Prognostic factors of intravesical recurrence after radical nephroureterectomy for upper tract urothelial carcinoma.

PURPOSE: To evaluate predictive factors of increasing intravesical recurrence (IVR) rate in patients with upper tract urothelial carcinoma (UTUC) after receiving radical nephroureterectomy (RNUx) with bladder cuff excision (BCE). MATERIALS AND METHODS: A total of 2114 patients were included from the updated data of the Taiwan UTUC Collaboration Group. It was divided into two groups: IVR-free and IVR after RNUx, with 1527 and 587 patients, respectively. To determine the factors affecting IVR, TNM stage, the usage of pre-operative ureteroscopy, and pathological outcomes were evaluated. The Kaplan-Meier estimator was used to estimate the rates of prognostic outcomes in overall survival (OS), cancer-specific survival (CSS), disease-free survival (DFS), and bladder recurrence-free survival (BRFS), and the survival curves were compared using the stratified log-rank test. RESULTS: Based on our research, ureter tumor, female, smoking history, age (< 70 years old), multifocal tumor, history of bladder cancer were determined to increase the risk of IVR after univariate analysis. The multivariable analysis revealed that female (BRFS for male: HR 0.566, 95% CI 0.469-0.681, p < 0.001), ureter tumor (BRFS: HR 1.359, 95% CI 1.133-1.631, p = 0.001), multifocal (BRFS: HR 1.200, 95% CI 1.001-1.439, p = 0.049), history of bladder cancer (BRFS: HR 1.480, 95% CI 1.118-1.959, p = 0.006) were the prognostic factors for IVR. Patients who ever received ureterorenoscopy (URS) did not increase the risk of IVR. CONCLUSION: Patients with ureter tumor and previous bladder UC history are important factors to increase the risk of IVR after RNUx. Pre-operative URS manipulation is not associated with higher risk of IVR and diagnostic URS is feasible especially for insufficient information of image study. More frequent surveillance regimen may be needed for these patients.

Deep Learning Fuzzy Inference: An Interpretable Model for Detecting Indirect Immunofluorescence Patterns Associated with Nasopharyngeal Cancer.

The detection of serum Epstein-Barr virus antibodies by immunofluorescence assay (IFA) is considered the gold standard screening test for nasopharyngeal cancer (NPC) in high-risk populations. Given the high survival rate after early detection in asymptomatic patients, compared to the poor prognosis in patients with late-stage NPC, screening using IFA has tremendous potential for saving lives in the general population. However, IFA requires visual interpretation of cellular staining patterns by trained pathology staff, making it labor intensive and hence nonscalable. In this study, an automated fuzzy inference (FI) system achieved high agreement with a human IFA expert in identifying cellular patterns associated with NPC (κ = 0.82). The integration of a deep learning module into FI further improved the performance of FI (κ = 0.90) and reduced the number of uncertain cases that required manual evaluation. The performance of the resulting hybrid model, termed deep learning FI (DeLFI), was then evaluated with a separate testing set of clinical samples. In this clinical validation, DeLFI outperformed human evaluation on the area under the curve (0.926 versus 0.821) and closely matched human performance on Youden J index (0.81 versus 0.80). Data from this study indicate that the combination of deep learning with FI in DeLFI has the potential to improve the scalability and accuracy of NPC detection.

A Rational Approach to JAK2 Mutation Testing in Patients with Elevated Hemoglobin: Results from the JAK2 Prediction Cohort (JAKPOT) Study.

