Postmortem redistribution of drugs: a literature review.

Postmortem drug analysis is crucial in identifying the potential cause and manner of death. However, it is threatened by a significant phenomenon called postmortem redistribution (PMR), which refers to the alterations in drug levels occurring after death. This review aims to describe the PMR phenomenon, the mechanisms involved in the PMR of drugs, the various methods used to predict it, and various artifacts of postmortem drug concentrations. Several mechanisms, including passive diffusion from solid organs that act as drug reservoirs to the surrounding tissues, cadaveric changes after death (e.g., cell death, blood coagulation, hypostasis, and movements), and the putrefactive process, can result in artifacts of postmortem drug concentrations. The drug's chemical and pharmacokinetic properties (such as acidic/basic properties, lipophilicity, protein binding, high volume of distribution, and residual metabolic activity) are additional factors. Several markers, including cardiac blood-to-peripheral blood ratio (C/P), liver-to-peripheral blood ratio (L/P), amino acid markers such as methionine, quantitative structure-activity relationship (QSAR) approach, and F factor, have been proposed for interpreting the liability of drugs to PMR. Several artifacts may affect the reliability of postmortem drug analysis. Peripheral blood is preferred for postmortem drug sample collection. Numerous laboratories evaluate the redistribution potential of drugs after death using the C/P concentration ratio. Nevertheless, the L/P concentration ratio is proposed to be a more reliable marker for PMR determination.

Selenium nanoparticles alleviate lead acetate-induced toxicological and morphological changes in rat testes through modulation of calmodulin-related genes expression.

Lead (Pb) is one of the most common toxic heavy metals. It is a well-known testicular toxicant. Selenium nanoparticles (SeNPs) are a more effective form of elemental selenium that reduces drug-induced toxicities. This study aimed to study the possible ameliorating effect of SeNPs on the toxicological and morphological changes in testes of lead acetate intoxicated rats. The study was conducted on 40 adult male albino rats divided into four groups; control, SeNPs-treated, lead acetate-treated, lead acetate and SeNPS treated groups. The concurrent treatment of lead acetate-exposed rats with SeNPs (0.1 mg/kg/day) for 12 weeks significantly lowered the blood and testicular lead levels, increased serum testosterone, and decreased luteinizing hormone and follicle-stimulating hormone to approach control values. In addition, it improved the histopathological, and ultrastructural alterations of the testes and improved the immunohistochemical expression of the c-kit. This was accompanied by maintenance of the testicular oxidant/antioxidant balance and reversing the lead-induced disrupted calmodulin-related genes expression in testicular tissue in the form of downregulation of CAMMK2 and MAP2K6 and upregulation of CXCR4 genes. There was a strong positive correlation between testicular malondialdehyde and MAP2K6 expression level as well as a strong positive correlation between CXCR4 gene expression and the C-kit area %. In conclusion, SeNPs can be considered as a potential therapy for a lead-induced testicular injury.

Vitamin D and Hypoxia-Inducible Factor (HIF-1α) Serum Levels as Markers for Progression of Nephropathy in Type 2 Diabetic Patients.

BACKGROUND: Vitamin D is a locally acting hormone, which plays a major role in skeletal health. Previous studies reported an important role of vitamin D in modulation of inflammatory response. We aimed to investigate the role of vitamin D deficiency and hypoxia-inducible factor (HIF-1α) as markers for the progression of diabetic nephropathy in Saudi patients with type 2 diabetes mellitus (T2DM). METHODS: We included 174 Saudi patients with T2DM in addition to 60 healthy control subjects. Patients were classified according to urinary Albumin to Creatinine Ratio (ACR) into three groups: Group AI: ACR < 30 µg/mg, Group AII: ACR levels of 30 - 300 µg/mg and Group AIII: ACR > 300 µg/mg. We estimated fasting blood glucose, HbA1c, lipid profile, serum creatinine, hemoglobin concentration (Hb), estimated glomerular filtration rate (eGFR), urine albumin/creatinine ratio, serum 25 hydroxyvitamin D, calcium, parathyroid hormone (PTH), tumor necrosis factor (TNF-α), C- reactive protein (CRP), and hypoxia-inducible factor (HIF-1α). RESULTS: There was a significant difference among studied groups regarding serum levels of vitamin D, calcium, PTH, TNF-α, CRP, and HIF-1α levels. The level of vitamin D was lower in diabetic patients in comparison to the controls and was significantly related to the severity of renal nephropathy as indicated by the level of albumin in urine. Moreover, vitamin D levels showed significant negative correlation with the inflammatory markers: TNF-α, CRP, and HIF-1α levels. CONCLUSIONS: Vitamin D deficiency and elevated HIF-1α serum levels showed a significant correlation to progression of nephropathy in Saudi patients with T2DM.

