Pregnancy induces significant adaptations in the cardio-autonomic nervous system, with additional cardiac stress in preeclampsia potentially impacting ventricular repolarization. Despite the widespread use of QT-prolonging drugs during pregnancy, the extent of heart rate (HR)-corrected QT (QTc) interval changes during normal pregnancy and preeclampsia remains unclear. This study aimed to quantify changes in QTc interval across different trimesters of normal pregnancy and third-trimester preeclampsia. Eight databases were systematically searched from their inception to January 13, 2025. Any type of study design, except case reports/series, reporting QT interval and HR or RR interval, and/or QTc interval for at least one trimester were included. Those reporting at least two trimesters or one trimester with nonpregnant controls were pooled in meta-analyses using random-effect models to calculate pooled mean differences (MD) across trimesters. Data from 57 studies (6,686 participants) were included with 33 studies (5,153 participants) pooled in meta-analyses. Compared with nonpregnant individuals, QTc intervals increased across trimesters of normal pregnancy and in third-trimester preeclampsia. Meta-analyses revealed significant increases in QTc interval during first (MD = 10.0 msec), second (MD = 20.2 msec), and third trimesters (MD = 23.0 msec) compared with nonpregnant individuals. Furthermore, preeclampsia increased the QTc interval by 21.7 msec during the third trimester compared to normal pregnancy. No publication bias was detected, and the overall quality scores of most studies were fair ( n  = 23) or poor ( n  = 33). A significant QTc interval lengthening throughout normal pregnancy was identified, and to a greater extent during preeclampsia. The arrhythmogenicity in third-trimester preeclampsia with a known risk for QTc interval prolongation, especially with using QT-prolonging drugs, warrants further investigation.

ABSTRACT:Testosterone and dihydrotestosterone (DHT) attenuate drug-induced lengthening of ventricular repolarization, but it is unknown whether they influence drug-induced torsades de pointes (TdP). We tested the hypothesis that DHT reduces the incidence and severity of drug-induced TdP. Male New Zealand white rabbits underwent orchiectomy and were implanted with 2 subcutaneous sustained-release pellets containing DHT 50 mg (100 mg, n = 23) or placebo (n = 20). After 7 days, hearts were excised, mounted, and perfused with modified Krebs-Henseleit solution. The atrioventricular node was destroyed manually. Dofetilide 100 nM was perfused for 30 minutes. Median (Q 1 , Q 3 ) serum DHT concentrations were higher in DHT-treated rabbits [314.0 (232.5, 388.6) vs. 32.5 (28.5, 36.2) ng/dL, P < 0.0001]. The incidence of TdP in the DHT and placebo groups was 6/23 (26%) versus 8/20 (40%), respectively ( P = 0.33). In hearts that developed TdP, the median (Q 1 , Q3) number of episodes was lower in the DHT group [2 (2, 3.5) vs. 18.5 (12.5, 20.5), P = 0.01]. The median time to first TdP episode was longer [18.5 (17.3, 21.3) vs. 10.5 (12.5, 20.5) minutes, P = 0.01], and the TdP burden (median total number of TdP beats per heart) was lower in the DHT group [11.5 (9.5-46.5) vs. 271 (99.3-440.3) beats, P = 0.007]. Pre-dofetilide Fridericia-corrected QT intervals were shorter in the DHT group [377 (366, 390) vs. 385 (378, 401) ms, P = 0.02]. Maximum Fridericia-corrected QT interval during dofetilide perfusion was shorter in the DHT group [399 (390, 410) vs. 414 (399, 442) ms, P = 0.007]. In conclusion, DHT diminishes the severity of dofetilide-induced TdP.

