Prolonged use of temozolomide leads to increased anxiety and decreased content of aggrecan and chondroitin sulfate in brain tissues of aged rats.

Chemotherapy with temozolomide (TMZ) is an essential part of anticancer therapy used for malignant tumors (mainly melanoma and glioblastoma); however, the long-term effects on patient health and life quality are not fully investigated. Considering that tumors often occur in elderly patients, the present study was conducted on long-term (4 months) treatment of adult Wistar rats (9 months old, n=40) with TMZ and/or dexamethasone (DXM) to investigate potential behavioral impairments or morphological and molecular changes in their brain tissues. According to the elevated plus maze test, long-term use of TMZ affected the anxiety of the adult Wistar rats, although no significant deterioration of brain morphology or cellular composition of the brain tissue was revealed. The expression levels of all studied heparan sulfate (HS) proteoglycans (HSPGs) (syndecan-1, syndecan-3, glypican-1 and HSPG2) and the majority of the studied chondroitin sulfate (CS) proteoglycans (CSPGs) (decorin, biglycan, lumican, brevican, neurocan aggrecan, versican, Cspg4/Ng2, Cspg5 and phosphacan) were not affected by TMZ/DXM, except for neurocan and aggrecan. Aggrecan was the most sensitive proteoglycan to TMZ/DXM treatment demonstrating downregulation of its mRNA and protein levels following TMZ (-10-fold), DXM (-45-fold) and TMZ-DXM (-80-fold) treatment. HS content was not affected by TMZ/DXM treatment, whereas CS content was decreased 1.5-2.5-fold in the TMZ- and DXM-treated brain tissues. Taken together, the results demonstrated that treatment of adult Wistar rats with TMZ had long-term effects on the brain tissues, such as decreased aggrecan core protein levels and CS chain content and increased anxiety of the experimental animals.

Dexamethasone Inhibits Heparan Sulfate Biosynthetic System and Decreases Heparan Sulfate Content in Orthotopic Glioblastoma Tumors in Mice.

Glioblastoma (GB) is an aggressive cancer with a high probability of recurrence, despite active chemoradiotherapy with temozolomide (TMZ) and dexamethasone (DXM). These systemic drugs affect the glycosylated components of brain tissue involved in GB development; however, their effects on heparan sulfate (HS) remain unknown. Here, we used an animal model of GB relapse in which SCID mice first received TMZ and/or DXM (simulating postoperative treatment) with a subsequent inoculation of U87 human GB cells. Control, peritumor and U87 xenograft tissues were investigated for HS content, HS biosynthetic system and glucocorticoid receptor (GR, Nr3c1). In normal and peritumor brain tissues, TMZ/DXM administration decreased HS content (5-6-fold) but did not affect HS biosynthetic system or GR expression. However, the xenograft GB tumors grown in the pre-treated animals demonstrated a number of molecular changes, despite the fact that they were not directly exposed to TMZ/DXM. The tumors from DXM pre-treated animals possessed decreased HS content (1.5-2-fold), the inhibition of HS biosynthetic system mainly due to the -3-3.5-fold down-regulation of N-deacetylase/N-sulfotransferases (Ndst1 and Ndst2) and sulfatase 2 (Sulf2) expression and a tendency toward a decreased expression of the GRalpha but not the GRbeta isoform. The GRalpha expression levels in tumors from DXM or TMZ pre-treated mice were positively correlated with the expression of a number of HS biosynthesis-involved genes (Ext1/2, Ndst1/2, Glce, Hs2st1, Hs6st1/2), unlike tumors that have grown in intact SCID mice. The obtained data show that DXM affects HS content in mouse brain tissues, and GB xenografts grown in DXM pre-treated animals demonstrate attenuated HS biosynthesis and decreased HS content.

Multiple Irradiation Affects Cellular and Extracellular Components of the Mouse Brain Tissue and Adhesion and Proliferation of Glioblastoma Cells in Experimental System In Vivo.

