gpIIIa interaction to occur. A set of interactions between TGF B and parasite pro teins as well as those between TGF B receptors and para site proteins are observed.

Regulation by parasite factors is through direct interactions involving TGF B or through interactions with platelets resulting in TGF B release. Experimental evidence suggests that PfTRAP activates latent TGF B. This activation is harmful during the early stages of the infec tion since TGF B down regulates the inflammatory cytokines resulting in reduced parasite clearance. How ever, in the later stages of the disease, TGF B activation may be protective through the down regulation of the systemic inflammation. Hence, the role of TGF B could depend on the time of its activation by parasite proteins, with activation in early stages resulting in increased para site clearance time. TGF B released by activated platelets transduces signals by binding to TGF B receptor type I and TGF B receptor type II.

TGF B receptor mediated signaling is crucial in endothe lial apoptosis. It is also known that the antagonistic binding of inhibitory proteins on TGF B receptors Dacomitinib inter feres with TGF B signaling and causes changes in the nor mal functioning of TGF B. Though the exact function of the parasite proteins involved in the PPI is currently unknown, it is possible that parasite proteins might interfere in TGF B receptor mediated signaling. Figure 2b shows PPI associated with platelet activation and their probable linkage to haemostasis dysfunction systemic inflammation.

Univariate survival analysis By univariate survival analysis, patients outcome was correlated with both tumor TNM and WHO stage. A borderline significance was observed for histological grade. AZD9291 The Kaplan Meier analysis of grouped Sirt1 expression was highly prognostic of poor overall survival for those patients with high Sirt1 expression with a mean postsurgical survival of 13. 0 vs. 54. 1 months. Multivariate survival analysis In multivariate Cox regression analysis, high Sirt1 expression was significantly related to shorter over all survival, in dependently of the degree of histological differentiation and WHO stage. Cellular effects of Sirt1 overexpression To test whether high Sirt1 expression also has a cellular ef fect in vitro, we performed overexpression experiments in both cell lines, MiaPaCa 2 and PANC 1, respectively, using cells upon transfection with flag tagged Sirt1 as determined by MTT assay and Xcelligence proliferation assays.

Nicotinamide and gefitinib treatment in cells with endogenous or overexpressed Sirt1 Inhibition of Sirt1 by increasing concentrations of nico tinamide led to a stepwise decrease of viable cells as depicted in Figure 5. Gefitinib treatment with concentra tions of 50 uM showed similar effects as observed for the application of 25 mM nicotinamide. Interestingly, combinatory treatment with 50 uM gefitinib and 25 mM or 40 mM nicotinamide showed a synergistic effect on cell viability, which was observed in both cell lines. Next, we asked whether inhibition of Sirt 1 by nicotina mide may counterbalance the beneficial effect on cell sur vival triggered by Sirt1 overexpression.

We found that application of 10 mM and lower concentrations of nicotina mide, which in untransfected cells already showed a strong flag tagged Sirt1. Overexpression of GFP served as control. Figure 3A shows immunoblots for endogenous and overexpressed Sirt1 in both cell lines. Cells overexpressing Sirt1 showed a markedly stronger immunosignal compared to their untransfected counterparts, which can also be depicted quantitatively as displayed in Figure 3B. Compared to GFP transfected cells, both cell lines showed statistically significantly increased amounts of viable, proliferating decrease of viable cell fractions compared to controls did not influence cell viability in cells overexpressing Sirt1, while higher concentrations of nicotinamide abrogated increased cell viability mediated by overexpressed Sirt1.

Cellular Cilengitide effects of cambinol, gemcitabine and gefitinib treatment Proliferation assay Real time proliferation assays revealed an inhibition of cell growth of Mia PaCa 2 cells and PANC 1 cells over a time period of 72 hrs upon treatment with cambinol. While for Mia PaCa 2 comparably lower concentrations of cambinol were necessary to achieve this effect, for PANC 1 cells concentrations up to 100 uM had to be applied.

These three regions contain consensus binding sequences for several transcription factors including AP2, PUF, STF, PEA3, E2F and PEA3. We analyzed the Cyclin D2 promoter by two indepen dent methods. The first was MCA Meth. The second method was MSP PCR. Unmethylated primers amplified the selleck chemicals region from 1616 to 1394 giving a product of 222 bp. methylated pri mers amplified the region from 1394 to 1152 producing a pro duct of 276 bp. PTCH1 promoter methylation analysis We analyzed the promoter region of PTCH1 from 238 to 62 bp, which corresponds with exon 1B. To assess methylation of the PTCH1 promoter, we fol lowed the MCA MSP method. This method gives us two melting peaks which are dependent on amplifica tion with unmethylated or methylated primers. Statistical analysis Statistical analyses were performed using Graphpad Prism 4.

