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Background: Differences have often been reported in the outcomes of bladder cancer (BC) patients according to gender., Objective: This study aims to provide data on patients undergoing radical cystectomy (RC) in a high-volume tertiary urologic center and to assess whether gender discrepancies do exist in terms of surgical options and clinical outcomes., Materials and Methods: Consecutive BC patients treated between 2016 and 2020 at a single center (Careggi University Hospital, Florence, Italy) were included in the study. The impact of gender on disease stage at diagnosis, overall survival (OS), and type of surgery was analyzed., Results: The study series comprised 447 patients (85 females and 362 males). At a median follow-up of 28.3 months (IQR: 33.5), OS was 52.6% and cancer-specific survival was 67.6%. Significant differences in OS emerged for age, acute myocardial infarction (AMI), Charlson Comorbidity Index (CCI), pT, and pN. OS rates were higher in patients undergoing robot-assisted surgery and in those receiving open orthotopic neobladder (ONB) (p = 0.0001). No statistically significant differences were found between male and female patients regarding surgical offer in any age group, surgical time, early postoperative complications, pathologic stage, and OS., Conclusions: After adjustment for pathologic tumor stage and treatment modalities, female and male patients showed similar oncologic outcomes. Further studies should be undertaken to evaluate functional results in women subjected to RC., Competing Interests: Declaration of competing interest All authors declare that there's no financial/personal interest or belief that could affect their objectivity. They also declare that there's no potential competing interests., (© 2023 Published by Elsevier Ltd.)

M pro represents one of the most promising drug targets for SARS-Cov-2, as it plays a crucial role in the maturation of viral polyproteins into functional proteins. HTS methods are currently used to screen M pro inhibitors, and rely on searching chemical databases and compound libraries, meaning that they only consider previously structurally clarified and isolated molecules. A great advancement in the hit identification strategy would be to set-up an approach aimed at exploring un-deconvoluted mixtures of compounds such as plant extracts. Hence, the aim of the present study is to set-up an analytical platform able to fish-out bioactive molecules from complex natural matrices even where there is no knowledge on the constituents. The proposed approach begins with a metabolomic step aimed at annotating the MW of the matrix constituents. A further metabolomic step is based on identifying those natural electrophilic compounds able to form a Michael adduct with thiols, a peculiar chemical feature of many M pro inhibitors that covalently bind the catalytic Cys145 in the active site, thus stabilizing the complex. A final step consists of incubating recombinant M pro with natural extracts and identifying compounds adducted to the residues within the M pro active site by bottom-up proteomic analysis (nano-LC-HRMS). Data analysis is based on two complementary strategies: (i) a targeted search applied by setting the adducted moieties identified as Michael acceptors of Cys as variable modifications; (ii) an untargeted approach aimed at identifying the whole range of adducted peptides containing Cys145 on the basis of the characteristic b and y fragment ions independent of the adduct. The method was set-up and then successfully tested to fish-out bioactive compounds from the crude extract of Scutellaria baicalensis, a Chinese plant containing the catechol-like flavonoid baicalin and its corresponding aglycone baicalein which are well-established inhibitors of M pro . Molecular dynamics (MD) simulations were carried out in order to explore the binding mode of baicalin and baicalein, within the SARS-CoV-2 M pro active site, allowing a better understanding of the role of the nucleophilic residues (i.e. His41, Cys145, His163 and His164) in the protein-ligand recognition process., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Giancarlo Aldini reports financial support was provided by Lombardy Region., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Introduction: We aimed to compare the outcomes of open vs robotic partial nephrectomy (PN), focusing on predictors of Trifecta failure in patients with highly complex renal masses., Patients and Methods: We queried the prospectively collected database from the SIB International Consortium, including 507 consecutive patients with cT1-2N0M0 renal masses treated at 16 high-volume referral centres, to select those with highly