Intensive adjuvant radiotherapy (RT) is a standard treatment for glioblastoma multiforme (GBM) patients; however, its effect on the normal brain tissue remains unclear. Here, we investigated the short-term effects of multiple irradiation on the cellular and extracellular glycosylated components of normal brain tissue and their functional significance. Triple irradiation (7 Gy*3 days) of C57Bl/6 mouse brain inhibited the viability, proliferation and biosynthetic activity of normal glial cells, resulting in a fast brain-zone-dependent deregulation of the expression of proteoglycans (PGs) (decorin, biglycan, versican, brevican and CD44). Complex time-point-specific (24-72 h) changes in decorin and brevican protein and chondroitin sulfate (CS) and heparan sulfate (HS) content suggested deterioration of the PGs glycosylation in irradiated brain tissue, while the transcriptional activity of HS-biosynthetic system remained unchanged. The primary glial cultures and organotypic slices from triple-irradiated brain tissue were more susceptible to GBM U87 cells' adhesion and proliferation in co-culture systems in vitro and ex vivo. In summary, multiple irradiation affects glycosylated components of normal brain extracellular matrix (ECM) through inhibition of the functional activity of normal glial cells. The changed content and pattern of PGs and GAGs in irradiated brain tissues are accompanied by the increased adhesion and proliferation of GBM cells, suggesting a novel molecular mechanism of negative side-effects of anti-GBM radiotherapy.

Chemotherapy-Induced Degradation of Glycosylated Components of the Brain Extracellular Matrix Promotes Glioblastoma Relapse Development in an Animal Model.

Adjuvant chemotherapy with temozolomide (TMZ) is an intrinsic part of glioblastoma multiforme (GBM) therapy targeted to eliminate residual GBM cells. Despite the intensive treatment, a GBM relapse develops in the majority of cases resulting in poor outcome of the disease. Here, we investigated off-target negative effects of the systemic chemotherapy on glycosylated components of the brain extracellular matrix (ECM) and their functional significance. Using an elaborated GBM relapse animal model, we demonstrated that healthy brain tissue resists GBM cell proliferation and invasion, thereby restricting tumor development. TMZ-induced [especially in combination with dexamethasone (DXM)] changes in composition and content of brain ECM proteoglycans (PGs) resulted in the accelerated adhesion, proliferation, and invasion of GBM cells into brain organotypic slices ex vivo and more active growth and invasion of experimental xenograft GBM tumors in SCID mouse brain in vivo. These changes occurred both at core proteins and polysaccharide chain levels, and degradation of chondroitin sulfate (CS) was identified as a key event responsible for the observed functional effects. Collectively, our findings demonstrate that chemotherapy-induced changes in glycosylated components of brain ECM can impact the fate of residual GBM cells and GBM relapse development. ECM-targeted supportive therapy might be a useful strategy to mitigate the negative off-target effects of the adjuvant GBM treatment and increase the relapse-free survival of GBM patients.

Single X-ray irradiation modulates proteoglycan expression in brain tissue: investigation using mouse model.

Radiotherapy is an integral part of glioblastoma treatment affecting both cancer cells and tumour microenvironment, where proteoglycans (PGs) are key extracellular components. However, the molecular effects of radiotherapy on PGs expression and functional activity in brain tissue are poorly understood. Here, we aimed to study the short-term effects of X-ray irradiation on PGs expression in normal brain tissue in mouse model in vivo. Two-month-old male CBL/6Bl mice (n = 54) were used in this study, animals' brains were irradiated using either research synchrotron VEPP-4 or clinical linear accelerator ElektaAxesse. Control (n = 18) and irradiated (n = 36) brain tissues were analysed at 24 h, 48 h and 72 h after irradiation. Morphology of the cortex and hippocampus was accessed by H&E staining, and expression of PGs (syndecan-1, glypican-1, HSPG2/perlecan, versican, brevican, neurocan, NG2/CSPG4, CD44, decorin, biglycan) was determined by RT-PCR. Single irradiation of mouse brain with a 7 Gy dose did not affect tissue morphology and mRNA levels of most highly-expressed PGs decorin and neurocan, although resulted in significant downregulation of brevican (3-10-fold) and NG2/CSPG4 (8-9-fold) expression both in cerebral cortex and subcortex. Research synchrotron and clinical linear accelerators demonstrated minor variability in their effects. Single X-ray irradiation with a 7 Gy dose does not significantly affect the mouse brain tissue morphology but selectively decreases expression levels of some PGs. The downregulation of brevican and NG2/CSPG4 but not decorin and neurocan reflects alteration of extracellular matrix in irradiated brain tissue, which might contribute to the formation of a permissive microenvironment for glioblastoma relapse development.

