The extracellular matrix molecules versican and hyaluronan in urethral and vaginal tissues in stress urinary incontinence.

PURPOSE: Abnormal extracellular matrix (ECM) changes are correlated with stress urinary incontinence (SUI). The ECM components versican (Vcan) and hyaluronan (HA) play key roles in regulating tissue inflammation and maintaining connective tissue homeostasis. We analyzed the localization and expression of these ECM components in urethral and vaginal tissues from a rat model of urinary incontinence and from human clinical specimens. METHODS: Nulliparous rats underwent vaginal distension (VD), a rodent model of SUI, or a sham procedure. Tissues were harvested from six rats per group at days 1, 4, and 21 for immunohistochemistry and RNA expression analysis of ECM components. Periurethral vaginal samples from female patients with SUI were also examined. RESULTS: High-intensity staining for Vcan was observed 1 day after procedure in both control and VD animals. This level of abundance persisted at day 4 in VD compared to control, with concurrent reduced messenger RNA (mRNA) expression of the Vcan-degrading enzymes ADAMTS5 and ADAMTS9 and reduced staining for the Vcan cleavage epitope DPEAAE. Abundance of HA was not different between VD and control, however mRNA expression of the HA synthase Has2 was significantly reduced in VD tissues at day 4. Abundant Vcan staining was observed in 60% of SUI patient samples, which was strongest in regions of disrupted elastin. CONCLUSION: Reduction of Vcan-degrading enzymes and HA synthases at day 4 postsurgery indicates a potential delay in ECM turnover associated with SUI. Abundant Vcan is associated with inflammation and elastin fiber network disruption, warranting further investigation to determine its role in SUI pathogenesis.

Practical use of the new American Urological Association adult urodynamics guidelines.

In 2012, the American Urological Association published guidelines on adult urodynamics. On the basis of a review of the available evidence, they established guideline recommendations for urodynamics in adults with stress urinary incontinence/pelvic organ prolapse, overactive bladder/urinary urgency incontinence/mixed urinary incontinence, neurogenic bladder, and lower urinary tract symptoms. This article reviews the practical use of these guidelines in a clinical practice.

Long-term follow-up of polypropylene bone anchored slings.

INTRODUCTION: The transvaginal bone anchored polypropylene sling (BAS) has proven to be a successful treatment for patients with SUI. However, there is limited data on long-term outcomes following BAS with polypropylene mesh. We report our series of patients who had at least 3 years of follow-up after placement of BAS. MATERIALS AND METHODS: A retrospective review of prospectively collected data of patients undergoing BAS for stress urinary incontinence (SUI) with minimum 3 year follow-up was performed. Outcomes and complications were determined from annual mailed post-operative questionnaires. RESULTS: 142 patients who had undergone BAS and had answered post-operative questionnaires at a minimum of 3 years were identified. Average follow-up was 58 months (range 36-97 months). The overall success rate was 71 % with a dry rate of 27 %. Complications occurred in 9 % of patients, more commonly in patients without a history of anti-incontinence procedure. CONCLUSIONS: Although less commonly used, BAS with polypropylene mesh is associated with an acceptable success rate at long term follow-up but a low completely dry rate.

Anterior pelvic organ prolapse repair using synthetic mesh.

Since the U.S. Food and Drug Administration (FDA) statement on mesh in July of 2011, there has been controversy regarding synthetic mesh repairs for vaginal prolapse. In this article, we review the biochemical basis for the use of synthetic mesh in prolapse repair as well as clinical results of anterior compartment prolapse repair with synthetic mesh. Finally, we discuss the FDA warning regarding mesh.

Overactive bladder and pain: management strategies.

Overactive bladder associated with pain (ie, interstitial cystitis/bladder pain syndrome [IC/BPS]) is a prevalent condition that can be very difficult to diagnose and even more difficult to treat. In this paper, we review the studied therapies for IC/BPS, including non-medication-based therapies, oral and intravesical-based medications, and surgical treatments for interstitial cystitis. We also review an approach to the treatment of a patient with IC/BPS using the previously published UPOINT clinical phenotyping template.