complex (PADUA score ≥10) tumors undergoing PN. RT was classified as enucleation, enucleoresection or resection according to the SIB score. Trifecta was defined as achievement of negative surgical margins, no acute kidney injury and no Clavien-Dindo grade ≥2 postoperative surgical complications. Multivariable logistic regression analysis was used to assess independent predictors of Trifecta failure., Results: 113 patients were included. Patients undergoing open PN (n = 47, 41.6%) and robotic PN (n = 66, 58.4%) were comparable in baseline characteristics. RT was classified as enucleation, enucleoresection and resection in 46.9%, 34.0% and 19.1% of open PN, and in 50.0%, 40.9% and 9.1% of robotic PN (p = 0.28). Trifecta was achieved in significantly more patients after robotic PN (69.7% vs. 42.6%, p = 0.004). On multivariable analysis, surgical approach (open vs robotic, OR: 2.62; 95%CI: 1.11-6.15, p = 0.027) and tumor complexity (OR for each additional unit of the PADUA score: 2.27; 95%CI: 1.27-4.06, p = 0.006) were significant predictors of Trifecta failure, while RT was not. The study is limited by lack of randomization; as such, selection bias and confounding cannot be entirely ruled out., Conclusions: Tumor complexity and surgical approach were independent predictors of Trifecta failure after PN for highly complex renal masses., Competing Interests: Declaration of competing interest All authors disclose any financial and personal relationships with other people or organizations that could inappropriately influence their work., (Copyright © 2021 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Due to the physiological properties of l-carnosine (l-1), supplementation of this dipeptide has both a nutritional ergogenic application and a therapeutic potential for the treatment of numerous diseases in which ischemic or oxidative stress are involved. Quantitation of carnosine and its analogs in biological matrices results to be crucial for these applications and HPLC-MS procedures with isotope-labeled internal standards are the state-of-the-art approach for this analytical need. The use of these standards allows to account for variations during the sample preparation process, between-sample matrix effects, and variations in instrument performance over analysis time. Although literature reports a number of studies involving carnosine, isotope-labeled derivatives of the dipeptide are not commercially available. In this work we present a fast, flexible, and convenient strategy for the synthesis of the 13 C-labeled carnosine analogs and their application as internal standards for the quantitation of carnosine and anserine in a biological matrix., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Competing Interests: Declaration of interests We declare no competing interests.

Introduction: Aim of the study was to evaluate the Florence intracorporeal neobladder (FloRIN) oncological and functional outcomes at the end of assessment phase (phase 3) IDEAL-Guidelines., Materials and Methods: This single-institution prospective series included consecutive patients treated with robot-assisted radical cystectomy (RARC) and FloRIN reconfiguration technique from February 2016 to June 2020. Functional features were evaluated six months after surgery. Patients were grouped into four quartiles according to time of radical cystectomy and impact of learning curve improvement was evaluated., Results: One-hundred FloRIN were completed with a median console time of 373 (IQR: 312-415) minutes. Two cases were converted to open surgery. No intraoperative complications occurred. At pathological examination, 30% of patients were staged as pT ≤ 1 and 47% as pT ≥ 3. Transitional cell carcinoma was present in 87% of cases. Carcinoma in situ (CIS) and nodal involvement were observed in 38% and 29% of patients, respectively. At a median follow-up time of 17 (IQR: 7-28) months, 20 clinically relevant events (Clavien-Dindo≥3) occurred. Operative time significantly decreased throughout the series (median minutes 435; 395; 365 and 330 in the four quartiles, respectively; p < 0.001). Similarly, early Clavien-Dindo≥3 postoperative complications rate significantly decreased across the series (number of events: 1; 4; 0; 0; p = 0.03). Overall, 75% and 65% of patients achieved day-time and nigh-time continence, respectively. Twenty-seven patients experienced disease recurrence. Cancer-specific and overall survival were equal to 80%., Conclusions: RARC with FloRIN reconfiguration showed worthy functional and survival outcomes, with learning curve improvement significantly influencing operative time and early complications rate across series., Competing Interests: Declaration of competing interest All authors disclose