Lipocalin-2 expression and serum levels as early predictors of type 2 diabetes mellitus in obese women.

Obesity and diabetes are increasing in epidemic proportions globally. Lipocalin-2 (LCN-2) is an inflammatory adipocytokine and obesity-related marker of low-grade inflammation. We aimed to investigate, for first time, the possible role of LCN-2 expression and serum levels in prediction of impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM) among obese Egyptian women. This study included 188 obese women and 180 controls. Obese women were subdivided into three subgroups according to their fasting blood glucose, normal glucose tolerance (NGT), IGT and T2DM. Circulating LCN-2 expression levels were determined by real time polymerase chain reaction. Serum LCN-2 concentrations were assessed by ELISA. Our findings revealed that LCN-2 expression and serum levels were higher in obese women compared to lean controls. They were higher in IGT and T2DM obese cases than in NGT obese women. Receiver operating characteristic analyses revealed that LCN-2 expression level was a useful biomarker discriminating IGT from NGT and T2DM from IGT obese women (AUC were 0.735 and 0.740, respectively). It was an independent predictor of IGT and T2DM among obese women. Serum LCN-2 level was a useful biomarker discriminating IGT from NGT and T2DM from IGT obese women (AUC were 0.705 and 0.728, respectively). It was independent predictor of T2DM without predicting IGT among obese women. The power of combined LCN-2 serum levels and expression in discriminating between IGT from NGT and T2DM from IGT obese women was high (AUC = 0.717 and 0.741, respectively). In conclusion, LCN-2 expression and serum levels could discriminate IGT from NGT and T2DM from IGT obese women and early predicting T2DM among obese women. While, LCN-2 expression level was the independent predictor of IGT in obese women. Combination of both LCN-2 expression and serum levels improved their diagnostic value in early detection of IGT and T2DM among obese women. © 2017 IUBMB Life, 69(2):88-97, 2017.

Association of estrogen receptor ß and estrogen-related receptor α gene polymorphisms with bone mineral density in postmenopausal women.

The aim of the study was to investigate the possible association of AluI and RsaI polymorphisms of estrogen receptor ß (ER-ß) gene and 23-bp nucleotide repeat polymorphism of estrogen-related receptor α (ERRα) gene with bone mineral density (BMD) in postmenopausal Egyptian women. Two-hundred postmenopausal osteoporotic women as cases and 180 healthy age-matched postmenopausal women as controls were genotyped by PCR fragment length polymorphism for AluI, allele-specific PCR for RsaI, and by sizing of PCR products on agarose gels for ERRα repeats. sRANKL levels were estimated by ELISA. BMD measurements for spine and femoral neck were performed by dual energy X-ray absorptiometry. A significant difference between women with osteoporosis and controls regarding allele and genotype distributions of AluI G/A (OR 2.37, 95 % CI 1.77-3.18 and p < 0.001 for A allele) and ERRα polymorphisms (for the two repeats allele OR 2.08, 95 % CI 1.09-4.00, and p = 0.02). Osteoporotic women with the AluI AA + GA genotype or with the EERα 2,2 genotype had significantly lower BMD than did women with the other genotypes. Moreover, there was a significant increase of the mean values of sRANKL in carriers of AluI A, RsaI A alleles and in patients having 2,2 genotypes of ERRα (p < 0.001, p < 0.001, p = 0.02, respectively). We demonstrated an association of ER-ß AluI G/A and ERRα 23-repeats polymorphisms with BMD in postmenopausal Egyptian women. A possible effect of ER-ß and ERRα polymorphisms on the levels of sRANKL was estimated.

Polymorphisms of DNA repair genes OGG1 and XPD and the risk of age-related cataract in Egyptians.

