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Pre-European tallgrass prairie covered approximately 260 million acres in the United States. Only 1-3% of remnant prairies remains and is threatened by loss of natural disturbances and introduction, intentional or accidental, of non-native invasive species. Introduced grasses often create unfavorable conditions for native species to thrive and reproduce. Cool-season non-native species deplete nutrient resources, crowd out native root systems, and reduce photosynthetically active radiation. This study examined the use of spring burns, glyphosate, and nitrogen (N) applications at various growth stages to stimulate native species [big bluestem (Andropogon gerardii) and sideoats grama (Bouteloua curtipendula)] growth and decrease competition with non-native species [smooth brome (Bromus inermis) and Kentucky bluegrass (Poa pratensis)] at two sites (Volga and Artesian) in eastern South Dakota. Treatments were applied in Oct 2009 (glyphosate and/or N applied) and spring 2010 (fire, glyphosate, and/or N applied). Biomass was collected at the peak of cool-season non-native (June, 2010) and warm-season native (August 2010) grass growth to evaluate results. Prescribed burn treatment increased native warm-season biomass a minimum of 60% compared with untreated control areas. Site and N timing influenced herbicide effectiveness. At Artesian, warm-season biomass doubled compared with the untreated control or when April N application was followed by a May herbicide application. May herbicide application followed by June N application had increased warm-season biomass by 30% at Volga. Compared with the untreated control, warm-season grass biomass increased 41% when N was applied in June at Artesian and increased 77% with when N was applied in both October and April at Volga. Prior management (haying and winter grazing vs lack of grazing) of sites may have influenced species response. [ABSTRACT FROM AUTHOR]

Silicone tubing is a frequently used material in pharmaceutical filling processes for parenteral formulations, as its characteristics like flexibility, chemical resistance and easy handling make it particularly suitable for these purposes. This study investigated the time-dependent interaction of phenol and m-cresol with silicone tubing and other broadly applied contact materials used during the filling and transport processes of parenteral formulations. Phenol losses could be observed after incubation in silicone tubing, depending on the inner diameter (ID). This has been demonstrated for process interruptions of up to 120 min. A loss of 40 % could be observed for a small ID of 3.2 mm which can be found close to filling needles, and up to 12 % for larger tubes with an ID of 9.5 mm commonly used for sterile filtration and transport processes. Analysis of tubes with varying ID revealed a linear relationship between the decrease of phenol and the surface-to-volume ratio. m-cresol showed an even more pronounced loss in silicone tubing. Fluorinated polymers and thermoplastic elastomers were also analyzed, and no loss of phenol and m-cresol was observed. Pumping tests revealed that shear forces in peristaltic pumps led to strong particle formation in selected tubing. A strong increase in particle concentration was observed in thermoplastic elastomers, particularly in PharMed® BPT tubing. In contrast, the C-Flex® tubing demonstrated minimal particle formation. Fluorinated polymers are not compatible with peristaltic pumps, which is why they were not analyzed regarding pumpability. Although silicone tubes are not impervious to preservatives such as phenol, they did not generate particles when pumped., Competing Interests: Declaration of competing interest The authors have declared no conflicts of interest. All authors are employees of Boehringer Ingelheim Pharma GmbH & Co. KG, which manufactures pharmaceutics., (Copyright © 2024. Published by Elsevier B.V.)