BACKGROUND: Erythrocytosis, most often measured as an increase in hemoglobin and/or hematocrit, is a common reason for referral to internal medicine and hematology clinics and a rational approach is required to effectively identify patients with polycythemia vera while avoiding over-investigation. AIM: We aimed to develop and validate a simple rule to predict JAK2 mutation positivity based on complete blood count parameters to aid in the diagnostic approach to patients referred for elevated hemoglobin. SETTING: Internal medicine and hematology clinics at an academic tertiary referral center. PARTICIPANTS: The JAK2 Prediction Cohort (JAKPOT), a large retrospective cohort (n = 901) of patients evaluated by internal medicine and hematology specialists for elevated hemoglobin. DESIGN: JAK2 mutation analysis was performed in all patients and clinical and laboratory variables were collected. Patients were randomly divided into derivation and validation cohorts. A prediction rule was developed using data from the derivation cohort and tested in the validation cohort. KEY RESULTS: The JAKPOT prediction rule included three variables: (i) red blood cell count >6.45×1012/L, (ii) platelets >350×109/L, and (iii) neutrophils >6.2×109/L; absence of all criteria was effective at ruling out JAK2-positivity with sensitivities 94.7% and 100%, and negative predictive values of 98.8% and 100% in the derivation and validation cohorts, respectively, with an overall low false negative rate of 0.4%. The rule was validated for three different methods of JAK2 testing. Applying this rule to our entire cohort would have resulted in over 50% fewer tests. CONCLUSION: In patients with elevated hemoglobin, the use of a simple prediction rule helps to accurately identify patients with a low likelihood of having a JAK2 mutation, potentially limiting costly over-investigation in this common referral population.

Cerebral Tau Deposition in Comorbid Progressive Supranuclear Palsy and Amyotrophic Lateral Sclerosis: An [18F]-Flortaucipir and 7T MRI Study.

INTRODUCTION: Progressive supranuclear palsy (PSP) is a four-repeat tauopathy characterized by multiple clinicopathologic subtypes. Advanced neuroimaging techniques have shown an early ability to distinguish PSP subtypes noninvasively for improved diagnosis. This study utilized tau PET imaging and MRI techniques at 7T to determine the neuroimaging profile of a participant with comorbid PSP and amyotrophic lateral sclerosis (ALS). METHOD: [18F]-flortaucipir PET imaging was performed on one participant with PSP-ALS, one participant with typical PSP (Richardson's syndrome; PSP-RS), and 15 healthy control volunteers. Standardized uptake value ratio (SUVR) in each brain region was compared between PSP participants and controls. Quantitative susceptibility mapping (QSM) and inflow-based vascular-space occupancy MRI at 7T were performed on the two PSP participants and on two age-matched healthy controls to evaluate for differences in regional brain iron content and arteriolar cerebral blood volume (CBVa), respectively. RESULTS: In the participant with PSP-ALS, the precentral gyrus demonstrated the highest [18F]-flortaucipir uptake of all brain regions relative to controls (z-score 1.94). In the participant with PSP-RS, [18F]-flortaucipir uptake relative to controls was highest in subcortical regions, including the pallidum, thalamus, hippocampus, and brainstem (z-scores 1.08, 1.41, 1.49, 1.32, respectively). Susceptibility values as a measure of brain iron content were higher in the globus pallidus and substantia nigra than in the midbrain and pons in each participant, regardless of group. CBVa values tended to be higher in the subcortical gray matter in PSP participants than in controls, although large measurement variability was noted in controls across multiple regions. CONCLUSION: In vivo tau PET imaging of an individual with PSP-ALS overlap demonstrated increased tau burden in the motor cortex that was not observed in PSP-RS or control participants. Consistent with prior PET studies, tau burden in PSP-RS was mainly observed in subcortical regions, including the brainstem and basal ganglia. QSM data suggest that off-target binding to iron may account for some but not all of the increased [18F]-flortaucipir uptake in the basal ganglia in PSP-RS. These findings support existing evidence that tau PET imaging can distinguish among PSP subtypes by detecting distinct regional patterns of tau deposition in the brain. Larger studies are needed to determine whether CBVa is sensitive to changes in brain microvasculature in PSP.

Impact of pathological response on oncological outcomes in patients with upper tract urothelial cancer receiving neo-adjuvant chemotherapy.