Prevalence of hyperoxaluria after bariatric surgery.

PURPOSE: Recent investigations have shown increased oxalate excretion in patients in whom kidney stones formed after contemporary bariatric surgery. We determined whether there is an increased prevalence of hyperoxaluria after such procedures performed in nonstone formers. MATERIALS AND METHODS: A total of 58 nonstone forming adults who underwent laparoscopic Roux-en-Y (52) or a biliopancreatic diversion-duodenal switch procedure (6) collected 24-hour urine specimens 6 months or greater after bariatric surgery. Standard stone risk parameters were assessed. Comparisons were made with a group of healthy nonstone forming adults and stone formers in a commercial database. RESULTS: The bariatric group had a significantly higher mean urinary oxalate excretion compared to that in controls and stone formers (67.2 vs 34.1 and 37.0 mg per day, respectively, p <0.001). Mean oxalate excretion of patients who underwent a biliopancreatic diversion-duodenal switch procedure was higher than in the Roux-en-Y group (90 vs 62 mg per day, p <0.05). There was a significant correlation between urine oxalate excretion on the 2 collection days but some patients showed significant variability. Of the patients 74% showed hyperoxaluria in at least 1, 24-hour urine collection and 26% demonstrated profound hyperoxaluria, defined as oxalate excretion more than 100 mg per day, in at least 1 collection. This occurred in 3 of the 6 patients in the biliopancreatic diversion-duodenal switch group and in 12 of the 52 in the Roux-en-Y cohort. Hyperoxaluria was not uniformly expressed. CONCLUSIONS: There is a high prevalence of hyperoxaluria in patients without a history of kidney stones who undergo bariatric surgery. A significant proportion of these patients have profound hyperoxaluria, which is not uniformly expressed.

Analysis of second-generation antidepressant drug, sertraline and its active metabolite, N-desmethyl sertraline in human plasma by a sensitive and selective liquid chromatography-tandem mass spectrometry method.

A precise, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of sertraline (SER) and its primary metabolite, N-desmethyl sertraline (NDS) in human plasma is developed and validated. The analytes and the internal standard-fluoxetine were extracted from 300 microL aliquots of human plasma via liquid-liquid extraction in methyl tert-butyl ether. Chromatographic separation was achieved in a run time of 2.5 min on a Betasil C8 column (100 mm x 2 .1 mm, 5 microm) under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for SER, NDS and IS were m/z 306.2-->159.0, 292.1-->159.0 and 310.6-->148.4, respectively. The method was fully validated for its sensitivity, selectivity, linearity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 0.5-150 ng/mL was established for both the analytes with mean correlation coefficient (r) of 0.9993 and 0.9980, respectively. The intra-batch and inter-batch precision (%CV) across five quality control levels was

An accurate, rapid and sensitive determination of tramadol and its active metabolite O-desmethyltramadol in human plasma by LC-MS/MS.

A rapid, sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous determination of tramadol and its active metabolite, O-desmethyltramadol in human plasma is developed using propranolol as internal standard (IS). The analytes and IS were extracted from 200 microL aliquots of human plasma via protein precipitation using acetonitrile. Chromatographic separation was achieved in a run time of 2.0 min on an Aquasil C18 (100mm x 2.1mm, 5microm) column under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The method was fully validated for its selectivity, sensitivity, linearity, precision and accuracy, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. A linear dynamic range was established from 1.0 to 600.0ng/mL for tramadol and 0.5-300.0ng/mL for O-desmethyltramadol. The method was successfully applied to a bioequivalence study of 200mg tramadol tablet formulation in 27 healthy Indian male subjects under fasting condition.

High throughput and sensitive determination of trazodone and its primary metabolite, m-chlorophenylpiperazine, in human plasma by liquid chromatography-tandem mass spectrometry.