PURPOSE: To analyze the association of the polymorphisms of xeroderma pigmentosum complementation group D (XPD) and 8-oxoguanine glycosylase-1 (OGG1) genes with the risk of age-related cataract (ARC) in an Egyptian population. METHODS: This case-control study included 150 patients with ARC and 50 controls. Genotyping of XPD Asp³¹²Asn was performed by amplification refractory mutation system PCR assay and genotyping of OGG1 Ser³²6Cys was carried out by PCR including confronting two-pair primers. RESULTS: The Asn/Asn genotype of XPD gene was significantly associated with increased risk of ARC (odds ratio [OR] = 2.74, 95% confidence interval [CI] = 1.01-7.43, p = 0.04) and cortical cataract (OR = 5.06, 95% CI = 1.70-15.05, p = 0.002). The Asn³¹² allele was significantly associated with an increased risk of ARC (OR = 1.75, 95% CI 1.06-2.89, p = 0.03) and cortical cataract (OR = 2.81, 95% CI = 1.56-5.08, p<0.001). The OGG1 Cys/Cys genotype frequency was significantly higher in ARC (OR = 4.13, 95% CI = 0.93-18.21, p = 0.04) and the Cys(³²6 allele (OR = 1.85, 95% CI = 1.07-3.20, p = 0.03). Moreover, the Cys/Cys genotype of the OGG1 gene was significantly higher in cortical cataract (OR = 6.00, 95% CI = 1.24-28.99, p = 0.01) and the Cys³²6 allele was also significantly associated with cortical cataract (OR = 2.45, 95% CI = 1.30-4.63, p = 0.005). CONCLUSIONS: The results suggest that the Asn/Asn genotype and Asn³¹² allele of XPD polymorphism, as well as the Cys/Cys genotype and Cys³²6 allele of the OGG1 polymorphism, may be associated with increased risk of the development of ARC, particularly the cortical type, in the Egyptian population.

Vitamin D, Parathormone and Associated Minerals among Students in Zagazig District, Sharkia Governorate, Egypt.

AIM: To determine the prevalence of vitamin D deficiency and associated factors among students of age 13-18 years. DESIGN: Community-based cross sectional survey. SETTING: Two schools were selected using multistage sampling techniques. SAMPLING: Cluster sampling of all enrolled students (550 students). OUTCOME MEASURES: Serum levels of 25-hydroxyvitamin D (25 OHD), parathyroid hormone and calcium. Data was collected about nutritional intake, physical activity and lifestyle variables that are potential risk factors for hypovitaminosis D. RESULTS: Hypovitaminosis D prevalence was 23.8%, of which 5.3% was deficiency and 18.5% insufficiency. Serum 25 OHD levels inversely correlated with parathyroid hormone levels (r=-0.206, P= 0.00). Low calcium and ionized calcium levels were 40.6% and 45.9%, respectively, and significantly correlated with vitamin D levels. Female students have significantly higher levels of hypovitaminosis D compared to males (29.3% to 15.0%, respectively) and the level of vitamin D significantly improved with increased age. Exposure to sun had a significant effect on vitamin D levels, and physical activity, soft drink consumption and smoking did not. Multinomial regression analysis revealed that age, sun exposure and Ca level were the only significant independent predictors of hypovitaminosis D among the studied group. CONCLUSION: Our findings revealed that hypovitaminosis D is a prevalent health problem in adolescents, especially girls, who were at higher risk, and increased age and sun exposure improved vitamin D status among the studied group. There is therefore a need to consider vitamin D supplementation for school children together with increased awareness through a health education program.

Evaluation of TET Family Gene Expression and 5-Hydroxymethylcytosine as Potential Epigenetic Markers in Non-small Cell Lung Cancer.