PURPOSE: The purpose of this study is to evaluate the rates of pathological complete response (ypT0N0/X) and pathological response (ypT1N0/X or less) in patients with upper tract urothelial cancer who were treated with neo-adjuvant chemotherapy and to examine their impact on oncological outcomes. METHODS: This study is a multi-institutional retrospective analysis of patients with high-risk upper tract urothelial cancer who underwent neoadjuvant chemotherapy and radical nephroureterectomy between 2002 and 2021. Logistic regression analyses were used to investigate all clinical parameters for response after neoadjuvant chemotherapy. Cox proportional hazard models were performed to assess the effect of the response on the oncological outcomes. RESULTS: A total of 84 patients with UTUC who received neo-adjuvant chemotherapy were identified. Among them, 44 (52.4%) patients received cisplatin-based chemotherapy, and 22 (26.2%) patients had a carboplatin-based regimen. The pathological complete response rate was 11.6% (n = 10), and the pathological response rate was 42.9% (n = 36). Multifocal tumors or tumors larger than 3 cm significantly reduced the odds of pathological response. In the multivariable Cox proportional hazard model, pathological response was independently associated with better overall survival (HR 0.38, p = 0.024), cancer-specific survival (HR 0.24, p = 0.033), and recurrence-free survival (HR 0.17, p = 0.001), but it was not associated with bladder recurrence-free survival (HR 0.84, p = 0.69). CONCLUSION: Pathological response after neo-adjuvant chemotherapy and radical nephroureterectomy is strongly associated with patient survival and recurrence, and it might be a good surrogate for evaluating the efficacy of neo-adjuvant chemotherapy in the future.

A colorimetric chemical tongue detects and distinguishes between multiple analytes.

The rate-limiting step for diagnostics development is the discovery and validation of biomarker analytes. We describe a new analyte-agnostic and label-free approach based on colorimetric reactions involving type I polymerization photoinitiators. We demonstrate that a chemically diverse array of hydrogels embedded with cleaved type I photoinitiators could act as microreactors, undergoing colorimetric reactions with bound analytes. The colorimetric signatures produced were visually distinctive and readable with a flatbed document scanner. Signatures of a broad range of sample types were accurately differentiated by unsupervised clustering without knowledge of any analytes bound to the array. The principles described have the potential to enable scalable and cost-effective analysis of complex samples.

Neurochemical correlates of functional decline in amyotrophic lateral sclerosis.

OBJECTIVE: To determine whether proton magnetic resonance spectroscopy (1H-MRS) can detect neurochemical changes in amyotrophic lateral sclerosis (ALS) associated with heterogeneous functional decline. METHODS: Nineteen participants with early-stage ALS and 18 age-matched and sex ratio-matched controls underwent ultra-high field 1H-MRS scans of the upper limb motor cortex and pons, ALS Functional Rating Scale-Revised (ALSFRS-R total, upper limb and bulbar) and upper motor neuron burden assessments in a longitudinal observational study design with follow-up assessments at 6 and 12 months. Slopes of neurochemical levels over time were compared between patient subgroups classified by the rate of upper limb or bulbar functional decline. 1H-MRS and clinical ratings at baseline were assessed for ability to predict study withdrawal due to disease progression. RESULTS: Motor cortex total N-acetylaspartate to myo-inositol ratio (tNAA:mIns) significantly declined in patients who worsened in upper limb function over the follow-up period (n=9, p=0.002). Pons glutamate + glutamine significantly increased in patients who worsened in bulbar function (n=6, p<0.0001). Neurochemical levels did not change in patients with stable function (n=5-6) or in healthy controls (n=14-16) over time. Motor cortex tNAA:mIns and ALSFRS-R at baseline were significantly lower in patients who withdrew from follow-up due to disease progression (n=6) compared with patients who completed the 12-month scan (n=10) (p<0.001 for tNAA:mIns; p<0.01 for ALSFRS-R), with a substantially larger overlap in ALSFRS-R between groups. CONCLUSION: Neurochemical changes in motor areas of the brain are associated with functional decline in corresponding body regions. 1H-MRS was a better predictor of study withdrawal due to ALS progression than ALSFRS-R.

Ultra-High Field Proton MR Spectroscopy in Early-Stage Amyotrophic Lateral Sclerosis.

A major hurdle in the development of effective treatments for amyotrophic lateral sclerosis (ALS) has been the lack of robust biomarkers for use as clinical trial endpoints. Neurochemical profiles obtained in vivo by high field proton magnetic resonance spectroscopy (1H-MRS) can potentially provide biomarkers of cerebral pathology in ALS. However, previous 1H-MRS studies in ALS have produced conflicting findings regarding alterations in the levels of neurochemical markers such as glutamate (Glu) and myo-inositol (mIns). Furthermore, very few studies have investigated the neurochemical abnormalities associated with ALS early in its course. In this study, we measured neurochemical profiles using single-voxel 1H-MRS at 7 T (T) and glutathione (GSH) levels using edited MRS at 3 T in 19 subjects with ALS who had relatively high functional status [ALS Functional Rating Scale-Revised (ALSFRS-R) mean ± SD = 39.8 ± 5.6] and 17 healthy controls. We observed significantly lower total N-acetylaspartate over mIns (tNAA/mIns) ratio in the motor cortex and pons of subjects with ALS versus healthy controls. No group differences were detected in GSH at 3 and 7 T. In subjects with ALS, the levels of tNAA, mIns, and Glu in the motor cortex were dependent on the extent of disease represented by El Escorial diagnostic subcategories. Specifically, combined probable/definite ALS had lower tNAA than possible ALS and controls (both p = 0.03), higher mIns than controls (p < 0.01), and lower Glu than possible ALS (p < 0.01). The effect of disease stage on MRS-measured metabolite levels may account for dissimilar findings among previous 1H-MRS studies in ALS.