Data graphed with error bars represent mean and SD from experiments performed in triplicate, unless otherwise noted. Fishers exact test was used to determine the sig nificance of any difference. Results Expression of GLI1 target genes after siRNA mediated GLI1 silencing We transfected Daoy and U87MG cell lines with GLI1 siRNA and scrambled siRNA. We monitored the effi ciency of transfection using lipofectamine through the delivery of the BLOCK iT Fluorescent Oligo. Images were captured using a fluorescent ima ging microscope and similar efficiencies of transfection were attained for both the GLI1 siRNA and the scrambled siRNA. To assess efficiency of silencing, expression of GLI1 was monitored by RT PCR after transfection.

Approximately 86% and 40 4 5% silencing of GLI1 transcript was achieved in Daoy and U87MG cell lines respectively. Silencing of GLI1 resulted in a 50% and 60% decrease in PTCH1 expression in Daoy and U87MG cell lines respectively. In contrast, Cyclin D2 showed a 17% decrease in Daoy and 113% increase in U87MG cells. Plakoglobin expression was decreased by 30% in Daoy cells and increased by 125% in U87MG cells. PAX6 expression was decreased by 35% in Daoy cells and increased by 100% in U87MG cells, and NKX2. 2 was decreased by 50% in Daoy cells and unaltered in U87MG cells. All changes listed were specific to GLI1 silenced cells and cells transfected with scrambled siRNA or untransfected cells did not show similar trends.

PTCH1 siRNA mediated silencing of GLI1 resulted in an 86% decrease in GLI1 transcript and a 50% decrease in PTCH1 transcript in the Daoy cell line compared with scrambled siRNA and untransfected Daoy cell lines. We subsequently sought to determine the pattern of PTCH1 transcript expression in 6 medulloblastoma cell lines and 14 primary medulloblastoma samples and observed that 50% of the cell lines and tumor samples showed high expression of PTCH1. Carfilzomib However, we were unable to find any significant correla tion between GLI1 and PTCH1 expression in the cell lines and tumor samples.

SNARE proteins, in particular syntaxin 2 and SNAP 23, are required for Enzalutamide regulated surfactant secretion. Both proteins are associated with the plasma membrane and to some degree with lamellar bodies. In parallel to secretion, AECII reinternalize and recycle surfactant components from the alveolar surface by means of endocytosis via clathrin dependent and clathrin inde pendent pathways, which include routing to early endo somes and multivesicular bodies. Interstitial lung disease is a heterogeneous group of diseases of known and unknown etiology. Several histological and clinical subtypes of ILD are linked to the SP C protein deficiency caused by muta tions of the corresponding SFTPC gene. Many SP C mutations cluster within the preproteins BRICHOS domain and lead to misfolding of the preprotein, aber rant trafficking and processing.

To date, all affected individuals with BRICHOS domain mutations have been heterozygous with no detectable mature SP C in their lungs, suggesting a dominant negative effect of the mutant allele. Moreover, in cell lines expressing BRICHOS domain mutations, proSP C forms perinuclear aggregates, consistent with the cells inability to clear aggregated misfolded proteins and a toxic gain of function. Various pathologic mechanisms for these mutations causing chronic accu mulation of misfolded proSP C have been proposed, such as induction of endoplasmic reticulum stress, cytotoxicity, and caspase 3 and caspase 4 mediated apoptosis. These factors might contribute to ILD through cell injury and death of AECII.

In addition to the BRICHOS domain mutations, a second class of SFTPC mutations has emerged. A heterozygous mis sense mutation, leading to a substitution of threonine for isoleucine at position 73 of the proSP C, is the most frequent SFTPC mutation. There is a strong variability in the phenotype of these patients, ranging from asymptomatic to early fatal cases. I73T SP C is marked by mistrafficking of the preprotein to the endosomal compartment and by preserved secre tion of both mature and aberrant proSP C and proSP B forms and their intra alveolar accumulation. Yet, current knowledge on SP CI73T lacks a precise understanding of the proSP C processing abnormalities, concurrent cell stress response and cytotoxicity, as well as perturbations of the surfactant composition and secretion.

Current treatment of the genetic interstitial lung dis eases in children is unfortunately empirical. Corticoster Drug_discovery oids are anti inflammatory and stimulate surfactant protein transcription. Chloroquine and its less toxic derivative hydroxychloroquine are used and believed to act on the lysosomal function, i. e. reduce vesicle fusion, exocytosis and proteolytic degra dation or stimulate lamellar body biogenesis. Thus, there is a need to define the cellular mechanism of the currently applied treatments.