Search

Radiotherapy affects not only malignant, but also a healthy tissue adjacent to tumor by increasing reactive oxygen species generation, with consequent damage to biomolecules, such as the oxidation of membrane lipids, known as lipid peroxidation. The end product of lipid peroxidation is malondialdehyde. Radioprotectors are compounds that could significantly protect normal cells from radiation, without changing the tumor cell radiosensitivity. Synthetic radioprotectors usually have side effects and are toxic. Natural radioprotectors exert protection without adverse effects. In this study, we examined the radioprotective ability of linden honey in rat blood, by detecting alterations in the activities of antioxidant enzymes catalase and glutathione peroxidase and malondialdehyde concentration after the exposure to a therapeutic dose of gamma rays. Sixteen rats were randomly divided into Control and Honey groups. Honey group received honey (1.5 mL(kgd-1)) orally for four weeks, while at the same time Control group were given distilled water. After four weeks, blood was sampled from all animals. Samples were halved, and one series of samples were gamma irradiated (2 Gy). Radiation induced decreased glutathione peroxidase activity and increased malondialdehyde level, while honey treatment attenuated those alterations, keeping glutathione peroxidase and malondialdehyde at physiological levels. These findings confirm radioprotective properties of linden honey.

Fundamental limits of thermal radiation are imposed by Kirchhoff’s law, which assumes the electromagnetic reciprocity of a material or material system. Thus, breaking reciprocity can enable breaking barriers in thermal efficiency engineering. In this work, we present a subwavelength, 1D photonic crystal composed of Weyl semimetal and dielectric layers, whose structure was optimized to maximize the nonreciprocity of infrared radiation absorptance in a planar and compact design. To engineer an ultra-compact absorber structure that does not require gratings or prisms to couple light, we used a genetic algorithm (GA) to maximize nonreciprocity in the design globally, followed by the application of the numerical gradient ascent (GAGA) algorithm as a local optimization to further enhance the design. We chose Weyl semimetals as active layers in our design as they possess strong, intrinsic nonreciprocity, and do not require an external magnetic field. The resulting GAGA-generated 1D magnetophotonic crystal offers high nonreciprocity (quantified by absorptance contrast) while maintaining an ultra-compact design with much fewer layers than prior work. We account for both s- and p-polarized absorptance spectra to create a final, eight-layer design suitable for thermal applications, which simultaneously minimizes the parasitic, reciprocal absorptance of s-polarized light.

Linden honey represents a unique honey variety valued for its nutritional benefits, distinctive taste and aroma. Phenols, polyphenols, flavonoids, ascorbic acid and phenolic acids in honey have antioxidant activities. This study aimed to investigate the effects of linden honey on the activities of antioxidant enzymes catalase (CAT) and glutathione peroxidase (GPx), as well as on the concentration of malondialdehyde (MDA) in individually housed animals. The investigated parameters were quantified using spectrophotometric method for determination of enzyme activities and MDA concentration in the blood. We found that treatment with linden honey in the socially isolated animals significantly increased the enzyme activities of CAT and GPx, and significantly decreased the concentration of MDA. The modulation of CAT and GPx activities in socially isolated animals treated with linden honey may be very important for understanding the role of honey in the capacity of antioxidant defense system to increase and maintain its stability in psychosocial stress conditions. Our results may be important in biomedical research for understanding the role of honey in the amelioration of oxidative stress.

The aim of this study was to examine whether there are differences in adrenomedullary function in respect to the left and right sides in chronic stress conditions. We investigated how chronic stress isolation (CSI 12 weeks) affected the protein levels of key enzymes involved in adrenaline (A) synthesis (phenyl ethanolamine N-methyltransferase -PNMT), storage (vesicular monoamine transporters 2 - VMAT2) and degradation (catechol-O-methyltransferase - COMT), as well as the concentrations of A as an index for adrenomedullary function in the left and right adrenal medulla. Also, we examined the concentrations of malondialdehyde (MDA), protein levels of nuclear factor κB (NF-κB), and activity of catalase (CAT) in the left and right adrenal medulla. The investigated parameters were quantified by Western blot analysis, assay of enzymatic activity, and CAT Research ELISA kits. We found that CSI pro duced significantly increased levels of PNMT protein, and VMAT2 protein, as well as increased concentrations of A in the right adrenal medulla. However, we recorded that CSI increased protein levels of COMT and NF-κB, as well as the concentrations of MDA in the left adrenal medulla. Also, CSI decreased the activity of CAT only in the left adrenal medulla. Based on these results, it may be concluded that adrenomedullary function is different in respect to the left and right sides in chronic stress conditions.