Chemoradiotherapy Increases Intratumor Heterogeneity of HPSE Expression in the Relapsed Glioblastoma Tumors.

Adjuvant chemoradiotherapy is a standard treatment option for glioblastoma multiforme (GBM). Despite intensive care, recurrent tumors developed during the first year are fatal for the patients. Possibly contributing to this effect, among other causes, is that therapy induces changes of polysaccharide heparan sulfate (HS) chains in the cancer cells and/or tumor microenvironment. The aim of this study was to perform a comparative analysis of heparanase (HPSE) expression and HS content in different normal and GBM brain tissues. Immunohistochemical analysis revealed a significant decrease of HPSE protein content in the tumor (12-15-fold) and paratumorous (2.5-3-fold) GBM tissues compared with normal brain tissue, both in cellular and extracellular compartments. The relapsed GBM tumors demonstrated significantly higher intertumor and/or intratumor heterogeneity of HPSE and HS content and distribution compared with the matched primary ones (from the same patient) (n = 8), although overall expression levels did not show significant differences, suggesting local deterioration of HPSE expression with reference to the control system or by the treatment. Double immunofluorescence staining of various glioblastoma cell lines (U87, U343, LN18, LN71, T406) demonstrated a complex pattern of HPSE expression and HS content with a tendency towards a negative association of these parameters. Taken together, the results demonstrate the increase of intratumor heterogeneity of HPSE protein in relapsed GBM tumors and suggest misbalance of HPSE expression regulation by the adjuvant anti-GBM chemoradiotherapy.

Chondroitin sulfate content and decorin expression in glioblastoma are associated with proliferative activity of glioma cells and disease prognosis.

Chondroitin sulfate proteoglycans (CSPGs) are important components of brain extracellular matrix (ECM), although their contribution in gliomagenesis remains underinvestigated. Here, both chondroitin sulfate (CS) content/distribution and expression of a number of CSPG core proteins were studied in glioblastoma multiforme (GBM) tumours with different prognosis (n = 40) using immunohistochemistry and RT-PCR analysis. Survival rates for clinically different patient groups were compared using the Kaplan-Meier analysis and univariate Cox model. CS content was increased in 60-65% of studied GBM tumours and distributed heterogeneously, mainly at perinecrotic and perivascular zones rather than tumour cells with specific morphology. CS accumulation, especially in the tumour extracellular matrix, was positively associated with the proliferative activity of GBM cells according to theKi67 index (p < 0.01) but revealed no significant association with age or sex of the patients, tumour localisation, relapse or disease outcome. The increase in CS content in GBM tumours was accompanied by upregulation of decorin (1.5-fold), biglycan (3-fold) and serglycin (2-fold) expression (p < 0.05), while only decorin expression level was negatively associated with the overall survival rate of the GBM patients (p < 0.05). These results demonstrate a contribution of CS to high intratumoural heterogeneity of GBM and suggest CS content and decorin expression for further investigation as potential microenvironmental glycomarkers/targets for GBM diagnostics and treatment.

Suitability of RNALater solution as a tissue-preserving reagent for immunohistochemical analysis.

Histological and immunohistochemical studies require high-quality paraffin blocks, where proper fixation of tissue samples with formalin is a key point. However, in some cases, the possibility to preserve biological samples prior to the formalin fixation or to use deposited tissues from biobanks is important. RNA-stabilizing reagent RNALater represents a potential option, but its suitability for pathological and immunohistochemical studies remains underinvestigated. Here, comparative study of formalin-fixed tissues and those had undergone preservation with RNALater was performed for different SCID mice tissues (brain, liver, kidney, and lung) using histological staining (hematoxylin-eosin and Weigert-van Gieson) or immunostaining for b-actin, glial fibrillary acidic protein, and glycosaminoglycan chondroitin sulfate. It was shown that RNALater preservation for 7-14 days was suitable for histological characterisation of mouse lung tissue, whereas all other tissues demonstrated some changes. Immunoreactivity of all the studied tissues was affected to a different extent, and the observed changes were detected at the 7th day already and continued to get worse by the 14th day. Overall, RNALater preservation affects immunogenicity of normal mouse tissues (brain, liver, kidney, and lung) making them unsuitable for immunohistochemistry. Some tissues retain their morphology (lung tissue) or demonstrate moderate changes (brain, liver, kidney), suggesting a restricted suitability of the RNALater-preserved tissues for histological analysis.