BACKGROUND/AIM: DNA methylation is the most studied epigenetic modification in cancer. Ten-eleven translocation enzymes (TET) catalyze the oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in the DNA. In the current research, we aimed to evaluate the role of 5-hmC and TET enzymes in non-small cell lung cancer (NSCLC) patients and their possible association with outcomes. PATIENTS AND METHODS: ELISA was used to measure the 5-hmC levels in genomic DNA and qRT-PCR was used to evaluate TET1, TET2, and TET3 mRNAs expression levels in NSCLC tissues and their paired normal controls. RESULTS: The levels of 5-hmC were significantly lower in NSCLC tissues than in normal tissues, with a mean ±SD of 0.28±0.37 vs. 1.84±0.58, respectively (t=22.77, p<0.0001), and this reduction was correlated with adverse clinical features. In addition, all TET genes were significantly down-regulated in NSCLC tissues in comparison to their matched normal tissues. The mean±SD level of TET1-mRNA was 38.48±16.38 in NSCLC vs. 80.65±11.25 in normal tissues (t=21.33, p<0.0001), TET2-mRNA level in NSCLC was 5.25±2.78 vs. 9.52±1.01 in normal tissues (t=14.48, p<0.0001), and TET3-mRNA level in NSCLC was 5.21±2.8 vs. 9.51±0.86 in normal tissues (t=14.75, p<0.0001). Downregulation of TET genes was correlated with poor clinical features. CONCLUSION: 5-HmC levels as well as TET1, TET2, and TET3 mRNA levels were reduced in NSCLC tissues. The reduced levels of 5-hmC and TET mRNAs were associated with adverse clinical features, suggesting that the level of 5-hmC may serve as a valuable prognostic biomarker for NSCLC.

Soluble Programmed Death-Ligand 1 (sPD-L1) as a Promising Marker for Head and Neck Squamous Cell Carcinoma: Correlations With Clinical and Demographic Characteristics.

Background and objective Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer type that affects the mucosal lining of the upper aerodigestive tract. Soluble programmed death-ligand 1 (sPD-L1) is a significant factor in hindering T cells' function, which prevents cancer cells from being detected by the immune system. This means that sPD-L1 is an essential component in the immune evasion of cancer. This study aimed to explore the potential of sPD-L1 as a prognostic biomarker for patients with HNSCC undergoing concurrent chemotherapy and radiation therapy. Methodology The study included 106 patients with locally advanced HNSCC who received three courses of induction chemotherapy followed by concurrent chemoradiation and 60 healthy subjects as controls. sPD-L1 levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit, and the cutoff value was determined based on receiver operating characteristic (ROC) curve analysis. Results The results showed that sPD-L1 levels were significantly higher in HNSCC patients compared to healthy controls, with a cutoff value of 31.51 pg/mL. Higher sPD-L1 levels were associated with poorer overall survival rates. Conclusions These findings suggest that sPD-L1 may serve as a valuable prognostic biomarker for HNSCC patients undergoing concurrent chemotherapy and radiation therapy. The study highlights the importance of exploring new biomarkers and therapeutic strategies for HNSCC to improve patient outcomes and reduce morbidity and mortality rates associated with this disease.

Association Between Serum Uric Acid Levels and Oxido-Inflammatory Biomarkers With Coronary Artery Disease in Type 2 Diabetic Patients.

BACKGROUND: Cardiovascular disease signifies a major cause of morbidity and mortality among patients with type 2 diabetes mellitus (T2DM). Serum uric acid (SUA) levels are elevated during the initial phases of impaired glucose metabolism. This work was designed to explore the association between SUA levels, serum oxido-inflammatory biomarkers, and the risk of coronary artery disease (CAD) in T2DM patients as the primary outcome. The secondary outcome was to assess the prognostic role of SUA in the prediction of the risk of CAD in T2DM patients. METHODS: In this case-control study, we enrolled 110 patients with T2DM who were further divided into patients with CAD and without CAD. In addition, 55 control participants were stringently matched to cases by age. RESULTS: Diabetic patients with CAD had significantly higher serum levels of the inflammatory biomarkers and the oxidative malondialdehyde but significantly lower levels of serum total antioxidant capacity (TAC) compared with the controls and diabetic patients without CAD. Significant positive correlations existed between SUA levels and serum levels of the inflammatory biomarkers and malondialdehyde, while a significant negative correlation existed between SUA levels and serum TAC. SUA demonstrated an accepted discrimination ability. SUA can differentiate between T2DM patients with CAD and patients without CAD, an area under the curve of 0.759. CONCLUSIONS: Elevated serum levels of SUA and oxido-inflammatory biomarkers are associated with an increased risk of CAD in T2DM. SUA levels reflect the body's inflammatory status and oxidant injury in T2DM. SUA could be utilized as a simple biomarker in the prediction of CAD risk in T2DM.