Introduction: Arterial hypertension is a major cardiovascular risk factor affecting about 10-40% of the adult population. Secondary endocrine hypertension most often results from excessive aldosterone secretion. Complications related to excessive aldosterone secretion include atrial fibrillation, myocardial infarction, myocardial fibrosis, left ventricular hypertrophy, stroke, and increased cardiovascular mortality. Case report: This report presents a hypotensive woman with hypertensive reactions, newly diagnosed unilateral hyperplasia of the left adrenal gland and secondary hyperaldosteronism. Due to good blood pressure and normalized electrolyte status as a result of antihypertensive drug therapy and absence of damage to target organs, surgical treatment of unilateral adrenal hyperplasia was postponed. Conclusion: In case of midlife and late-life hypertension, it is necessary to consider a cause in the patient's endocrine system. AUTHORS SUMMARY SRPSKI 2021; 50 (1,2) 51-54

Background/Aim. Pseudoexfoliation syndrome (XPS) is an age-related systemic disorder characterized by increased production and accumulation of elastic microfibrillar material in different tissues of the body: skin, connective tissue portions of visceral organs, periphery blood vessels and the eye, as well. The aim of our study was to determine the significance of atherosclerotic changes in the carotid arteries in the development of XFS and pseudoexfoliation glaucoma (XFG). Methods. The study included 120 patients – 40 patients per each of the three defined groups: XFS group, XFG group and age- and sex-matched control subjects (control group) without XFG. Blood samples were collected from the patients before cataract surgery. Serum levels of total cholesterol, low-density lipoprotein – LDL, high density lipoprotein – HDL and triglycerides were analyzed by standard laboratory techniques. Standard ultrasonography of the carotid blood vessels was performed in all the participants. Results. Lipid’s profile was disturbed in the patients with XFS and XFG with statistical significance p control group (p < 0.01). Systolic and diastolic pressure was elevated in the patients with XFS and XFG (p < 0.01). Resistance index was increased in the patients with XFG (p < 0.01). Intima-media thickness was prolonged in patients with XFG (p < 0.01). Conclusion. A disturbed lipid profile with elevated resistancy index and intima-media thickness and increased systolic and diastolic pressure were compulsory findings in patients with developed XFG. So, these factors could be considered as risk. It seems to be difficult to inhibit the process of pseudoexfolation production in the whole body, but it appears that with proper therapy (antihypertnesive, cardiotoncs, etc.) and adequate nourishing, the process of XFG development could be interrupted.

Parathyroid glands are small endocrine gland in the neck that men secrete parathyroid hormone , or PTH ( PTH) , which together with calcitonin and vitamin - D has a primary role in regulating the concentration of calcium and phosphate in the body. The most common disease of the parathyroid gland presents increased and uncontrolled secretion of PTH , which can be defined as primary hyperparathyroidism, if occurs as a result of enhanced functions of one or more of the parathyroid glands , or secondary hyperparathyroidism, which occurs most often in chronic renal failure or as a result of the deficiency of vitamin D. In our case report we describe a patient with cancer paratioidnih gland , which is a very rare disease and is the rarest malignant endocrine tumor.

The paper examines the complex relationship between a lauded American animated sitcom South Park on the one side, and literature, especially satire, on the other side. Upon asserting the bond between literature and popular culture, numerous references presented in the show are pointed out, namely the stance the authors take on literature and the act of reading, the types and subtypes of humor exhibited, and topicality. Finally, using the writing of Mikhail Bakhtin, South Park itself is treated as literature as its type of humor is a continuation of an age-old literary tradition of laughter. The conclusion asserts this as it is revealed that South Park owes much of its popularity to the literary aspect of the humor exhibited in the show.