Test-retest reproducibility of neurochemical profiles with short-echo, single-voxel MR spectroscopy at 3T and 7T.

PURPOSE: To determine the test-retest reproducibility of neurochemical concentrations obtained with a highly optimized, short-echo, single-voxel proton MR spectroscopy (MRS) pulse sequence at 3T and 7T using state-of-the-art hardware. METHODS: A semi-LASER sequence (echo time = 26-28 ms) was used to acquire spectra from the posterior cingulate and cerebellum at 3T and 7T from six healthy volunteers who were scanned four times weekly on both scanners. Spectra were quantified with LCModel. RESULTS: More neurochemicals were quantified with mean Cramér-Rao lower bounds (CRLBs) ≤20% at 7T than at 3T despite comparable frequency-domain signal-to-noise ratio. Whereas CRLBs were lower at 7T (P < 0.05), between-session coefficients of variance (CVs) were comparable at the two fields with 64 transients. Five metabolites were quantified with between-session CVs ≤5% at both fields. Analysis of subspectra showed that a minimum achievable CV was reached with a lower number of transients at 7T for multiple metabolites and that between-session CVs were lower at 7T than at 3T with fewer than 64 transients. CONCLUSION: State-of-the-art MRS methodology allows excellent reproducibility for many metabolites with 5-min data averaging on clinical 3T hardware. Sensitivity and resolution advantages at 7T are important for weakly represented metabolites, short acquisitions, and small volumes of interest. Magn Reson Med 76:1083-1091, 2016. © 2015 Wiley Periodicals, Inc.

Elucidating Bacterial Spore Dynamics through Lanthanide-Enhanced Live Imaging.

Identifying and distinguishing dormant and active bacterial spores are vital for biosecurity, food safety, and space exploration. Yet, there is a lack of simple, quick, and nondestructive methods to achieve this. The common Schaeffer-Fulton method is both sample-destructive and requires significant operator involvement. In this study, we employed lanthanide-beta-diketonate complexes to directly observe both dormant and germinated single spores. Staining is instantaneous and requires minimal sample processing. The complex stains areas outside the core of dormant spores, leaving the core hollow and nonfluorescent. However, upon germination, the complex enters the core, making it brightly fluorescent. This difference was noted in five bacterial species including Bacillus, Clostridium, and Clostridioides. Various lanthanides and beta-diketonates can be mixed to form a range of spore-visualizing complexes. Due to their low toxicity, these complexes allow for live imaging of single germinating spores. We demonstrate low-cost imaging using a USB microscope as well as imaging of spores in milk matrices. This method provides a valuable tool for studying bacterial spores.

Risk of secondary primary malignancies in survivors of upper tract urothelial carcinoma: A nationwide population-based analysis.