Evaluation of CDCP1 (CD318) and endoglin (CD105) expression as prognostic markers in acute myeloid leukemia.

BACKGROUND: The most commonly used prognostic factors in acute myeloid leukemia (AML) are cytogenetic, molecular, and morphological markers. However, AML prognosis is still unfavorable particularly in adults. So, further reliable markers are urgently needed to improve the risk stratification and treatment decisions. CUB domain-containing protein 1 (CDCP1; CD318) and endoglin (CD105) are new markers correlated with poor prognosis in different solid tumors, but their role in AML prognosis is not fully evaluated. OBJECTIVES: This work aimed to evaluate the prognostic role of CD318 and CD105 in AML and their impact on the outcomes. METHODS: Sixty-five newly diagnosed AML patients were included in this study. CD318 and CD105 expression was assessed by quantitative real-time polymerase chain reaction. Patients were followed up for ∼ 2 years to evaluate the prognostic impact of gene expression on the outcomes. RESULTS: Patients with high CD318 and CD105 showed higher white blood cell (WBC) count, M2 subtype, poor cytogenetic risk, reduced complete remission, and a greater number of deaths compared to low CD318 and CD105. CD318 was correlated with CD105, and both were correlated with WBC count, bone marrow blasts, and peripheral blood blasts. After a follow-up period of up to 24 months, relapse-free survival for high CD318 and CD105 was significantly different (42.1% and 52.6% vs. 64.5% and 58.1% for low CD318 and CD105, respectively). Survival was worse in patients with high CD318 and CD105, as the mean survival time was 13.9 and 13.3 months compared to 24 and 22.7 months in low CD318 and CD105, respectively. CONCLUSIONS: CD318 and CD105 are upregulated in AML patients. Their overexpression was associated with poor response to treatment and poor outcomes. Therefore, CD318 and CD105 can be useful prognostic markers in AML.

Association between circulating interleukin-1 beta (IL-1ß) levels and IL-1ß C-511T polymorphism with cervical cancer risk in Egyptian women.

Cancer cervix is one of the leading causes of cancer-related mortality among women worldwide. It is believed that the host genetic factors such as inflammation-induced cytokines may play a role in cervical carcinogenesis. The interleukin-1ß (IL-1ß) gene contains several single nucleotide polymorphisms. One of them, C-511T, which in the promoter region has been associated with increased IL-1ß production and with increased risk of developing cancers. We assessed the association between the IL-1ß C-511T polymorphism and cervical cancer risk in a case-control study among 100 histopathologically confirmed Egyptian women with cervical cancer and 50 age-matched, cervical cytology negative, healthy controls by polymerase chain reaction-restriction fragment length polymorphism. Plasma levels of IL-1ß were assayed by enzyme-linked immunosorbent assay. There was significant increase in the mean plasma IL-1ß level in cervical cancer cases (43.40 ± 25.95 pg/ml) when compared with controls (30.51 ± 18.28 pg/ml, P = 0.002). The plasma levels above the 75th percentile of controls (IL-1ß ≥ 45.74 pg/ml) were significantly associated with a 2.49-fold increased risk of cervical cancer. The significant increase in IL-1ß concentration in cervical cancer cases was observed only among cervical cancer cases carrying C-511T variant genotypes. T/T genotype of IL-1ß polymorphism was significantly higher in cervical cancer cases compared with controls (57 vs. 38%; OR = 2.16; P = 0.028) and the T allele carriage was significantly associated with cervical cancer risk (OR = 2.00, 95% CI = 1.19-3.38, and P = 0.008). In conclusion, plasma IL-1ß level and IL-1ß C-511T polymorphism may be considered as candidate biomarkers for cervical cancer in Egyptian women.

Association of L55M and Q192R polymorphisms in paraoxonase 1 (PON1) gene with breast cancer risk and their clinical significance.