Conventional Anti-glioblastoma Chemotherapy Affects Proteoglycan Composition of Brain Extracellular Matrix in Rat Experimental Model in vivo.

Temozolomide (TMZ) is a conventional chemotherapy drug for adjuvant treatment of glioblastoma multiforme (GBM), often accompanied by dexamethasone (DXM) to prevent brain oedema and alleviate clinical side effects. Here, we aimed to investigate an ability of the drugs to affect normal brain tissue in terms of proteoglycan (PG) composition/content in experimental rat model in vivo. Age- and brain zone-specific transcriptional patterns of PGs were demonstrated for 8, 60, and 120 days old rats, and syndecan-1, glypican-1, decorin, biglycan, and lumican were identified as the most expressed PGs. DXM treatment affected both PG core proteins expression (mainly syndecan-1, glypican-1, decorin, biglycan, lumican, versican, brevican, and NG2) and heparan sulphate (HS)/chondroitin sulphate (CS) content in organotypic brain slice culture ex vivo and experimental animals in vivo in a dose-dependent manner. TMZ treatment did not result in the significant changes in PG core proteins expression both in normal rat brain hippocampus and cortex in vivo (although generics did), but demonstrated significant effects onto polysaccharide HS/CS content in the brain tissue. The effects were age- and brain zone-specific and similar with the age-related PGs expression changes in rat brain. Combination of TMZ with DXM resulted in the most profound deterioration in PGs composition and content in the brain tissue both at core protein and glycosaminoglycan levels. Taken together, the obtained results demonstrate that conventional anti-glioblastoma therapy affects proteoglycan structure and composition in normal brain tissue, potentially resulting in deterioration of brain extracellular matrix and formation of the favourable tumorigenic niche for the expansion of the residual glioma cells. During the TMZ chemotherapy, dose and regimen of DXM treatment matter, and repetitive low DXM doses seem to be more sparing treatment compared with high DXM dose(s), which should be avoided where possible, especially in combination with TMZ.

Heparan sulfate accumulation and perlecan/HSPG2 up-regulation in tumour tissue predict low relapse-free survival for patients with glioblastoma.

Glycosaminoglycans are major components of brain extracellular matrix (ECM), although heparan sulfate (HS) contribution in brain physiology and carcinogenesis remains underinvestigated. This study examined HS content and distribution in glioblastoma multiforme (GBM) tissues in the context of potential molecular mechanisms underlying its deregulation in brain tumours. Totally, 42 tissue samples and paraffin-embedded tissues for 31 patients with different prognosis were investigated. HS expression was demonstrated in 50-55% of the GBM tumours by immunohistochemistry (IHC), while almost no HS content was detected in the surrounding paratumourous brain tissues. Heterogeneous HS distribution in the HS-positive tumours was more related to the necrosis or glandular-like brain zones rather than glioma cells with high or low Ki-67 index. According the Kaplan-Meier curves, HS accumulation in glioma cells was associated with low relapse-free survival (RS) of the GBM patients (p < 0.05) and was likely to be due to the increased transcriptional activity of HSPG core proteins (syndecan-1, 2-3 fold; glypican-1, 2,5 fold; perlecan/HSPG2, 13-14 fold). Activation of perlecan/HSPG2 expression correlated with the patients' survival according Kaplan-Meier (p = 0.0243) and Cox proportional-hazards regression (HR = 3.1; P(Y) = 0.03) analyses, while up-regulation of syndecan-1 and glypican-1 was not associated with the patients survival. Taken together, the results indicate that increase of HS content and up-regulation of perlecan/HSPG2 expression in glioblastoma tissues contribute to tumour development through the transformation of brain extracellular matrix into tumour microenvironment, and represent negative prognostic factors for glioblastoma progression.

Heparan Sulfate Biosynthetic System Is Inhibited in Human Glioma Due to EXT1/2 and HS6ST1/2 Down-Regulation.