any financial and personal relationships with other people or organizations that could inappropriately influence their work., (Copyright © 2021 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Introduction: Multicenter retrospective analysis of robotic partial nephrectomy for completely endophytic renal tumors (i.e. 3 points for the 'E' domain of the R.E.N.A.L. nephrometry score) was performed., Materials and Methods: Patients' demographics, tumor characteristics, perioperative, functional, pathological and oncological data were analyzed and compared with those of patients with exophytic and mesophytic masses (i.e. 1 and 2 points for the 'E' domain, respectively). Multivariable logistic regression analysis was used to assess variables for trifecta achievement (negative margin, no postoperative complications, and 90% estimated glomerular filtration rate [eGFR] recovery)., Results: Overall, 147 patients were included in the study group. Patients with a completely endophytic mass had bigger tumors (mean 4.2 vs. 4.1 vs. 3.2 cm; p < 0.001) on preoperative imaging and higher overall R.E.N.A.L. score. There was no difference in mean operative time. Estimated blood loss was higher in the endophytic group (mean 177.75 vs. 185.5 vs. 130 ml; p = 0.001). Warm ischemia time was shorter for the exophytic group (median 16 vs. 21 vs. 22 min; p < 0.001). Postoperative complications were more frequent in patients with endophytic tumor (24.8% vs. 19.5% vs. 14.8%; p < 0.001). Six (4.5%) patients had positive surgical margins, there was no difference between groups. Trifecta was achieved in 44 patients in endophytic group (45.4 vs. 68.8 and 50.9%, p < 0.001). Multivariable analysis for trifecta revealed that clinical tumor size (odds ratio: 0.667, 95% confidence interval: 0.56-0.79, p < 0.001) was only significant predictor for trifecta achievement., Conclusions: Our findings confirm that RAPN in case of completely endophytic renal masses can be performed with acceptable outcomes in centers with significant robotic expertise., (Copyright © 2020 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

The possible role of JC virus in determining urinary tract involvement has only recently been recognized. The case of a man with laboratory-confirmed JC virus replication in the urine after a maintenance schedule of rituximab administered for a lymphoproliferative disorder is reported herein. The patient developed severe renal and urinary tract impairment, characterized by the onset of nephropathy, bilateral ureteral strictures, and a serious reduction in vesical compliance, ultimately requiring an ileal neobladder configuration. The renal and urinary tract involvement was finally attributed to JC virus reactivation. This observation suggests that renal and urinary tract diseases related to JC virus might be associated with long-term rituximab treatment., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: To investigate the perioperative and morbidity outcomes after partial nephrectomy (PN) in patients with short life expectancy (SLE) (≥95% 10-year expected mortality (10y-EM)), to assess the main predictors of outcomes in this population and to compare these results with those of a group at the opposite upper range with long LE (LLE, ≤5% 10y-EM) relying on a multicenter Italian prospective registry of kidney surgery (the RECORD 2 project)., Methods: Clinical data of 4,325 patients undergone kidney surgery were collected at 26 urological Italian Centers from 2013 to 2016. SLE was defined as a ≥95% 10y-EM (assessed using the age-adjusted Charlson comorbidity index [CCI]). A multivariable logistic regression for overall postoperative complications, acute kidney injury (AKI), positive surgical margins (SM) and ∆ estimated glomerular filtration rate (eGFR) ≥25% at 2 years from surgery was performed in patients with SLE including clinically relevant variables. Adjusted outcomes reported as mean (SD) of the 2 groups were generated using separate multivariable logistic regression models and compared., Results: Overall, 559 patients with SLE were selected. Patients had an ASA score ≥3 in 58.4% of cases. A clinical T1a, T1b, and T2 stage was found in 412 (74.5%), 124 (22.4%), and 17 (3.1%) patients. The median PADUA score was 7 (6-8). Surgical and medical postoperative complication rates were registered in 14.8% and 6% cases. Postoperative AKI was reported in 27.3% cases, positive surgical margins (PSM) in 9.3% cases. In this subgroup of patients, ASA score, cerebrovascular disease, surgery in low volume centers, and open surgery were independent predictors of overall complications. ASA and PADUA scores, renal clamping, resection technique and lower eGFR at baseline were independent predictors of