The aim of this study was to examine how acute immobilization stress affects the concentrations of catecholamines in the plasma and the expression of the splenic catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-Я-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in chronically socially isolated rats. We found that acute immobilization increases the plasma catecholamine levels and splenic PNMT protein levels in chronically socially isolated rats. These results show that acute stress of chronically stressed animals activates the sympatho-adrenomedullary system and increases synthesis of splenic PNMT by 37%, both of which can modulate the immune function. [Projekat Ministarstva nauke Republike Srbije, br. III 41027, br. III 41022 and br. ON 173044]

In this study we examined how chronic forced running (CFR) affects the expression of catecholamine biosynthetic enzymes and cAMP response element-binding (CREB) in the adrenal medulla and the weight of adrenal glands of rats. Also, we examined how CFR and additional acute immobilization stress affect the expression of catecholamine biosynthetic enzymes in the adrenal medulla and the concentration of catecholamines and corticosterone (CORT) in the blood plasma. In this experiment we used as a model forced exercise in rats (treadmill running). We used the most advanced method for determining the level of gene expression, Real-time PCR with TaqMan probes, as well as Western blot analysis (ECL). We found that CFR decreases tyrosine hydroxylase (TH), and dopamine-β-hydroxylase (DBH) mRNA and protein levels in the adrenal medulla. The decreased TH and DBH mRNA levels coincide with the reduced expression of CREB in the adrenal medulla and with the reduced plasma CORT level. Additionally, CFR reduces the level of phenylethanolamine N-methyltransferase (PNMT) mRNA, but elevates its protein level in the adrenal medulla and increases the concentration of adrenaline (A) in the plasma. Reduced level of PNMT mRNA in the adrenal medulla coincides with reduced plasma CORT level. The additional acute immobilization stress increases gene expression of catecholamine biosynthetic enzymes in the adrenal medulla, as well as catecholamines and CORT levels in the plasma. The increased synthesis of PNMT enzyme in the adrenal medulla may result in an increased biosynthesis of A under chronic stress conditions. Additionally, increased level of catecholamines in the plasma after chronic physical stress is the allostatic load that may induce numerous diseases and pathological conditions.

We investigated the biochemical and ultrastructural changes in the liver of the freshwater fish, European perch (Perca fluviatilis), in response to Aphanizomenon flos-aquae bloom in the Gruža Reservoir, Serbia. The activities of total manganese- and copper zinc-containing superoxide dismutase (Tot SOD, Mn-SOD, Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and biotransformation phase II enzyme glutathione-S-transferase (GST), as well as concentrations of total glutathione (GSH) and sulfhydryl (-SH) groups were examined before and during the bloom period. Mn-SOD activity was significantly higher, while the activities of Cu/Zn-SOD, CAT and GSH-Px and the concentration of the -SH groups were significantly lower during the bloom. The ultrastructure of the liver revealed necrotic and apoptotic damage to the hepatocytes during the bloom period. Our work represents the first study to report the influences of an Aphanizomenon flos-aquae bloom in the Gruža Reservoir on antioxidant biomarkers and on histopathological alterations in the liver of the freshwater fish European perch (Perca fluviatilis).

In the present study we examined gross changes in the mass of whole adrenal glands and that of the adrenal cortex, as well as the serum corticosterone and glucose level of mature male Wistar rats subjected to three different treatments: animals subjected to chronic restraint-stress, animals injected with lithium (Li) and chronically stressed rats treated with Li. Under all three conditions we observed hypertrophy of whole adrenals, as well as the adrenal cortices. Chronic restraint stress, solely or in combination with Li treatment, significantly elevated the corticosterone level, but did not change the blood glucose level. Animals treated only with Li exhibited an elevated serum corticosterone level and blood glucose level. The aim of our study was to investigate the modulation of the chronic stress-induced effect on the blood glucose level by lithium, as a possible mechanism of avoiding the damage caused by chronic stress. Our results showed that lithium is an agent of choice which may help to reduce stress-elevated corticosterone and replenish exhausted glucose storages in an organism.