A precise, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of trazodone (TRZ) and its primary metabolite, m-chlorophenylpiperazine (mCPP), in human plasma was developed and validated. The analytes and the internal standard-nefazodone were extracted from 500 microL aliquots of human plasma via liquid-liquid extraction in n-hexane. Chromatographic separation was achieved in a run time of 2.5 min on a Betabasic cyano column (100 mm x 2.1 mm, 5 microm) under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for TRZ, mCPP and IS were m/z 372.2-->176.2, 197.2-->118.1 and 470.5-->274.6 respectively. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 10.0-3000.0 ng/mL for TRZ and 0.2-60.0 ng/mL for mCPP was evaluated with mean correlation coefficient (r) of 0.9986 and 0.9990 respectively. The intra-batch and inter-batch precision (%CV) across five validation runs (LLOQ, lower limit of quantitation; LQC, low quality control; MQC, middle quality control; HQC, high quality control and ULOQ, upper limit of quantitation) was < or =8.4% for both the analytes. The method was successfully applied to a bioequivalence study of 100mg trazodone tablet formulation in 36 healthy Indian male subjects under fasting and fed conditions.

Liquid chromatography tandem mass spectrometry assay for the simultaneous determination of venlafaxine and O-desmethylvenlafaxine in human plasma and its application to a bioequivalence study.

A rapid, simple and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for simultaneous determination of venlafaxine (VEN) and its active metabolite, O-desmethylvenlafaxine (ODV) in human plasma was developed using nadolol as internal standard (IS). The analytes and IS were extracted from 200 microl aliquots of human plasma via protein precipitation using 0.43% formic acid in acetonitrile and separated on a Hypurity cyano (50 mm x 4.6 mm, 5 microm) column. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring (MRM) and positive ion mode. The precursor to product ion transitions monitored for VEN, ODV and IS were m/z 278.3-->58.1, 264.3-->58.1 and 310.4-->254.1, respectively. The total chromatographic runtime was 3 min with retention time for VEN, ODV and IS at 1.93, 1.50 and 1.29 min, respectively. The method was fully validated for its sensitivity, accuracy and precision, linearity, recovery, matrix effect, dilution integrity and stability studies. The linear dynamic range of 2.0-500 ng/ml was established for both VEN and ODV with mean correlation coefficient (r), 0.9994 and 0.9990, respectively. The intra-batch and inter-batch precision (%CV) in three validation batches across five concentration levels (LLOQ, LQC, MQC, HQC and ULOQ) was less than 12.6% for both the analytes. The accuracy determined at these levels was within -9.8 to +3.9% in terms of %bias. The method was successfully applied to a bioequivalence study of 150 mg venlafaxine extended release capsule formulation in 22 healthy Indian male subjects under fed condition.

High-throughput LC-MS/MS assay for 6-methoxy-2-naphthylacetic acid, an active metabolite of nabumetone in human plasma and its application to bioequivalence study.

A simple, precise and accurate assay for the determination of 6-methoxy-2-naphthylacetic acid (6-MNA), an active metabolite of nabumetone in human plasma, was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte (6-MNA) and propranolol (internal standard, IS) were extracted from 200 microL aliquot of human plasma via solid-phase extraction employing HLB Oasis cartridges and separated on a Discovery HS C18 (50 x 4.6 mm, 5 microm) column. Detection of analyte and IS was done by tandem mass spectrometry with a turbo ion spray interface operating in positive ion and multiple reaction monitoring acquisition mode. The total chromatographic runtime was 3.0 min with retention time for 6-MNA and IS at 1.97 and 1.26 min, respectively. The method was validated over a dynamic linear range of 0.20-60.00 microg/mL for 6-MNA with mean correlation coefficient r > or = 0.9986. The intra-batch and inter-batch precision (%CV) across five validation runs (lower limit of quantiation, low-, medium- and high-quality controls and upper limit of quantitation) was less than 7.5%. The accuracy determined at these levels was within -5.8 to +0.2% in terms of percentage bias. The method was successfully applied for a bioequivalence study of 750 mg nabumetone tablet formulation in 12 healthy Indian male subjects under fasted condition.