Background – Enoxaparin is an anticoagulant used for preventing and treating venous thromboembolism. However, there is limited information on optimal enoxaparin dosing and an absence of evidence-based dosage regimens in neonates and infants, especially in critically ill patients. This project aims to develop a population pharmacokinetic model to describe the anti-factor Xa (anti-Xa) activity-time profile and to identify sources of variation in anti-Xa activity following enoxaparin subcutaneous administration in neonates and infants.Method – Anti-Xa activity, dosing, demographic and clinical covariates were obtained from the electronic health records of neonates and infants admitted between 2016 to 2021 for cardiac indications to the intensive care unit Riley Hospital for Children at Indiana University Health in Indianapolis, IN. Demographic data included date of birth, body weight, and laboratory measurements for serum creatinine (SCr), hemoglobin, and platelet count. PK modeling was performed using NONMEM 7.4 and Pirana 2.10 with the FOCE-I method. Goodness-of-fit plots were generated using xpose package in R 4.3. Stepwise covariate modeling was used for covariate selection based on model objective function value, relative standard error, BIC, and condition number. Results – This analysis included 499 patients with a median age of 2.2 (IQR, 0.9 – 4.9) mos, 24098 enoxaparin doses, and 2721 plasma samples analyzed for anti-Xa activity. The PK of enoxaparin was best characterized by a one-compartment model with first-order absorption and elimination. Inter- and intra-individual variability were modeled exponentially, and proportionally, respectively. The model that fit the data best integrated weight scaled allometrically with exponents of 0.75 and 1.0 on apparent clearance (CL/F) and apparent volume of distribution (Vd/F), respectively. The estimated population CL/F, Vd/F, and Ka were 1.27 L/h/70 kg (95% CI: 1.17 to 1.37), 26.9 L/70 kg (95% CI: 23.2 to 30.6), and 0.77 h-1 (95% CI: 0.36 to 1.18), respectively. The disposition of enoxaparin using anti-Xa activity as a surrogate was influenced by SCr and postnatal age (PNA). The Vd/F was notably affected by PNA, with higher exposure observed as age increased until about 1.5 mos PNA, at which point the effect plateaus. The final model incorporated SCr on CL/F using a power function with estimated effect of -0.21 (95% CI: -0.31 to -0.09). A piecewise function was used to model PNA on Vd/F with an inflection point of 1.5 mos, and an estimated effect of -0.57 (95% CI: -0.77 to -0.37).Conclusions – The estimated CL/F, and Vd/F of enoxaparin in neonates and infants in this study are higher than reported values in adults (CL/F ≈ 0.74 L/h, Vd/F ≈ 5.0 L). The inverse relationship between SCr and CL/F indicates dose adjustment may be required in this population. Additionally, pediatric patients less than 1.5 mos of age require higher body-weight adjusted doses of enoxaparin than older infants and adults.Citations: N/A

Heart failure (HF) with reduced ejection fraction (HFrEF) is a risk factor for drug-induced QT interval prolongation. It is unknown if HF with preserved ejection fraction (HFpEF) is also associated with an increased risk. Dofetilide and sotalol are potent QT interval-prolonging agents that are frequently used in patients with HFpEF, in whom atrial fibrillation is a common comorbidity. We tested the hypothesis that the risk of QT interval prolongation associated with dofetilide and sotalol is increased in patients with HFpEF. We conducted a retrospective cohort study conducted using electronic health records from the Indiana Network for Patient Care (January 31, 2010 -May 3, 2021). After removing patients with overlapping diagnoses of HFpEF and HFrEF, no diagnosis code, and absence of QT interval records, we identified patients taking dofetilide or sotalol among three groups: HFrEF (n = 138), HFpEF (n = 109), and no HF (n = 729). QT prolongation was defined as heart rate-corrected QT (QTc) > 500 ms during dofetilide/sotalol therapy. Unadjusted odds ratios (OR) for QT prolongation were determined by univariate analysis. Adjusted ORs were determined by generalized estimating equations (GEE) with logit link to account for an individual cluster with different times of hospitalization and covariates. QTc prolongation associated with dofetilide or sotalol occurred in 53.2%, 71.7% and 30.0% of patients with HFpEF, HFrEF, and patients with no HF, respectively. After adjusting for age, sex, race, serum potassium and magnesium concentrations, kidney function, concomitant drug therapy, and comorbid conditions, the adjusted odds of QTc prolongation were significantly higher in patients with HFpEF [OR = 1.98 (95% CI 1.17-3.33)], and in those with HFrEF [OR = 5.23, (3.15-8.67)], compared to those with no evidence of HF. The odds of QT prolongation among inpatients receiving dofetilide or sotalol were increased in patients with HFpEF and HFrEF compared to those who did not have HF.