In this study we investigated the effects of breast cancer radiotherapy on the antioxidant (AO) enzyme activities of copper, zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), as well as on the concentration of reduced glutathione (GSH) and lipid peroxides (LP) in blood of patients aged 45-58 years and older than 60 years. The results show that in blood of patients aged 45-58 years, radiotherapy increased the activities of CuZnSOD, CAT, and GR, as well as the concentration of GSH, without affecting the activity of GPx and concentration of LP. In patients older than 60 years, radiotherapy increased the activities of CuZnSOD and CAT, lowered the activity of GPx and concentration of GSH, and increased the concentration of LP. Our results indicate that the response to radiotherapy involves age-related impairment of AO capacity for elimination of H2O2, causing oxidative damage to blood cells. This suggests that cytotoxic effects of radiation on healthy tissues might be more pronounced during the aging of breast cancer patients, and should be considered in the further development of individualization protocols in cancer radiotherapy.

We investigated activities of glutathione peroxidase (GSH-Px), glutathione reductase (GR), and the phase II biotransformation enzyme glutathione-S-transferase (GST) in the foot of three freshwater mussel species: Unio pictorum (Up), Unio tumidus (Ut), and Sinanodonta woodiana (Sw) from the Sava River. Specific and total GSH-Px activity was lower in Sw than in Up and Ut. Total GR activity was higher in Up than in Sw. Specific GST activity was higher in Up than in Ut. Total GST activity was higher in Up than in Ut and Sw. Electrophoretic analysis of proteins shows species specifities between the investigated mussel species. Our study represents the first comprehensive report of the investigated glutathione-dependent enzyme activities in the foot of three freshwater mussel species from the Sava River, Serbia. .

The authors give a historical overview of the origin and development of terrorism in Latin America describing the forms in which it appears in this region of the world (political, military, state and narco terrorism). They also explore to what degree the attacks on the USA launched on 11 September 2001 will affect the governments of Latin American countries to harmonize their positions with those of the US government in taking joint actions in their combat to eliminate terrorist activities on the American continent.

Ionizing radiation increases intracellular production of reactive oxygen species (ros), which can damage cell structure and function. The brain is particularly vulnerable to oxidative injury, and in an area-dependent manner. In order to elucidate differences in enzymatic antioxidative responses of the rat hippocampus and cortex, we measured the activities of cytosol superoxide dismutase (CuZnSOD), mitochondrial superoxide dismutase (MnSOD), and catalase (CAT) in those two brain regions, isolated 1 h and 24 h after exposure to 2 Gy of γ-rays. Our results indicate that the lower MnSOD activity and inducibility found in the hippocampus are probably among the main reasons for particularly great oxidative vulnerability of this brain region.

Background: The goal of this study was to introduce a new predictive biomarker assay that might improve a clinical irradiation treatment of malignant diseases. Methods: Thirty-two peripheral blood samples obtained from breast cancer patients were analyzed for superoxide dismutase (SOD) after irradiation with gamma rays (60 Co). SOD was measured in subcellular fractions prepared from unirradiated and irradiated blood samples (McCord and Fridovich). The activity of SOD was measured by the method of Misra and Fridovich and protein concentration by the method of Lowry et al. Results Antioxidant radiation response of patients' blood cells was very variable and specific for each individual. The results indicated that the radiation response during radiotherapy directly depends on the initial state of antioxidant activity in the blood of cancer patients. In the blood samples with high level of SOD activity the irradiation decreased enzymatic activity while in the samples with medium or low level of SOD, the SOD activity was preserved or increased by irradiation with 2 Gy of gamma rays. Conclusion We showed that the modulation of SOD activity in blood cells after irradiation in vitro might be used as predictive biomarker in individualization of therapy protocols.