Background: Perianal fistulas and abscesses occur commonly as complications of pediatric Crohn's disease (CD). A validated imaging assessment tool for quantification of perianal disease severity and activity is needed to evaluate treatment response. We aimed to identify magnetic resonance imaging (MRI)-based measures of perianal fistulizing disease activity and study design features appropriate for pediatric patients., Methods: Seventy-nine statements relevant to MRI-based assessment of pediatric perianal fistulizing CD activity and clinical trial design were generated from literature review and expert opinion. Statement appropriateness was rated by a panel (N = 15) of gastroenterologists, radiologists, and surgeons using modified RAND/University of California Los Angeles appropriateness methodology., Results: The modified Van Assche Index (mVAI) and the Magnetic Resonance Novel Index for Fistula Imaging in CD (MAGNIFI-CD) were considered appropriate instruments for use in pediatric perianal fistulizing disease clinical trials. Although there was concern regarding the use of intravascular contrast material in pediatric patients, its use in clinical trials was considered appropriate. A clinically evident fistula tract and radiologic disease defined as at least 1 fistula or abscess on pelvic MRI were considered appropriate trial inclusion criteria. A coprimary clinical and radiologic end point and inclusion of a patient-reported outcome were also considered appropriate., Conclusion: Outcomes of treatment of perianal fistulizing disease in children must include MRI. Existing multi-item measures, specifically the mVAI and MAGNIFI-CD, can be adapted and used for children. Further research to assess the operating properties of the indices when used in a pediatric patient population is ongoing., (© The Author(s) 2023. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The advent of Personalized Medicine (PM) holds significant promise in revolutionizing healthcare by tailoring treatments to individual patients based on their data. However, its successful implementation requires the seamless integration of innovative technologies and presents formidable challenges in terms of sustainability. To tackle these challenges head-on, the International Consortium for Personalized Medicine (ICPerMed) was established, and the IC2PerMed project, as part of this consortium, seeks to foster collaboration between the European Union (EU) and China in the field of Personalized Medicine. Based on the results collected by the project, the objective of this study is to discern the key priorities for the implementation of Personalised Medicine concerning Information and Communication Technologies (ICT) and Big Data and digital solutions, with a particular emphasis on data management and protection., Methods: A Delphi survey was conducted to gather expert's consensus on the main priorities for actions on Information and Communication Technologies (ICT) and Big Data and digital solutions in the field of Personalized Medicine., Results: The survey identified seven priorities in the area of Big Data and digital solutions, including data interoperability, standards, security measures, and international partnerships. Additionally, twelve priorities were identified for the innovation-to-market process, emphasizing cost-effectiveness, need assessment, and value definition in resource allocation., Conclusions: The effective implementation of new technologies in Personalized Medicine research and practice is essential for the advancement of healthcare systems in both the European and Chinese contexts. The identified priorities play a pivotal role in promoting the sustainability of health systems and driving innovation in the implementation of Personalized Medicine. Addressing challenges related to data interoperability, standards, security, international collaboration, cost-effectiveness, and value assessment is of utmost importance in order to propel the progress of Personalized Medicine in healthcare systems., (© 2024. The Author(s).)

Members of the family Prevotellaceae are Gram-negative, obligate anaerobic bacteria found in animal and human microbiota. In Prevotella bryantii, the Na + -translocating NADH:quinone oxidoreductase (NQR) and quinol:fumarate reductase (QFR) interact using menaquinone as electron carrier, catalyzing NADH:fumarate oxidoreduction. P. bryantii NQR establishes a sodium-motive force, whereas P. bryantii QFR does not contribute to membrane energization. To elucidate the possible mode of function, we present 3D structural models of NQR and QFR from P. bryantii to predict cofactor-binding sites, electron transfer routes and interaction with substrates. Molecular docking reveals the proposed mode of menaquinone binding to the quinone site of subunit NqrB of P. bryantii NQR. A comparison of the 3D model of P. bryantii QFR with experimentally determined structures suggests alternative pathways for transmembrane proton transport in this type of QFR. Our findings are relevant for NADH-dependent succinate formation in anaerobic bacteria which operate both NQR and QFR., (© 2023. The Author(s).)