BACKGROUND: To investigate the cancer types and risk factors of secondary primary malignancy (SPM) in patients with upper tract urothelial carcinoma (UTUC) in Taiwan. METHODS: Using National Health Insurance Research Dataset and catastrophic illness registry, we enrolled newly diagnosed UTUC patients from 2000 to 2013. Those without catastrophic illness registration were excluded from the study. The cancer types and hazard ratios (HRs) of subsequent SPMs were calculated according to the antecedent malignancy. We analyzed the risk factors for developing SPMs using multivariate Cox proportional hazard models. RESULTS: A total of 9050 UTUC patients were registered and 2187 (24.2%) patients developed SPMs during the study period. As compared with primary UTUC, the relative risk ratios of SPM was 2.5 folds and 18% higher in those with antecedent non-UC malignancy and with bladder cancer history, respectively. Totally, 387 (37.8%) of 1022 UTUC patients with antecedent non-UC malignancy developed subsequent SPM after UTUC diagnosis. The antecedent and subsequent cancer types are similar and kidney cancer is most common, followed by hepatoma. Multivariate analysis showed that a history of antecedent non-UC malignancy is the most unfavorable factor for SPM development (HR, 2.50; 95% CI, 2.23-2.81), followed by liver disease, male gender, antecedent bladder cancer history, age ≥ 75 years, and chronic kidney disease. CONCLUSIONS: Our study, conducted in Taiwan and involving 9050 UTUC patients, meticulously examined the types of SPM and the associated risk factors. Our research unearthed several pivotal discoveries: a preceding history of non-UC malignancies emerged as the single most influential factor contributing to the occurrence of subsequent cancers, followed by liver disease, male gender, antecedent bladder cancer history, age ≥75 years, and chronic kidney disease. Futhermore, kidney cancer emerged as the predominant subsequent malignancy, closely trailed by hepatoma..

Clinical performance of a prefabricated immunofluorescence assay for nasopharyngeal cancer screening.

BACKGROUND: Epstein-Barr virus (EBV) IgA serology for viral capsid antigen (VCA) and early antigen (EA) aids early detection of nasopharyngeal cancer (NPC), resulting in improved survival. We evaluated the diagnostic performance of a prefabricated immunofluorescent assay (IFA) for NPC screening in high-risk individuals. METHODS: Sera from 96 biopsy-proven patients with NPC diagnosed at the outpatient clinic and 96 healthy family members were tested for EBV-VCA IgA and EBV-EA IgA using the prefabricated IFA from EUROIMMUN (EI) and the traditional immunofluorescence method. RESULTS: The AUC of EI EBV-VCA IgA and EBV-EA IgA was 0.907 (95% confidence interval [CI]: 0.894-0.965) and 0.898 (95% CI: 0.848-0.947), respectively. Combined testing with the prefabricated assay at a threshold of VCA ≥1:320 or EA ≥1:10 showed 92.7% sensitivity and 81.2% specificity. Overall, the traditional EBV-EA IgA assay demonstrated the best accuracy (sensitivity 91.7% and specificity 96.9%) at a threshold of ≥1:5. CONCLUSION: While the traditional IFA method was more accurate, the prefabricated IFA test kit can be a useful tool for NPC screening in high-risk populations.

Mutant PIK3CA promotes cell growth and invasion of human cancer cells.

PIK3CA is mutated in diverse human cancers, but the functional effects of these mutations have not been defined. To evaluate the consequences of PIK3CA alterations, the two most common mutations were inactivated by gene targeting in colorectal cancer (CRC) cells. Biochemical analyses of these cells showed that mutant PIK3CA selectively regulated the phosphorylation of AKT and the forkhead transcription factors FKHR and FKHRL1. PIK3CA mutations had little effect on growth under standard conditions, but reduced cellular dependence on growth factors. PIK3CA mutations resulted in attenuation of apoptosis and facilitated tumor invasion. Treatment with the PI3K inhibitor LY294002 abrogated PIK3CA signaling and preferentially inhibited growth of PIK3CA mutant cells. These data have important implications for therapy of cancers harboring PIK3CA alterations.

Genetic inactivation of AKT1, AKT2, and PDPK1 in human colorectal cancer cells clarifies their roles in tumor growth regulation.

Phosphotidylinositol-3-kinase (PI3K) signaling is altered in the majority of human cancers. To gain insight into the roles of members of this pathway in growth regulation, we inactivated AKT1, AKT2, or PDPK1 genes by targeted homologous recombination in human colon cancer cell lines. Knockout of either AKT1 or AKT2 had minimum effects on cell growth or downstream signaling. In contrast, knockout of both AKT1 and AKT2 resulted in markedly reduced proliferation in vitro when growth factors were limiting and severely affected experimental metastasis in mice. Unexpectedly, AKT1 and AKT2 appeared to regulate growth through FOXO proteins, but not through either GSK3beta or mTOR. In contrast, inactivation of PDPK1 affected GSK3beta and mTOR activation. These findings show that the PI3K signaling pathway is wired differently in human cancer cells than in other cell types or organisms, which has important implications for the design and testing of drugs that target this pathway.