Background:Heart failure (HF) with reduced ejection fraction (HFrEF) is a risk factor for drug-induced arrhythmias. It is unknown whether HF with preserved ejection fraction (HFpEF) also increases the risk. Objectives:The purpose of this study was to determine if the risk of ventricular tachycardia (VT) and sudden cardiac arrest (SCA) is increased in patients with HFpEF prescribed dofetilide or sotalol. Methods:Using Medicare claims and pharmacy benefits from 2014 to 2016, we identified patients taking dofetilide or sotalol and non-dofetilide/sotalol users among 3 groups: HFrEF (n = 26,176), HFpEF (n = 33,304), and no HF (n = 580,249). Multinomial propensity score matching was performed. We compared baseline characteristics using Cochran-Mantel-Haenszel statistics and standardized differences, and tested associations of VT and SCA among dofetilide/sotalol users and those with HFpEF, HFrEF, or no HF using a generalized Cox proportional hazards model. Results:VT and SCA occurred 166 (10.68%) and 16 (1.03%) of 1,554 dofetilide/sotalol users with HFpEF, 543 (38.76%) and 40 (2.86%) of 1,401 dofetilide/sotalol users with HFrEF, and 245 (5.06%) and 13 (0.27%) of 4,839 dofetilide/sotalol users without HF. Overall VT risk was increased in HFrEF and HFpEF patients (HR: 7.00 [95% CI: 6.10-8.02] and 1.99 [95% CI: 1.70-2.32], respectively). The risk of VT in patients prescribed dofetilide/sotalol was increased in HFrEF and HFpEF patients (1.53 [95% CI: 1.07-2.20] and 2.34 [95% CI: 1.11-4.95], respectively). While the overall SCA risk was increased in HFrEF and HFpEF patients (5.19 [95% CI: 4.10-6.57] and 2.53 [95% CI: 1.98-3.23], respectively), dofetilide/sotalol use was not associated with an increased SCA risk. Conclusions:In patients with HF who are prescribed dofetilide or sotalol, the risk of VT, but not SCA, was increased.

Background Heart failure (HF) with preserved ejection fraction (HFpEF) is associated with enhanced response to drug-induced QT interval lengthening. We determined the influence of HFpEF on drug-induced lengthening of dispersion of repolarization, a measure of proarrhythmic risk. Methods We administered intravenous ibutilide 0.003 mg/kg to 10 patients with HFpEF and 10 age- and sex-matched controls without HF. Twelve‑lead electrocardiograms were obtained prior to ibutilide and serially for 8 h post-ibutilide. Tpeak-Tend, a measure of dispersion of ventricular repolarization, and heart rate-corrected J-Tpeak (J-Tpeakc), representing early repolarization, were measured by an investigator blinded to study groups. Results Baseline (pre-ibutilide) Tpeak-Tend and J-Tpeakc were not significantly different in the HFpEF and control groups. Maximum Tpeak-Tend was longer in the HFpEF group than in the control group (85 ± 10 vs 73 ± 8 ms, p = 0.01). Additionally, % change from baseline in Tpeak-Tend was greater in the HFpEF group [median (IQR) 17 (11) vs 8 (3)%, p = 0.003]. The area under the effect curve from 0 to 8 hours following ibutilide (including the 10-minute infusion) (AUEC0-8.17) for Tpeak-Tend was also larger in the HFpEF group (600 ± 42 vs. 543 ± 49 ms•hr, p = 0.03). Maximum J-Tpeakc, % change from baseline in J-Tpeakc and AUEC0-8.17 for J-Tpeakc in the two groups were not significantly different. Conclusion HFpEF is associated with enhanced response to drug-induced increases in dispersion of repolarization.

Background: Variability in the pharmacokinetics and pharmacodynamics of oxycodone in children undergoing surgery could be due to genetic polymorphisms. Materials & methods: The authors studied the association between clinical outcomes and pharmacogenes in children undergoing major surgery. A total of 89 children (35 undergoing pectus excavatum repair and 54 undergoing spinal fusion) were recruited. Results: OPRM1 SNP rs6902403 showed an association with maximum pain score and total morphine equivalent dose (p < 0.05). Other polymorphisms in OPRM1 SNP, PXR, COMT and ABCB1 were also shown to be associated with average morphine equivalent dose, length of hospital stay and maximum surgical pain (p < 0.05). Conclusion: This study demonstrates novel associations between the above pharmacogenes and oxycodone's pharmacokinetics as well as postoperative outcomes in children. Clinical trial registration: NCT03495388 (ClinicalTrials.gov).