The time-resolved impact of monensin on the active rumen microbiome was studied in a rumen-simulating technique (Rusitec) with metaproteomic and metabolomic approaches. Monensin treatment caused a decreased fibre degradation potential that was observed by the reduced abundance of proteins assigned to fibrolytic bacteria and glycoside hydrolases, sugar transporters and carbohydrate metabolism. Decreased proteolytic activities resulted in reduced amounts of ammonium as well as branched-chain fatty acids. The family Prevotellaceae exhibited increased resilience in the presence of monensin, with a switch of the metabolism from acetate to succinate production. Prevotella species harbour a membrane-bound electron transfer complex, which drives the reduction of fumarate to succinate, which is the substrate for propionate production in the rumen habitat. Besides the increased succinate production, a concomitant depletion of methane concentration was observed upon monensin exposure. Our study demonstrates that Prevotella sp. shifts its metabolism successfully in response to monensin exposure and Prevotellaceae represents the key bacterial family stabilizing the rumen microbiota during exposure to monensin., (© 2022 The Authors. Proteomics published by Wiley-VCH GmbH.)

To achieve minimum DNA input requirements for next-generation sequencing (NGS), pathologists visually estimate macrodissection and slide count decisions. Unfortunately, misestimation may cause tissue waste and increased laboratory costs. We developed an artificial intelligence (AI)-augmented smart pathology review system (SmartPath) to empower pathologists with quantitative metrics for accurately determining tissue extraction parameters. SmartPath uses two deep learning architectures, a U-Net based network for cell segmentation and a multi-field-of-view convolutional network for tumor area segmentation, to extract features from digitized H&E-stained formalin-fixed paraffin-embedded slides. From the segmented tumor area, SmartPath suggests a macrodissection area. To predict DNA yield per slide, the extracted features from within the macrodissection area are correlated with known DNA yields to fit a regularized linear model (R = 0.85). Then, a pathologist-defined target yield divided by the predicted DNA yield per slide gives the number of slides to scrape. Following model development, an internal validation trial was conducted within the Tempus Labs molecular sequencing laboratory. We evaluated our system on 501 clinical colorectal cancer slides, where half received SmartPath-augmented review and half traditional pathologist review. The SmartPath cohort had 25% more DNA yields within a desired target range of 100-2000 ng. The number of extraction attempts was statistically unchanged between cohorts. The SmartPath system recommended fewer slides to scrape for large tissue sections, saving tissue in these cases. Conversely, SmartPath recommended more slides to scrape for samples with scant tissue sections, especially those with degraded DNA, helping prevent costly re-extraction due to insufficient extraction yield. A statistical analysis was performed to measure the impact of covariates on the results, offering insights on how to improve future applications of SmartPath. With these improvements, AI-augmented histopathologic review has the potential to decrease tissue waste, sequencing time, and laboratory costs by optimizing DNA yields, especially for samples with scant tissue and/or degraded DNA., (© 2022. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Objectives: Fatigue is a frequent and often disabling symptom in patients with post-COVID syndrome. To better understand and evaluate the symptom of motor fatigue in the context of the post-COVID syndrome, we conducted treadmill walking tests to detect the phenomenon of motor fatigability or to evaluate whether evidence of organic lesions of the motor system could be found, similar to patients with multiple sclerosis., Method: Twenty-nine non-hospitalized patients with post-COVID syndrome completed the Fatigue Scale for Motor and Cognitive Function (FSMC) questionnaire to determine the trait component of subjective fatigue before they were tested on a treadmill walking at a moderate speed for up to 60 min or until exhaustion. During the walking test oxygen uptake, ventilation and acceleration data of both feet were collected. To determine motor performance fatigability, the Fatigue Index Kliniken Schmieder (FKS) was calculated using the attractor method., Results: The average walking duration was 42.7 ± 18.6 min with 15 subjects stopping the walking test prematurely. The FSMC score revealed a severe cognitive (37.6 ± 8.2) and motor (37.1 ± 7.8) fatigue averaged over all subjects but only two subjects showed an FKS above the normal range (>4), representing performance fatigability. There was no significant correlation between subjective fatigue (FSMC) and FKS as well as walking time. Absolute values of oxygen uptake and ventilation were in the normal range reported in literature ( r = 0.9, p < 0.05), although eight subjects did not produce a steady-state behavior., Conclusion: Almost all patients with post-COVID syndrome and subjectively severe motor fatigue, did not show motor fatigability nor severe metabolic anomalies. This is argued against organic, permanent damage to the motor system, as is often seen in MS. Many of the patients were - to our and their own surprise - motorically more exertable than expected., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Weich, Dettmers, Saile, Schleicher, Vieten and Joebges.)