Determination of asenapine in presence of its inactive metabolites in human plasma by LC-MS/MS.

A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been described for the determination of asenapine (ASE) in presence of its inactive metabolites N-desmethyl asenapine (DMA) and asenapine-N-glucuronide (ASG). ASE, and ASE 13C-d3, used as internal standard (IS), were extracted from 300 µL human plasma by a simple and precise liquid-liquid extraction procedure using methyl tert-butyl ether. Baseline separation of ASE from its inactive metabolites was achieved on Chromolith Performance RP8e (100 mm × 4.6 mm) column using acetonitrile-5.0 mM ammonium acetate-10% formic acid (90:10:0.1, v/v/v) within 4.5 min. Quantitation of ASE was done on a triple quadrupole mass spectrometer equipped with electrospray ionization in the positive mode. The protonated precursor to product ion transitions monitored for ASE and ASE 13C-d3 were m/z 286.1 → 166.0 and m/z 290.0 → 166.1, respectively. The limit of detection (LOD) and limit of quantitation (LOQ) of the method were 0.0025 ng/mL and 0.050 ng/mL respectively in a linear concentration range of 0.050-20.0 ng/mL for ASE. The intra-batch and inter-batch precision (% CV) and mean relative recovery across quality control levels were ≤ 5.8% and 87.3%, respectively. Matrix effect, evaluated as IS-normalized matrix factor, ranged from 1.03 to 1.05. The stability of ASE under different storage conditions was ascertained in presence of the metabolites. The developed method is much simpler, matrix free, rapid and economical compared to the existing methods. The method was successfully used for a bioequivalence study of asenapine in healthy Indian subjects for the first time.

Highly sensitive LC-MS/MS method to estimate doxepin and its metabolite nordoxepin in human plasma for a bioequivalence study.

A selective, sensitive and rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been developed for the simultaneous determination of doxepin (Dox) and its pharmacologically active metabolite, nordoxepin (NDox) in human plasma. The analytes and their internal standards (IS) were extracted from 500 µL of human plasma by liquid-liquid extraction using methyl tert-butyl ether. Chromatographic separation was achieved on Hypurity C8 column (100 mm × 4.6 mm, 5 µm) using a mixture of acetonitrile-methanol (95:5, v/v) and 2.0 mM ammonium formate in 93:7 (v/v) ratio. Detection was accomplished by tandem mass spectrometry in the positive ionization and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions studied for Dox, NDox, and their corresponding ISs, propranolol and desipramine, were m/z 280.1→107.0, 266.0 →107.0, 260.1→116.1 and 267.1→72.1, respectively. A linear dynamic range of 15.0-3900 pg/mL for Dox and 5.00-1300 pg/mL for NDox was established with mean correlation coefficient (r 2) of 0.9991 and 0.9993, respectively. The extraction recovery ranged from 86.6%-90.4% and 88.0%-99.1% for Dox and NDox, respectively. The intra-batch and inter-batch precision (% CV) across quality control levels was ≤ 8.3% for both the analytes. Stability evaluated under different storage conditions showed no evidence of degradation and the % change in stability samples compared to nominal concentration ranged from 4.7% to 12.3%. The method was successfully applied to a bioequivalence study of 6 mg doxepin hydrochloride orally disintegrating tablet in 41 healthy Indian subjects under fasting and fed conditions.

Wireless teleradiology for renal colic and renal trauma.