Abstract Background Most human cancers show inactivation of both pRB- and p53-pathways. While retinoblastomas are initiated by loss of the RB1 tumor suppressor gene, TP53 mutations have not been found. High expression of the p53-antagonist MDM2 in human retinoblastomas may compromise p53 tumor surveillance so that TP53 mutations are not selected for in retinoblastoma tumorigenesis. We previously showed that p14ARF protein, which activates p53 by inhibiting MDM2, is low in retinoblastomas despite high mRNA expression. Methods In human fetal retinas, adult retinas, and retinoblastoma cells, we determined endogenous p14ARF mRNA, ARF protein, and miR-24 expression, while integrity of p53 signalling in WERI-Rb1 cells was tested using an adenovirus vector expressing p14ARF. To study p14ARF biogenesis, retinoblastoma cells were treated with the proteasome inhibitor, MG132, and siRNA against miR-24. Results In human retinoblastoma cell lines, p14ARF mRNA was disproportionally high relative to the level of p14ARF protein expression, suggesting a perturbation of p14ARF regulation. When p14ARF was over-expressed by an adenovirus vector, expression of p53 and downstream targets increased and cell growth was inhibited indicating an intact p14ARF-p53 axis. To investigate the discrepancy between p14ARF mRNA and protein in retinoblastoma, we examined p14ARF biogenesis. The proteasome inhibitor, MG132, did not cause p14ARF accumulation, although p14ARF normally is degraded by proteasomes. miR-24, a microRNA that represses p14ARF expression, is expressed in retinoblastoma cell lines and correlates with lower protein expression when compared to other cell lines with high p14ARF mRNA. Transient over-expression of siRNA against miR-24 led to elevated p14ARF protein in retinoblastoma cells. Conclusions In retinoblastoma cells where high levels of p14ARF mRNA are not accompanied by high p14ARF protein, we found a correlation between miR-24 expression and low p14ARF protein. p14ARF protein levels were restored without change in mRNA abundance upon miR-24 inhibition suggesting that miR-24 could functionally repress expression, effectively blocking p53 tumor surveillance. During retinal tumorigenesis, miR-24 may intrinsically compromise the p53 response to RB1 loss.

Abstract Background Oxidative stress and impaired antioxidant system have been proposed as a potential factors involved in the pathophysiology of diverse disease states, including carcinogenesis. In this study, we explored the lipid peroxidation levels and antioxidant enzyme activities in women diagnosed with different forms of gynecological diseases in order to evaluate the antioxidant status in endometrium of such patients. Methods Endometrial tissues of gynecological patients with different diagnoses were collected and subjected to assays for superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and lipid hydroperoxides. Results Superoxide dismutase activity was significantly decreased (50% in average) in hyperplastic and adenocarcinoma patients. Activities of both glutathione peroxidase and glutathione reductase were increased 60% and 100% on average, in hyperplastic patients, while in adenocarcinoma patients only glutathione reductase activity was elevated 100%. Catalase activity was significantly decreased in adenocarcinoma patients (47%). Lipid hydroperoxides level was negatively correlated to superoxide dismutase and catalase activities, and positively correlated to glutathione peroxidase and glutathione reductase activities. Conclusions This study provided the first comparison of antioxidant status and lipid peroxidation in endometrial tissues of patients with polyps, myoma, hyperplasia and adenocarcinoma. The results showed that patients with premalignant (hyperplastic) and malignant (adenocarcinoma) lesions had enhanced lipid peroxidation and altered uterine antioxidant enzyme activities than patients with benign uterine diseases, polyps and myoma, although the extent of disturbance varied with the diagnosis. Further investigation is needed to clarify the mechanisms responsible for the observed alterations and whether lipid hydroperoxide levels and antioxidant enzyme activities in uterus of gynecological patients might be used as additional parameter in clinical evaluation of gynecological disorders.

Colloidal self-assembly has attracted significant interest in numerous applications including optics, electrochemistry, thermofluidics, and biomolecule templating. To meet the requirements of these applications, numerous fabrication methods have been developed. However, these are limited to narrow ranges of feature sizes, are incompatible with many substrates, and/or have low scalability, significantly limiting the use of colloidal self-assembly. In this work, we study the capillary transfer of colloidal crystals and demonstrate that this approach overcomes these limitations. Enabled by capillary transfer, we fabricate 2D colloidal crystals with nano-to-micro feature sizes spanning 2 orders of magnitude and on typically challenging substrates including those that are hydrophobic, rough, curved, or structured with microchannels. We developed and systemically validated a capillary peeling model, elucidating the underlying transfer physics. Due to its high versatility, good quality, and simplicity, this approach can expand the possibilities of colloidal self-assembly and enhance the performance of applications using colloidal crystals.