Replacement of the Lactobacillus dominated vaginal microbiome by a mixed bacterial population including Prevotella bivia is associated with bacterial vaginosis (BV). To understand the impact of P. bivia on this microbiome, its growth requirements and mode of energy production were studied. Anoxic growth with glucose depended on CO 2 and resulted in succinate formation, indicating phosphoenolpyruvate carboxylation and fumarate reduction as critical steps. The reductive branch of fermentation relied on two highly active, membrane-bound enzymes, namely the quinol:fumarate reductase (QFR) and Na + -translocating NADH:quinone oxidoreductase (NQR). Both enzymes were characterized by activity measurements, in-gel fluorography, and VIS difference spectroscopy, and the Na + -dependent build-up of a transmembrane voltage was demonstrated. NQR is a potential drug target for BV treatment since it is neither found in humans nor in Lactobacillus . In P. bivia , the highly active enzymes L-asparaginase and aspartate ammonia lyase catalyze the conversion of asparagine to the electron acceptor fumarate. However, the by-product ammonium is highly toxic. It has been proposed that P. bivia depends on ammonium-utilizing Gardnerella vaginalis , another typical pathogen associated with BV, and provides key nutrients to it. The product pattern of P. bivia growing on glucose in the presence of mixed amino acids substantiates this notion.

Ruminants such as cattle and sheep depend on the breakdown of carbohydrates from plant-based feedstuff, which is accomplished by the microbial community in the rumen. Roughly 40% of the members of the rumen microbiota belong to the family Prevotellaceae , which ferments sugars to organic acids such as acetate, propionate, and succinate. These substrates are important nutrients for the ruminant. In a metaproteome analysis of the rumen of cattle, proteins that are homologous to the Na + -translocating NADH:quinone oxidoreductase (NQR) and the quinone:fumarate reductase (QFR) were identified in different Prevotella species. Here, we show that fumarate reduction to succinate in anaerobically growing Prevotella bryantii is coupled to chemiosmotic energy conservation by a supercomplex composed of NQR and QFR. This s odium-translocating N ADH: f umarate oxido r eductase (SNFR) supercomplex was enriched by blue native PAGE (BN-PAGE) and characterized by in-gel enzyme activity staining and mass spectrometry. High NADH oxidation (850 nmol min -1  mg -1 ), quinone reduction (490 nmol min -1  mg -1 ), and fumarate reduction (1,200 nmol min -1  mg -1 ) activities, together with high expression levels, demonstrate that SNFR represents a charge-separating unit in P. bryantii . Absorption spectroscopy of SNFR exposed to different substrates revealed intramolecular electron transfer from the flavin adenine dinucleotide (FAD) cofactor in NQR to heme b cofactors in QFR. SNFR catalyzed the stoichiometric conversion of NADH and fumarate to NAD + and succinate. We propose that the regeneration of NAD + in P. bryantii is intimately linked to the buildup of an electrochemical gradient which powers ATP synthesis by electron transport phosphorylation. IMPORTANCE Feeding strategies for ruminants are designed to optimize nutrient efficiency for animals and to prevent energy losses like enhanced methane production. Key to this are the fermentative reactions of the rumen microbiota, dominated by Prevotella spp. We show that succinate formation by P. bryantii is coupled to NADH oxidation and sodium gradient formation by a newly described supercomplex consisting of Na + -translocating NADH:quinone oxidoreductase (NQR) and fumarate reductase (QFR), representing the s odium-translocating N ADH: f umarate oxido r eductase (SNFR) supercomplex. SNFR is the major charge-separating module, generating an electrochemical sodium gradient in P. bryantii . Our findings offer clues to the observation that use of fumarate as feed additive does not significantly increase succinate production, or decrease methanogenesis, by the microbial community in the rumen.

Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B 1 4 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P. bryantii , as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P. bryantii B 1 4 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na + -translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P. bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.