Heparan sulfate (HS) is an important component of the extracellular matrix and cell surface, which plays a key role in cell-cell and cell-matrix interactions. Functional activity of HS directly depends on its structure, which determined by a complex system of HS biosynthetic enzymes. During malignant transformation, the system can undergo significant changes, but for glioma, HS biosynthesis has not been studied in detail. In this study, we performed a comparative analysis of the HS biosynthetic system in human gliomas of different grades. RT-PCR analysis showed that the overall transcriptional activity of the main HS biosynthesis-involved genes (EXT1, EXT2, NDST1, NDST2, GLCE, HS2ST1, HS3ST1, HS3ST2, HS6ST1, HS6ST2, SULF1, SULF2, HPSE) was decreased by 1.5-2-fold in Grade II-III glioma (p < 0.01) and by 3-fold in Grade IV glioma (glioblastoma multiforme, GBM) (p < 0.05), as compared with the para-tumourous tissue. The inhibition was mainly due to the elongation (a decrease in EXT1/2 expression by 3-4-fold) and 6-O-sulfation steps (a decrease in 6OST1/2 expression by 2-5-fold) of the HS biosynthesis. Heparanase (HPSE) expression was identified in 50% of GBM tumours by immunostaining, and was characterised by a high intratumoural heterogeneity of the presence of the HPSE protein. The detected disorganisation of the HS biosynthetic system in gliomas might be a potential molecular mechanism for the changes of HS structure and content in tumour microenvironments, contributing to the invasion of glioma cells and the development of the disease.

Prognostic relevance of NG2/CSPG4, CD44 and Ki-67 in patients with glioblastoma.

Neuron-glial antigen 2 (NG2, also known as CSPG4) and hyaluronic acid receptor CD44 are chondroitin sulphate proteoglycans actively involved in brain development and its malignant transformation. Here, we aimed to compare prognostic significances of NG2, CD44 and Ki-67 expression in glioblastoma multiforme patients. Totally, 45 tissue samples and 83 paraffin-embedded tissues for 75 patients were analysed. The prognostic values of the genes were analysed using Kaplan-Meier survival curves. Grade III gliomas showed 2-fold difference in NG2 expression between anaplastic astrocytoma and oligoastrocytoma (10.1 ± 3.5 and 25.5 ± 14.5, respectively). For grade IV gliomas, upregulated NG2 expression (21.0 ± 6.8) was associated with poor glioblastoma multiforme prognosis (overall survival < 12 months) compared with glioblastoma multiforme patients with good prognosis (4.4 ± 3.2; overall survival > 12 months). Multivariate survival analysis using Cox proportional hazards model confirmed that high NG2 expression was associated with low survival of the patients (hazard ratio: 3.43; 95% confidence interval: 1.18-9.93; p = 0.02), whereas age (hazard ratio: 1.02; 95% confidence interval: 0.96-1.09; p = 0.42), tumour resection (hazard ratio: 1.03; 95% confidence interval: 0.98-1.08; p = 0.25) and sex (hazard ratio: 0.62; 95% confidence interval: 0.21-1.86; p = 0.40) did not show significant association with prognosis. Although the positive correlation was shown for NG2 and CD44 expression in the glioblastomas (Pearson coefficient = 0.954), Kaplan-Meier and multivariate survival analyses did not revealed a significant association of the increased CD44 expression (hazard ratio: 2.18; 95% confidence interval: 0.50-9.43; p = 0.30) or high Ki-67 proliferation index (hazard ratio: 1.10; 95% confidence interval: 1.02-1.20; p = 0.02) with the disease prognosis. The results suggest that upregulation of NG2/CSPG4 rather than changes in CD44 or Ki-67 expression is associated with low overall survival in glioblastoma multiforme patients, supporting NG2/CSPG4 as a potential prognostic marker in glioblastoma.

Proteoglycans as potential microenvironmental biomarkers for colon cancer.