Histone H3 mutations at amino acids 27 (H3K27M) and 34 (H3G34R) are recurrent drivers of pediatric-type high-grade glioma (pHGG). H3K27M mutations lead to global disruption of H3K27me3 through dominant negative PRC2 inhibition, while H3G34R mutations lead to local losses of H3K36me3 through inhibition of SETD2. However, their broader oncogenic mechanisms remain unclear. We characterized the H3.1K27M, H3.3K27M and H3.3G34R interactomes, finding that H3K27M is associated with epigenetic and transcription factor changes; in contrast H3G34R removes a break on cryptic transcription, limits DNA methyltransferase access, and alters mitochondrial metabolism. All 3 mutants had altered interactions with DNA repair proteins and H3K9 methyltransferases. H3K9me3 was reduced in H3K27M-containing nucleosomes, and cis-H3K9 methylation was required for H3K27M to exert its effect on global H3K27me3. H3K9 methyltransferase inhibition was lethal to H3.1K27M, H3.3K27M and H3.3G34R pHGG cells, underscoring the importance of H3K9 methylation for oncohistone-mutant gliomas and suggesting it as an attractive therapeutic target., (© 2022. The Author(s).)

Chromatin structure, transcription, DNA replication, and repair are regulated via locus-specific incorporation of histone variants and posttranslational modifications that guide effector chromatin-binding proteins. Here we report unbiased, quantitative interactomes for the replication-coupled (H3.1) and replication-independent (H3.3) histone H3 variants based on BioID proximity labeling, which allows interactions in intact, living cells to be detected. Along with a significant proportion of previously reported interactions detected by affinity purification followed by mass spectrometry, three quarters of the 608 histone-associated proteins that we identified are new, uncharacterized histone associations. The data reveal important biological nuances not captured by traditional biochemical means. For example, we found that the chromatin assembly factor-1 histone chaperone not only deposits the replication-coupled H3.1 histone variant during S-phase but also associates with H3.3 throughout the cell cycle in vivo. We also identified other variant-specific associations, such as with transcription factors, chromatin regulators, and with the mitotic machinery. Our proximity-based analysis is thus a rich resource that extends the H3 interactome and reveals new sets of variant-specific associations., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

High-grade diffuse glioma (HGG) is the leading cause of brain tumour death. While the genetic drivers of HGG have been well described, targeting these has thus far had little impact on survival suggesting other mechanisms are at play. Here we interrogate the alternative splicing landscape of pediatric and adult HGG through multi-omic analyses, uncovering an increased splicing burden compared with normal brain. The rate of recurrent alternative splicing in cancer drivers exceeds their mutation rate, a pattern that is recapitulated in pan-cancer analyses, and is associated with worse prognosis in HGG. We investigate potential oncogenicity by interrogating cancer pathways affected by alternative splicing in HGG; spliced cancer drivers include members of the RAS/MAPK pathway. RAS suppressor neurofibromin 1 is differentially spliced to a less active isoform in >80% of HGG downstream from REST upregulation, activating the RAS/MAPK pathway and reducing glioblastoma patient survival. Overall, our results identify non-mutagenic mechanisms by which cancers activate oncogenic pathways which need to accounted for in personalized medicine approaches., (© 2022. The Author(s).)

Histone H3 lysine 27 (H3K27M) mutations represent the canonical oncohistone, occurring frequently in midline gliomas but also identified in haematopoietic malignancies and carcinomas. H3K27M functions, at least in part, through widespread changes in H3K27 trimethylation but its role in tumour initiation remains obscure. To address this, we created a transgenic mouse expressing H3.3K27M in diverse progenitor cell populations. H3.3K27M expression drives tumorigenesis in multiple tissues, which is further enhanced by Trp53 deletion. We find that H3.3K27M epigenetically activates a transcriptome, enriched for PRC2 and SOX10 targets, that overrides developmental and tissue specificity and is conserved between H3.3K27M-mutant mouse and human tumours. A key feature of the H3K27M transcriptome is activation of a RAS/MYC axis, which we find can be targeted therapeutically in isogenic and primary DIPG cell lines with H3.3K27M mutations, providing an explanation for the common co-occurrence of alterations in these pathways in human H3.3K27M-driven cancer. Taken together, these results show how H3.3K27M-driven transcriptome remodelling promotes tumorigenesis and will be critical for targeting cancers with these mutations.