Short-chain fatty acids (SCFAs) are bacterial products that are known to be used as energy sources in eukaryotic hosts, whereas their role in the metabolism of intestinal microbes is rarely explored. In the present study, acetic, propionic, butyric, isobutyric, valeric, and isovaleric acid, respectively, were added to a newly defined medium containing Prevotella bryantii B 1 4 cells. After 8 h and 24 h, optical density, pH and SCFA concentrations were measured. Long-chain fatty acid (LCFA) profiles of the bacterial cells were analyzed via gas chromatography-time of flight-mass spectrometry (GC-ToF MS) and proteins were quantified using a mass spectrometry-based, label-free approach. Cultures supplemented with single SCFAs revealed different growth behavior. Structural features of the respective SCFAs were identified in the LCFA profiles, which suggests incorporation into the bacterial membranes. The proteomes of cultures supplemented with acetic and valeric acid differed by an increased abundance of outer membrane proteins. The proteome of the isovaleric acid supplementation showed an increase of proteins in the amino acid metabolism. Our findings indicate a possible interaction between SCFAs, the lipid membrane composition, the abundance of outer membrane proteins, and a modulation of branched chain amino acid biosynthesis by isovaleric acid.

Respiratory NADH oxidation in the rumen bacterium Prevotella bryantii is catalyzed by the Na + -translocating NADH:quinone oxidoreductase (NQR). A method for cell disruption and membrane isolation of P. bryantii under anoxic conditions using the EmulisFlex-C3 homogenizer is described. We compared NQR activity and protein yield after oxic and anoxic cell disruption by the EmulsiFlex, by ultrasonication, and by glass beads treatment. With an overall membrane protein yield of 50 mg L -1 culture and a NADH oxidation activity of 0.8 µmol min -1  mg -1 , the EmulsiFlex was the most efficient method. Anoxic preparation yielded fourfold higher NQR activity compared to oxic preparation. P. bryantii lacks genes coding for superoxide dismutases and cell extracts do not exhibit superoxide dismutase activity. We propose that inactivation of NQR during oxic cell rupture is caused by superoxide, which accumulates in P. bryantii extracts exposed to air. Anoxic cell rupture is indispensable for the preparation of redox-active proteins and enzymes such as NQR from P. bryantii.

Strictly anaerobic Prevotella spp. are characterized by their vast metabolic potential. As members of the Prevotellaceae family, they represent the most abundant organisms in the rumen and are typically found in monogastrics such as pigs and humans. Within their largely anoxic habitats, these bacteria are considered to rely primarily on fermentation for energy conservation. A recent study of the rumen microbiome identified multiple subunits of the Na + -translocating NADH:quinone oxidoreductase (NQR) belonging to different Prevotella spp. Commonly, the NQR is associated with biochemical energy generation by respiration. The existence of this Na + pump in Prevotella spp. may indicate an important role for electrochemical Na + gradients in their anaerobic metabolism. However, detailed information about the potential activity of the NQR in Prevotella spp. is not available. Here, the presence of a functioning NQR in the strictly anaerobic model organism P. bryantii B 1 4 was verified by conducting mass spectrometric, biochemical, and kinetic experiments. Our findings propose that P. bryantii B 1 4 and other Prevotella spp. retrieved from the rumen operate a respiratory NQR together with a fumarate reductase which suggests that these ruminal bacteria utilize a sodium motive force generated during respiratory NADH:fumarate oxidoreduction., Competing Interests: The authors declare no conflict of interest. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. There is no conflict of interest and there are no commercial and other relationships to declare.