Glycosylation changes occur widely in colon tumours, suggesting glycosylated molecules as potential biomarkers for colon cancer diagnostics. In this study, proteoglycans (PGs) expression levels and their transcriptional patterns are investigated in human colon tumours in vivo and carcinoma cells in vitro. According to RT-PCR analysis, normal and cancer colon tissues expressed a specific set of PGs (syndecan-1, perlecan, decorin, biglycan, versican, NG2/CSPG4, serglycin, lumican, CD44), while the expression of glypican-1, brevican and aggrecan was almost undetectable. Overall transcriptional activity of the PGs in normal and cancer tissues was similar, although expression patterns were different. Expression of decorin and perlecan was down-regulated 2-fold in colon tumours, while biglycan and versican expression was significantly up-regulated (6-fold and 3-fold, respectively). Expression of collagen1A1 was also increased 6-fold in colon tumours. However, conventional HCT-116 colon carcinoma and AG2 colon cancer-initiating cells did not express biglycan and decorin and were versican-positive and -negative, respectively, demonstrating an extracellular origin of the PGs in cancer tissue. Selective expression of heparan sulfate (HS) proteoglycans syndecan-1 and perlecan in the AG2 colon cancer-initiating cell line suggests these PGs as potential biomarkers for cancer stem cells. Overall transcriptional activity of the HS biosynthetic system was similar in normal and cancer tissues, although significant up-regulation of extracellular sulfatases SULF1/2 argues for a possible distortion of HS sulfation patterns in colon tumours. Taken together, the obtained results suggest versican, biglycan, collagen1A1 and SULF1/2 expression as potential microenvironmental biomarkers and/or targets for colon cancer diagnostics and treatment.

Transcriptional Activity of Heparan Sulfate Biosynthetic Machinery is Specifically Impaired in Benign Prostate Hyperplasia and Prostate Cancer.

Heparan sulfates (HSs) are key components of mammalian cells surface and extracellular matrix. Structure and composition of HS, generated by HS-biosynthetic system through non-template-driven process, are significantly altered in cancer tissues. The aim of this study was to investigate the involvement of HS-metabolic machinery in prostate carcinogenesis. Transcriptional patterns of HS-metabolic enzymes (EXT1, EXT2, NDST1, NDST2, GLCE, 3OST1/HS3ST1, SULF1, SULF2, HPSE) were determined in normal, benign, and cancer human prostate tissues and cell lines (PNT2, LNCaP, PC3, DU145). Stability of the HS-metabolic system patterns under the pressure of external or internal stimuli was studied. Overall impairment of transcriptional activity of HS-metabolic machinery was detected in benign prostate hyperplasia, while both significant decrease in the transcriptional activity and changes in the expression patterns of HS metabolism-involved genes were observed in prostate tumors. Prostate cancer cell lines possessed specific transcriptional patterns of HS metabolism-involved genes; however, expression activity of the system was similar to that of normal prostate PNT2 cells. HS-metabolic system was able to dynamically react to different external or internal stimuli in a cell type-dependent manner. LNCaP cells were sensitive to the external stimuli (5-aza-deoxycytidin or Trichostatin A treatments; co-cultivation with human fibroblasts), whereas PC3 cells almost did not respond to the treatments. Ectopic GLCE over-expression resulted in transcriptional activation of HS-biosynthetic machinery in both cell lines, suggesting an existence of a self-regulating mechanism for the coordinated transcription of HS metabolism-involved genes. Taken together, these findings demonstrate impairment of HS-metabolic system in prostate tumors in vivo but not in prostate cancer cells in vitro, and suggest that as a potential microenvironmental biomarker for prostate cancer diagnostics and treatment.

Proteoglycan expression in normal human prostate tissue and prostate cancer.

Proteoglycans (PGs) are expressed on the cell surface and extracellular matrix of all mammalian cells and tissues, playing an important role in cell-cell and cell-matrix interactions and signaling. Changes in the expression and functional properties of individual PGs in prostate cancer are shown, although common patterns of PGs expression in normal and tumour prostate tissues remain unknown. In this study, expression of cell surface and stromal proteoglycans (glypican-1, perlecan, syndecan-1, aggrecan, versican, NG2, brevican, decorin, and lumican) in normal tissue and prostate tumours was determined by RT-PCR analysis and immunostaining with core protein- and GAG-specific antibodies. In normal human prostate tissue, versican, decorin, and biglycan were predominant proteoglycans localised in tissue stroma, and syndecan-1 and glypican-1 were expressed mainly by epithelial cells. In prostate tumours, complex changes in proteoglycans occur, with a common trend towards decrease of decorin and lumican expression, overall increase of syndecan-1 and glypican-1 expression in tumour stroma along with its disappearance in tumour epithelial cells, and aggrecan and NG2 expressions in some prostate tumours. All the changes result in the highly individual proteoglycan expression patterns in different prostate tumours, which may be potentially useful as molecular markers for prostate cancer personalised diagnosis and treatment.