BACKGROUND PURPOSE: Image transmission is an integral part of telemedicine, allowing evaluation of patients at remote sites. We developed a simple method of wireless transmission of digital images to a hand-held computer (PDA) and evaluated its feasibility and diagnostic accuracy in patients with acute renal colic or renal trauma. MATERIALS AND METHODS: The CT images from 11 patients with suspected renal colic and one patient with renal trauma were transmitted using a cellular telephone with a wireless modem link to a PDA (Sony Clie 615C). A diagnostic interpretation was recorded for the presence/absence of an upper-tract stone, stone location, estimated stone size, and signs of upper-tract obstruction. Radiologic staging of trauma was provided in the patient with a renal injury. Comparison was made with the final dictated report of a staff radiologist. RESULTS: Ten CT sets of patients with renal colic were used for comparison, the remaining one being excluded because of ambiguity in the final radiology report. An average of 5.9+/-1.6 images, average size 32.2+/-5.2 kb (range 21-42 kb) for each patient were sent at an average speed of 1 kb/sec. Interpretation correctly identified stone presence in 80%, hydronephrosis in 100%, and perinephric stranding in 80% and stone size within 1+/-1 mm. A stage-3 renal trauma was correctly identified and staged. CONCLUSION: Wireless teleradiology to PDA units provides image quality sufficient for diagnostic interpretation. Anticipated improvements in wireless transmission and PDA screen image resolution will enhance the speed, quality, and quantity of images transmitted. Wireless teleradiology may facilitate convenient rapid evaluation of patients at remote sites.

Robot-assisted laparoscopic radical prostatectomy after heart transplantation.

Prostate cancer (CaP) is the leading visceral malignancy in males. Patients who undergo cardiac transplantation are immune compromised, thus presenting a therapeutic challenge. Immunosuppresion could accelerate tumor growth, while medical intervention may be associated with increased treatment mortality or morbidity. Due to paucity of such cases, there are no randomized trials that address the treatment algorithm for cardiac transplant patients with CaP, with only a few scattered reports in the literature. Treatment options range from hormonal manipulation to radiation therapy to radical prostatectomy. To our knowledge, we report the first successful robot-assisted laparoscopic radical prostatectomy in a heart transplant patient with CaP.

Quantitation of nitrofurantoin in human plasma by liquid chromatography tandem mass spectrometry.

A reliable, selective and sensitive LC-MS/MS assay has been proposed for the determination of nitrofurantoin in human plasma. The analyte and nitrofurazone were extracted from 100 µL of human plasma via SPE on Strata-X 33 µm extraction cartridges. Chromatography was done on a BDS Hypersil C18 (100 mm × 4.6 mm, 5 µm) column under isocratic conditions. Quantitation was done using the multiple reaction monitoring (MRM) mode for deprotonated precursor to product ion transitions of nitrofurantoin (m/z 237.0 → 151.8) and nitrofurazone (m/z 197.0 → 123.9). The limit of detection and the lowest limit of quantitation of the method were 0.25 ng mL-1 and 5.00 ng mL-1, respectively, with a linear dynamic range of 5.00-1500 ng mL-1 for nitrofurantoin. The intra- -batch and inter-batch precision (RSD, %) was ≤ 5.8 %, while the mean extraction recovery was > 92 %. The method was successfully applied to a bioequivalence study of a 100 mg nitrofurantoin capsule formulation in 36 healthy subjects.

Sensitive and selective determination of diflunisal in human plasma by LC-MS.

A reliable and sensitive liquid chromatography-tandem mass spectrometry assay has been proposed for the selective determination of diflunisal in the presence of its glucuronide metabolites. The analyte and clofibric acid as internal standard (IS) are extracted from 50 µL of human plasma by solid-phase extraction. Chromatographic separation is conducted on a Prodigy ODS 3V column (150 × 4.6 mm, 5 µm) under isocratic conditions. The possible interference of acyl glucuronide and phenolic glucuronide, the two major inactive metabolites of diflunisal, is also checked in plasma samples. Detection of the analyte and IS is achieved by tandem mass spectrometry, operating in negative ionization and multiple reaction monitoring acquisition mode. The limits of detection and quantitation of the method are 0.10 and 1.00 µg/mL, respectively, with a linear dynamic range of 1.00-160 µg/mL for diflunisal. The intra-batch and inter-batch precision (percent coefficient of variation) is ≤4.2% and the mean recovery is >92% for diflunisal across quality control levels. The method is successfully applied to a bioequivalence study of a 500 mg diflunisal tablet formulation in 30 healthy Indian male subjects under fasting conditions. The reproducibility in the measurement of study data is demonstrated by the reanalysis of 120 incurred samples.