Abstract: Remdesivir, approved for the treatment of COVID-19, has been associated with heart-rate corrected QT interval (QTc) prolongation and torsade de pointes in case reports. However, data are conflicting regarding the ability of remdesivir to inhibit the human ether-a-go-go-related gene (hERG)-related current. The objective of this study was to investigate the effects remdesivir and its primary metabolite, GS-441524, on hERG-related currents. Human embryonic kidney 293 cells stably expressing hERG were treated with various concentrations of remdesivir and GS-441524. The effects of acute and prolonged exposure on hERG-related current were assessed using whole-cell configuration of voltage-clamp protocols. Acute exposure to remdesivir and GS-441524 had no effect on hERG currents and the half-activation voltage (V1/2). Prolonged treatment with 100 nM and 1 µM remdesivir significantly reduced peak tail currents and hERG current density. The propensity for remdesivir to prolong QTc intervals and induce torsade de pointes in predisposed patients warrants further investigation.

Introduction: KCNH2 codes for a potassium channel that mediates the primary cardiac repolarizing current, I Kr . Calcium/calmodulin-dependent protein kinase II (CaMKII) modulates several ion channels, is enhanced during sustained β-adrenergic receptor (βAR) stimulation, and is proarrhythmic. Furthermore, states of enhanced βAR stimulation/CaMKII activity, such as heart failure, alter microRNA (miR) expression profiles that suppress protein expression. Hypothesis: CaMKII decreases KCNH2-related current during sustained βAR stimulation through miR regulation. Methods: Putative miR binding sites (n=327) on KCNH2 3′-UTR were identified with bioinformatic tools. Binding sites were then constructed into a pIS0 plasmid, amplified, and transfected into hiPSC-cardiomyocytes. Cells were subjected to sustained βAR stimulation (Isoproterenol: 100 nM, 1 μM), constitutively active δCaMKII (Thr287Asp), or control conditions. Next-generation sequencing was used to identify altered miR binding sites expression and endogenous miR alterations. Whole-cell, voltage-clamp experiments were performed in HEK293 cells expressing KCNH2 and δCaMKII T287D . Cells were treated with isoproterenol, and miR mimics were used for validation of the bioassay. Results: Nine and eight predicted miR binding sites were downregulated in the CaMKII group (Δlog fold: -0.287 to -0.59, p<0.05) and sustained βAR group (Δlog fold: -0.29 to -0.72, p<0.05), respectively. Thirty miRs were predicted to bind to these sites on KCNH2 3′-UTR, of which seven were selected for validation based on a Δlog fold < -0.6 in one group (n=4) or < -0.5 in both treatment groups (n=3). Mimics of all seven miRs reduced KCNH2 protein expression, and three decreased KCNH2 currents compared to control (p<0.05). Sustained βAR stimulation decreased peak activation current in cells expressing both KCNH2 and δCaMKII T287D by 25% (17±7 pA/pF, p = 0.03) and tail current density by 27% (21±9 pA/pF, p = 0.03), but had no effect on currents in cells expressing only KCNH2. Conclusions: CaMKII hyperactivity alters miR expression profiles and decreases KCNH2 function during sustained βAR activation. The effect of the identified miRs on arrhythmia susceptibility with enhanced CaMKII activity warrants further study.

IntroductionDrug-induced prolongation of the heart rate-corrected QT interval (QTc) is associated with increased risk for the potentially fatal arrhythmia torsades de pointes. Due to arrhythmia risk, clinical trials with cancer therapeutics often exclude patients based on thresholds for QTc prolongation. Our objective was to assess associations between prescriptions for QT-prolonging drugs and the odds of meeting cancer trial exclusionary QTc thresholds in a cohort of adults with advanced cancer.MethodsElectronic health records were retrospectively reviewed for 271 patients seen at our institutional molecular solid tumor clinic. Collected data included demographics, QTc measurements, ventricular arrhythmia-related diagnoses, and all inpatient and outpatient prescriptions. Potential associations were assessed between demographic and clinical variables, including prescriptions for QT-prolonging drugs, and QTc measurements.ResultsWomen had longer median QTc measurements than men (p = 0.030) and were prescribed more QT-prolonging drugs during the study (p = 0.010). In all patients, prescriptions for QT-prolonging drugs were associated with longer median and maximum QTc measurements at multiple assessed time points (i.e., for QT-prolonging drugs prescribed within 10, 30, 60, and 90 days of QTc measurements). Similarly, the number of QT-prolonging drugs prescribed was correlated with longer median and maximum QTc measurements at multiple time points. Common QTc-related exclusionary criteria were collected from a review of ClinicalTrials.gov for recent cancer clinical trials. Based on common exclusion criteria, prescriptions for QT-prolonging drugs increased the odds of trial exclusion.ConclusionThis study demonstrates that prescriptions for QT-prolonging drugs were associated with longer QTc measurements and increased odds of being excluded from cancer clinical trials.