AKI. PADUA score, open approach and resection technique were independent predictors of PSM. Cardiovascular disease, hilar clamping, and resection technique were independent predictors of eGFR decrease >25% at 2 years from surgery. Patients with SLE were compared with those with LLE (n = 302). All analyzed parameters at baseline were significantly different among the groups with the exception of cancer laterality. After adjusting for several clinical variables, the SLE group had a significantly higher risk rate of adjusted overall postoperative complication rate compared to the LLE group (20.6% ± 0.36 vs. 9.9% ± 0.65, P < 0.0001), while the overall intraoperative complications (4.1% ±0.13 vs. 2.3% ± 0.23), overall postoperative major complications (3.8% ± 0.09 vs. 1.9% ± 0.14) adjusted AKI (24.2% ± 0.37 vs. 22.6% ± 0.92), positive surgical margins (8% ± 0.22 vs. 6.4% ± 0.49), and 2-year RF loss (13.4% ± 0.17 vs. 12.4% ± 0.74)., Conclusion: In selected patients with SLE, PN is feasible with an acceptable safety profile that is overall comparable to patients with no LE limitations. While a robotic approach and surgery performed in high volume centers could reduce the risk of complications, an off-clamp approach and a SE surgical technique may decrease the risk of postoperative AKI and of longer term eGFR decrease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Purpose: to evaluate clinical predictors of positive surgical margins (PSMs) in a large multicenter prospective observational study and to develop a clinic nomogram to predict the likelihood of PSMs after partial nephrectomy (PN)., Materials and Methods: We prospectively evaluated 4308 patients who had surgical treatment for renal tumors between January 2013 and December 2016 at 26 urological Italian Centers (RECORd 2 project). Two multivariable logistic models were evaluated to predict the likelihood of PSMs. Center caseload was dichotomized using a visual assessment adjusted for several predictors of PSMs. A nomogram predicting PSMs was developed., Results: Overall, 2076 patients treated with PN were evaluated. pT1a, pT1b, pT2 and pT3a were recorded in 68.7%, 22.6%, 2.1% and 6.6% of the patients, respectively. PSMs were recorded in 342 (16.5%) patients. From a null multivariable model against number of PN/year, 60 PN/year were identified as the best cut-off to define a high-volume centre. At multivariable analysis, clinical stage (cT1a vs. cT2 [OR 1.94]; p = 0.03), volume centre (≤60 PN/year) (OR 2.22; p < 0.0001), imperative vs elective indication (OR 2.10; p = 0.04), surgical technique (laparoscopic vs. open [OR 1.62; p = 0.002), lymphovascular invasion (OR 2.27; p = 0.01) and upstaging to pT3a (OR 2.81; p < 0.0001) were independent predictors of PSMs. The final nomogram included age, ASA score, Charlson score, clinical tumor stage, surgical indication, surgical approach, surgical technique, PADUA score, clamp procedure and volume centre., Conclusions: PSMs after PN were significantly more likely in patients with lower clinical stage, higher PADUA score, in individuals referred to laparoscopic PN and in those treated at lower volume centers. We used these data to develop a nomogram to predict such risk., (Copyright © 2020 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Objectives: Surgically treated clinical T1 (cT1) kidney cancer has in general a good prognosis, but there is a risk of upstaging that can potentially jeopardize the oncological outcomes after partial nephrectomy (PN). Aim of this study is to analyze the outcomes of robot-assisted PN (RAPN) for cT1 kidney cancer upstaged to pT3a, and to identify predictors of upstaging., Material and Methods: The study cohort included 1,640 cT1 patients who underwent RAPN between 2005 and 2018 at 10 academic institutions. Multivariate logistic regression model was used to assess the predictors of upstaging. Kaplan-Meier curves and multivariable Cox regression analyses were used to evaluate recurrence-free survival and overall survival., Results: Overall, 74 (4%) were upstaged cases (cT1/pT3a). Upstaged patients presented larger renal tumors (3.1 vs. 2.4 cm; P = 0.001), and higher R.E.N.A.L. score (8.0 vs. 6.0; P = 0.004). cT1/pT3a group had higher rate of intraoperative complications (5 vs. 1% P = 0.032), higher pathological tumor size (3.2 vs. 2.5 cm; P < 0.001), higher rate of Fuhrman grade ≥3 (32 vs. 17%; P = 0.002), and higher number of sarcomatoid differentiation (4 vs. 1%; P = 0.008). Chronic kidney disease (CKD) stage ≥3 (OR: 2.54; P < 0.014), and clinical tumor size (OR: 1.07; P < 0.001) were independent predictors of upstaging. cT1/pT3a group had worse 2-year (94% vs. 99%) recurrence-free survival (P < 0.001)., Conclusions: Upstaging to pT3a in