Escherichia coli is a convenient host for the expression of proteins, but the heterologous production of large membrane protein complexes often is hampered by the lack of specific accessory genes required for membrane insertion or cofactor assembly. In this study we introduce the non-pathogenic and fast-growing Vibrio natriegens as a suitable expression host for membrane-bound proteins from Vibrio cholerae . We achieved production of the primary Na + pump, the NADH:quinone oxidoreductase (NQR), from V. cholerae in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed V. natriegens , and the sensitivity toward Ag + , a specific inhibitor of the NQR. Complete assembly of V. cholerae NQR expressed in V. natriegens was demonstrated by BN PAGE followed by activity staining. The secondary transport system Mrp from V. cholerae , another membrane-bound multisubunit complex, was also produced in V. natriegens in a functional state, as demonstrated by in vivo Li + transport. V. natriegens is a promising expression host for the production of membrane protein complexes from Gram-negative pathogens.

Objective: To identify key organizational approaches associated with underuse of breast cancer care., Setting: Nine New York City area safety-net hospitals., Study Design: Mixed qualitative-quantitative, cross-sectional cohort., Methods: We used qualitative comparative analysis (QCA) of key stakeholder interviews, defined organizational "conditions," calibrated conditions, and identified solution pathways. We defined underuse as no radiation after lumpectomy in women <75 years or mastectomy in women with ≥4 positive nodes, or no systemic therapy in women with tumors ≥1 cm. We used hierarchical models to assess organizational and patient factors' impact on underuse., Principal Findings: Underuse varied by hospital (8-29 percent). QCA found lower underuse sites designated individuals to track and follow-up no-shows; shared clinical information during handoffs; had fully integrated electronic medical records enabling transfer of responsibility across specialties; had strong system support; allocated resources to cancer clinics; had a patient-centered culture paying close organizational attention to clinic patients. High underuse sites lacked these characteristics. Multivariate modeling found that hospitals with strong approaches to follow-up had low underuse rates (RR = 0.28; 0.08-0.95); individual patient characteristics were not significant., Conclusions: At safety-net hospitals, underuse of needed cancer therapies is associated with organizational approaches to track and follow-up treatment. Findings provide varying approaches to safety nets to improve cancer care delivery., (© Health Research and Educational Trust.)

Objective: A major focus of health care today is a strong emphasis on improving the health and quality of care for entire patient populations. One common approach utilizes electronic clinical alerts to prompt clinicians when certain interventions are due for individual patients being seen. However, these alerts have not been consistently effective, particularly for less visible (though important) conditions such as hearing loss (HL) screening., Materials and Methods: We conducted hour-long cognitive task analysis interviews to explore how family medicine clinicians view, perceive, and use electronic clinical alerts, and to utilize this information to design a more effective alert using HL identification and referral as a model diagnosis., Results: Four key direct barriers were identified that impeded alert use: poor standardization and formatting, time pressures in primary care, clinic workflow variations, and mental models of the condition being prompted (in this case, HL). One indirect barrier was identified: electronic health record and institution/government regulations. We identified that clinicians' mental model of the condition being prompted was probably the major barrier, though this was often expressed as time pressure. We discuss solutions to each of the 5 identified barriers, such as addressing physicians' mental models, by focusing on physicians' expertise rather than knowledge to improve their comfort when caring for patients with the conditions being prompted., Conclusions: To unleash the potential of electronic clinical alerts, electronic health record and health care institutions need to address some key barriers. We outline these barriers and propose solutions., (© The Author 2017. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Background: A 67-year-old woman with a history of smoking presented with abdominal pain and hematuria. On physical examination, she had a palpable pelvic mass. Imaging revealed a large pelvic mass situated on the dome of the bladder, extending from the urachus, without evidence of other sites of metastases. After resection, urachal adenocarcinoma was histologically confirmed. No adjuvant therapy was administered. Repeat imaging obtained 3 months after resection revealed a large left pelvic mass consistent with recurrence., Investigations: Physical examination, pelvic and complete lymph-node examination, laboratory tests, urine analysis, CT scan of the abdomen and pelvis, chest X-ray, bone scan, cystoscopy, histologic examination of tumor morphology, and MRI of the abdomen and pelvis., Diagnosis: Metastatic urachal adenocarcinoma., Management: Surgical excision combined with cystectomy and pelvic lympadenectomy, chemotherapy with 5-fluorouracil, leucovorin, and irinotecan.