Introduction: Heart failure (HF) with reduced ejection fraction (HFrEF) is a risk factor for drug-induced proarrhythmia. It is unknown whether patients with HF with preserved ejection fraction (HFpEF) are also at increased risk. Hypothesis: The risk of drug-associated ventricular tachycardia (VT) and sudden cardiac arrest (SCA) is increased in patients with HFpEF. Methods: Using Medicare enrollment in fee-for-service medical and pharmacy benefits (2014 to 2016) and ICD-9/10 codes, we identified patients taking drugs known to cause torsades de pointes (TdP drugs; www.crediblemeds.org) and non-TdP drug users among three groups: HFrEF (n=31,422), HFpEF (n=40,012), and no HF (n=633,558). Multinomial propensity score-matching was performed to minimize baseline differences in covariates (patient demographics, comorbidities, health care utilization and drug history). Cochran-Mantel-Haenszel statistics and standardized differences were used to compare baseline characteristics. A generalized Cox proportional hazards model was used to estimate hazard ratios (HRs) and test the association of VT and SCA among TdP drug users with HFpEF, HFrEF, and no HF. Results: Of 23,910 TdP drug users with HFrEF, VT and SCA occurred in 4,263 (17.8%) and 493 (2.1%) patients, respectively. In comparison, among 31,359 TdP drug users with HFpEF, VT and SCA occurred in 1,570 (5.0%) and 340 (1.1%) patients. VT and SCA occurred in 3,154 (0.8%) and 528 (0.1%) of 384,824 TdP drug users without HF. The overall risk of both VT and SCA was increased in patients with HFrEF and in those with HFpEF (Table). The risk of VT associated with TdP drugs was increased across the overall population. Use of TdP drugs significantly increased the risk of VT and SCA in patients with HFrEF, but not in patients with HFpEF. Conclusions: The risk of VT and SCA associated with drugs known to cause TdP was increased in patients with HFrEF but not in those with HFpEF.

Introduction: Heart failure (HF) with reduced ejection fraction (HFrEF) is a risk factor for drug-induced QT interval prolongation. It is unknown if HF with preserved ejection fraction (HFpEF) also increases the risk. Dofetilide (D) and sotalol (S) are potent QT interval-prolonging agents that are commonly used in patients with HFpEF, in whom atrial fibrillation is a common comorbidity. Hypothesis: The risk of QTc prolongation associated with D or S (D/S) is increased in patients with HFpEF. Methods: The data source was electronic health records from the Indiana Network for Patient Care (February 2010 to May 2021). After removing patients with overlapping diagnoses of HFpEF and HFrEF, no diagnosis code, absence of QT interval records, and no validated record of using D/S, we identified patients taking D/S among three groups: HFrEF (n=138), HFpEF (n=109), and no HF (n=729). Cochran-Mantel-Haenszel statistics were used to compare baseline characteristics. QT interval prolongation was defined as heart rate-corrected QT (QTc) > 500 ms during D/S therapy. Unadjusted odds ratios (OR) of QT interval prolongation were determined by univariate analysis, and adjusted ORs were determined by generalized estimating equations (GEE) with logit link to account for an individual cluster with different times of hospitalization and covariates. Results: QTc prolongation associated with D/S occurred in 51.2% of patients with HFpEF, 70.1% of patients with HFrEF, and 29.4% of patients with no HF. After adjusting for age, sex, race, serum potassium and magnesium concentrations, kidney function, concomitant drug therapy, and comorbid conditions, the adjusted odds of QTc prolongation were significantly higher in patients with HFpEF, as well as in those with HFrEF, compared to those with no evidence of HF (Table). Conclusions: The odds of QT interval prolongation among inpatients receiving D/S were increased in patients with HFpEF and HFrEF compared to those who did not have HF.