Iatrogenic obstruction after sling surgery.

Sling surgery has supplanted other anti-incontinence procedures as the treatment of choice for stress urinary incontinence. Iatrogenic obstruction after sling surgery is increasingly reported as the procedure becomes more and more popular. The rate of retention (defined as catheter-dependency for at least 28 days) has been estimated at 1-10%. Iatrogenic obstruction after sling surgery has a variable presentation and can include urinary urgency, urgency incontinence, hesitancy, straining to void, weak urinary stream, nocturia, incomplete emptying, frequency, dysuria or urinary tract infections. The evaluation and diagnosis rely upon a thorough patient history, physical examination, a urine flow test and postvoid residual volume. Cystoscopy and pressure-flow studies can also be considered. The single most important factor in the diagnosis of sling-related obstruction or voiding dysfunction is the temporal relationship between the sling procedure and onset of symptoms. Transient urinary retention can be managed with indwelling or intermittent catheter drainage. For those patients with moderate or symptomatic retention, surgical options for treatment include sling loosening, sling incision, sling excision, and urethrolysis.

Application of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for determination of bumetanide in human plasma for a bioequivalence study.

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been proposed for the determination of bumetanide in human plasma using tamsulosin as internal standard (IS). The analyte and IS were extracted from 200 µL of human plasma via solid phase extraction and the chromatographic separation was achieved on Peerless Basic C18 (100 mm × 4.6 mm, 3 µm) column under isocratic conditions. Detection of bumetanide and IS was done by tandem mass spectrometry, operating in positive ionization and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for bumetanide and IS were m/z 365.2→240.2 and 409.2→228.2 respectively. The method was fully validated as per the US FDA guidelines. The limit of detection and lower limit of quantitation of the method were 0.03 and 0.30 ng/mL respectively with a linear dynamic range of 0.30-200.0 ng/mL for bumetanide. The intra-batch and inter-batch precision (% CV) was ≤6.9% while the mean extraction recovery was >90% across quality control levels. The method is selective in presence of four diuretic drugs and some commonly used medications by healthy volunteers. It was successfully applied to a bioequivalence study of 2mg bumetanide tablet formulation in 10 healthy Indian male subjects under fasting condition. The reproducibility in the measurement of study data was demonstrated by reanalysis of 42 incurred samples.

Development and validation of a selective and sensitive LC-MS/MS method for determination of cycloserine in human plasma: application to bioequivalence study.

A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the determination of cycloserine in human plasma is developed using niacin as internal standard (IS). The analyte and IS were extracted from 500 µL of human plasma via solid phase extraction on Waters Oasis MCX cartridges. Chromatographic separation was achieved on a Peerless Basic C18 (100 mm × 4.6mm, 3 µm) column under isocratic conditions. Detection of analyte and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for cycloserine and niacin were at m/z 103.1 → 75.0 and 124.1 → 80.1 respectively. The method was fully validated for its selectivity, interference check, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The limit of detection (LOD) and lower limit of quantitation of the method were 0.0013 and 0.20 µg/mL respectively with a linear dynamic range of 0.20-30.00 µg/mL for cycloserine. The intra-batch and inter-batch precision (%CV) across six quality control levels was less than 8.0% for cycloserine. The method was successfully applied to a bioequivalence study of 250 mg cycloserine capsule formulation in 24 healthy Indian male subjects under fasting condition.