A design of experiments screen reveals that Clostridium novyi-NT spore germinant sensing is stereoflexible for valine and its analogs.

Although Clostridium novyi-NT is an anti-cancer bacterial therapeutic which germinates within hypoxic tumors to kill cancer cells, the actual germination triggers for C. novyi-NT are still unknown. In this study, we screen candidate germinants using combinatorial experimental designs and discover by serendipity that D-valine is a potent germinant, inducing 50% spore germination at 4.2 mM concentration. Further investigation revealed that five D-valine analogs are also germinants and four of these analogs are enantiomeric pairs. This stereoflexible effect of L- and D-amino acids shows that spore germination is a complex process where enantiomeric interactions can be confounders. This study also identifies L-cysteine as a germinant, and hypoxanthine and inosine as co-germinants. Several other amino acids promote (L-valine, L-histidine, L-threonine and L-alanine) or inhibit (L-arginine, L-glycine, L-lysine, L-tryptophan) germination in an interaction-dependent manner. D-alanine inhibits all germination, even in complex growth media. This work lays the foundation for improving the germination efficacy of C. novyi-NT spores in tumors.

Preoperative hydronephrosis is an independent protective factor of renal function decline after nephroureterectomy for upper tract urothelial carcinoma.

Objectives: To evaluate the predictive role of pre-nephroureterectomy (NU) hydronephrosis on post-NU renal function (RF) change and preserved eligibility rate for adjuvant therapy in patients with upper tract urothelial carcinoma (UTUC). Patients and methods: This retrospective study collected data of 1018 patients from the Taiwan UTUC Collaboration Group registry of 26 institutions. The patients were divided into two groups based on the absence or presence of pre-NU hydronephrosis. Estimated glomerular filtration rate (eGFR) was calculated pre- and post-NU respectively. The one month post-NU RF change, chronic kidney disease (CKD) progression, and the preserved eligibility rate for adjuvant therapy were compared for each CKD stage. Results: 404 (39.2%) patients without and 614 (60.8%) patients with pre-NU hydronephrosis were enrolled. The median post-NU change in the eGFR was significantly lower in the hydronephrosis group (-3.84 versus -12.88, p<0.001). Pre-NU hydronephrosis was associated with a lower post-NU CKD progression rate (33.1% versus 50.7%, p< 0.001) and was an independent protective factor for RF decline after covariate adjustment (OR=0.46, p<0.001). Patients with pre-NU hydronephrosis had a higher preserved eligibility rate for either adjuvant cisplatin-based chemotherapy (OR=3.09, 95%CI 1.95-4.69) or immune-oncology therapy (OR=2.31, 95%CI 1.23-4.34). Conclusion: Pre-NU hydronephrosis is an independent protective predictor for post-NU RF decline, CKD progression, and eligibility for adjuvant therapy. With cautious selection for those unfavorably prognostic, non-metastatic UTUC patients with preoperative hydronephrosis, adjuvant rather than neoadjuvant therapy could be considered due to higher chance of preserving eligibility.

A frequent kinase domain mutation that changes the interaction between PI3Kalpha and the membrane.

Mutations in oncogenes often promote tumorigenesis by changing the conformation of the encoded proteins, thereby altering enzymatic activity. The PIK3CA oncogene, which encodes p110alpha, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kalpha), is one of the two most frequently mutated oncogenes in human cancers. We report the structure of the most common mutant of p110alpha in complex with two interacting domains of its regulatory partner (p85alpha), both free and bound to an inhibitor (wortmannin). The N-terminal SH2 (nSH2) domain of p85alpha is shown to form a scaffold for the entire enzyme complex, strategically positioned to communicate extrinsic signals from phosphopeptides to three distinct regions of p110alpha. Moreover, we found that Arg-1047 points toward the cell membrane, perpendicular to the orientation of His-1047 in the WT enzyme. Surprisingly, two loops of the kinase domain that contact the cell membrane shift conformation in the oncogenic mutant. Biochemical assays revealed that the enzymatic activity of the p110alpha His1047Arg mutant is differentially regulated by lipid membrane composition. These structural and biochemical data suggest a previously undescribed mechanism for mutational activation of a kinase that involves perturbation of its interaction with the cellular membrane.