Methadone is a synthetic opioid used as an analgesic and for the treatment of opioid abuse disorder. The analgesic dose in the pediatric population is not well-defined. The pharmacokinetics (PKs) of methadone is highly variable due to the variability in alpha-1 acid glycoprotein (AAG) and genotypic differences in drug-metabolizing enzymes. Additionally, the R and S enantiomers of methadone have unique PK and pharmacodynamic properties. This study aims to describe the PKs of R and S methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in pediatric surgical patients and to identify sources of inter- and intra-individual variability. Children aged 8-17.9 years undergoing orthopedic surgeries received intravenous methadone 0.1 mg/kg intra-operatively followed by oral methadone 0.1 mg/kg postoperatively every 12 h. Pharmacokinetics of R and S methadone and EDDP were determined using liquid chromatography tandem mass spectrometry assays and the data were modeled using nonlinear mixed-effects modeling in NONMEM. R and S methadone PKs were well-described by two-compartment disposition models with first-order absorption and elimination. EDDP metabolites were described by one compartment disposition models with first order elimination. Clearance of both R and S methadone were allometrically scaled by bodyweight. CYP2B6 phenotype was a determinant of the clearance of both the enantiomers in an additive gene model. The intronic CYP3A4 single-nucleotide polymorphism (SNP) rs2246709 was associated with decreased clearance of R and S methadone. Concentrations of AAG and the SNP of AAG rs17650 independently increased the volume of distribution of both the enantiomers. The knowledge of these important covariates will aid in the optimal dosing of methadone in children.

Dear Editor, The majority of allograft rejection occurs within 1 month after liver transplantation; with the highest incidence around 7–10 days. In this study, we demonstrate the impact of donor and recipient genotypes on tacrolimus clearance and dosing requirements during the first 28 days following liver transplantation. Tacrolimus is primarily metabolized by cytochrome P450 (CYP) 3A isozymes, CYP3A4 and CYP3A5, which mediate hepatic and intestinal biotransformation.1 However, it is unknown how the influence of CYP3A5 genotype of the donor and recipient contribute to tacrolimus variability as liver performance improves with time in the early post-operative phase.2–7 There remains an unmet medical need to find an optimal dose regimen for immunosuppressants within the first few weeks after transplantation to avoid potential toxicities due to overdose or acute rejection.2 Thus, the goal of our work is to establish personalized immunosuppressive regimens following liver transplantation. By using genetics and patient-related factors, individualized dosing regimens can be initiated and used with current drugmonitoring protocols to decrease toxicity and graft rejection during the early phases of post-transplant. We enrolled adult patients in two independent cohorts undergoing orthotopic liver transplantation. Tacrolimus and mycophenolate mofetil were administered following transplant without steroids. Patients were excluded from undergoing multi-organ transplantation or had incomplete data. Cohort A (index set) comprised 115 from Shanghai General Hospital Affiliated to Shanghai Jiao Tong University. Cohort B (validation set) comprised 95 patients from First Affiliated Hospital of Zhengzhou University. The patient demographics are displayed in Table 1. The research was carried out in accordance with the Declaration of Helsinki and was approved by the Ethics Commit-

Aim: Methadone exhibits significant variability in clinical response. This study explores the genetic influence of variable methadone pharmacokinetics. Methods: This is a prospective study of methadone in children undergoing major surgery. CYP2B6 genotyping, plasma methadone and metabolite levels were obtained. Clinical outcomes include pain scores and postoperative nausea and vomiting (PONV). Results: CYP2B6 poor metabolizers (*6/*6) had >twofold lower methadone metabolism compared with normal/rapid metabolizers. The incidence of PONV was 4.7× greater with CYP2B6 rs1038376 variant. AG/GG variants of rs2279343 SNP had 2.86-fold higher incidence of PONV compared with the wild variant ( AA). Nominal associations between rs10500282, rs11882424, rs4803419 and pain scores were observed. Conclusion: We have described novel associations between CYP2B6 genetic variants and perioperative methadone metabolism, and associations with pain scores and PONV.