The HDL-associated paraoxonase (PON) activities play a role in decreasing oxidative stress, which is known to contribute to cancer development. The aim of this study was to examine the relation between the PON1 L55M and Q192R polymorphisms and breast cancer (BC) risk in Egyptian females and to analyze their relation to clinicopathological parameters of BC. Both polymorphisms were characterized in 100 BC Egyptian females and 100 healthy women who had no history of any malignancy by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, using DNA from peripheral blood in a case control study. With respect to PON1 L55M, the mutated allele (M) frequency was found in 70.5% in BC patients and in 53.5% in controls; the M allele was significantly associated with an increased risk of BC (adjusted odds ratio (OR(adj)) 2.07, 95% confidence interval (95% CI) 1.37-3.13; P = 0.011). The homozygous mutant genotype (MM) significantly increased the risk of BC (OR(adj) 2.07, 95% CI 1.17-3.64, P = 0.011). However, as regard PON1 Q192R, the R mutated allele frequency was found in 28.5% in BC patients and in 33% in controls, the women who were QR heterozygotes (OR(adj) 0.96, 95% CI 0.55-1.68) or RR homozygotes (OR(adj) 0.64, 95% CI 0.25-1.63), and R allele (OR(adj) 0.81, 95% CI 0.53-1.42) did not show any risk for BC. Both PON1 L55M and Q192R polymorphisms genotype frequencies were not related to patient's age (P = 0.94 and 0.72, respectively). M allele genotypes (LM/MM) carriers showed significant association only with nodal metastases (P = 0.02) but not with other clinicopathologic parameters. However, R allele genotype (QR/RR) carriers showed insignificant correlation with clinicopathological parameters. In conclusion, our results suggest that the M allele of L55M polymorphism could be a suitable marker for BC susceptibility and tumor prognosis in Egyptian women.

The melanoma-associated antigen-A3, -A4 genes: relation to the risk and clinicopathological parameters in breast cancer patients.

This study aimed to evaluate the clinical reliability and accuracy of two MAGE transcripts (MAGE-A3, MAGE-A4 mRNA) in the peripheral blood (PB) of patients with breast cancer (BC), and to evaluate their potential limits and utility to detect BC. We aimed also to analyze their relation to clinicopathological characteristics of the tumor. This study is a prospective, controlled, double-blinded study conducted on 100 BC women and 100 age-matched control women. There were 52 patients with localized breast mass with no evidence of nodal affection or distant metastases and 48 patients suffering from metastatic BC. MAGE-A3 and MAGE-A4 mRNA in the PB were assayed using reverse transcriptase-polymerase chain reaction (RT-PCR). None of the control women was positive for either MAGE-A3, MAGE-A4. In BC women, positivity for MAGE-A3 in PB was observed in 37 patients (37%), and MAGE-A4 positivity was observed in 11 patients (11%); with 100% specificity for both transcripts. The presence of MAGE-A3 was significantly associated with nodal status (P = 0.009), tumor size (P = 0.009), and American Joint Committee on Cancer stage (P = 0.009), whereas MAGE-A4 positivity was significantly associated with histological grade (P = 0.020). RT-PCR assays of MAGE-A3 and MAGE-A4 in the PB of BC patients may have prognostic and predictive implications, and they are promising specific tumor markers of BC.

Serum copper is a simple but valuable prognostic marker in B-cell chronic lymphocytic leukemia.

We investigated the relationship between serum copper and various prognostic factors, time to start treatment, and treatment response in patients with B-cell chronic lymphocytic leukemia (B-CLL) and related disorders. Fifty newly diagnosed CLL patients aged 36-70 years were included. Patients were studied for serum lactate dehydrogenase (LDH), serum copper, direct Coombs' test, serum ß(2) microglobulin (ß(2)M), immunophenotyping for diagnosis of B-CLL, evaluation of CD38 and zeta-associated protein (ZAP-70) expression, and fluorescence in situ hybridization technique for cytogenetic analysis. Fourteen of 50 patients had high serum copper level; they had a significant increase in LDH, serum ß(2)M, incidence of positive Coombs' test, CD38 and ZAP-70, incidence of 17p del, and a decrease in hemoglobin concentration, lymphocyte doubling time and time to start treatment with a lower treatment response rate. No significant difference was found with regard to Rai staging for CLL. These results indicate that serum copper level, a cheap and simple laboratory test, is of great value in CLL patients as it showed a significant association with some important adverse prognostic markers such as increased expression of ZAP-70 and CD38, shorter time to start treatment and poor response to treatment.