patients with cT1 renal mass undergoing RAPN represents an uncommon event, involving less than 5% of cases. Pathologic upstaging might translate into worse oncological outcomes, and therefore strict follow-up protocols should be applied in these cases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: To investigate the influence of the body weight on peri- and postoperative outcome in a series of pediatric patients with a diagnosis of uretero-pelvic junction obstruction (UPJO) treated with robot-assisted laparoscopic pyeloplasty (RALP) at a single tertiary referral center., Objective: In this study, outcomes of RALP in children divided according to the weight are evaluated., Study Design: Sixty-one consecutive patients treated with RALP from January 2016 to May 2019 were recorded retrospectively and divided according to their weight: group A < 15 kg, group B ≥ 15 kg and were included in this study. Eligible criteria for surgery were symptomatic UPJO, worsening of hydronephrosis, or obstructive pattern at renogram. Success criteria were the reduction of the hydronephrosis at imaging and the absence of flank pain. All procedures were performed by one expert robotic surgeon., Results: 18 patients were included in group A (median weight 12 kg) and 43 patients in group B (median weight 33 kg). The median (IQR) operative was 95 (90-120) for group A compared to 90 (85-110) of control group. No significant difference has been found (P = 0.93). We registered one (5.6%) Clavien 3b complication (omental hernia after removal of the drainage requiring surgical correction) and two (4.7%) Clavien 2 complication in group B (urinary infections). No difference has been found in the length of hospital stay, length of catheterization, and duration of procedure between the groups (P > 0.05). At a median follow-up of 23 months (IQR 9-27 vs 9-33), the success rate was comparable between the two groups (94.4% vs 97.7%; P = 0.51). Relapse was recorded in one child per group and both required nephrostomy placement., Discussion: Our study tried to fill the gaps in the evidence on the feasibility of RALP in low-weight children reporting favorable peri-operative and long-term outcomes; however, this study shows some intrinsic limitations. The relatively small numbers of patients in the <15 kg may have underpowered the comparison with heavier patients. Moreover, only two patients were <10 kg so that no definitive conclusions on the safety and feasibility of RALP in this cohort of patients can be drawn., Conclusion: RALP in children <15 kg was feasible and effective to treat UPJO with superimposable results to heavier counterparts. In our experience, the need for a different trocar placement and limited space in patients <15 kg did not affect peri-operative and functional outcomes., (Copyright © 2019. Published by Elsevier Ltd.)

Background: In search of novel prognostic biomarkers for clear cell renal carcinoma (ccRCC), we analysed the expression of several proteins related to angiogenesis and hypoxia., Methods: A monocentric study on 30 consecutive surgical samples from surgically-treated ccRCC patients with a 10-year follow up was performed. The following proteins were analysed by immunohistochemistry: Vascular Endothelial Growth Factor- A (VEGF-A), Platelet-Derived Growth Factor β Receptor (PDGFRβ), VEGF-receptor 1 (Flt1), VEGF-receptor 2 (KDR), Glucose Transporter 1 (GLUT1), Carbonic anhydrase IX (CA-IX) and the hERG1 potassium channel. Data were analysed in conjunction with the clinico-pathological characteristics of the patients and follow up., Results: All the proteins were expressed in the samples, with statistically significant associations of VEGF-A with PDGFRβ and Flt1 and hERG1 with CA IX. Notably, hERG1 and CAIX co-immunoprecipitated in primary ccRCC samples and survival analysis showed that the positivity for hERG1 and CA IX had a negative impact on Recurrence Free Survival (RFS) at the univariate analysis. At the multivariate analysis only hERG1 maintained its statistically significant negative impact., Conclusions: hERG1 expression can be exploited to predict recurrence in surgically-treated ccRCC patients. hERG1 channels form a multiprotein complex with the pH regulator CA IX in primary ccRCC samples their potential use as therapeutic target might be suggested., (Copyright © 2019 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Introduction: Open Simple Enucleation (OSE) has been demonstrated to be an oncologically safe alternative to standard partial nephrectomy. We assessed the mid-term oncologic outcomes and predictors of disease recurrence in patients treated with Endoscopic Robot-Assisted Simple Enucleation (ERASE) verified through a standardized reporting system at a single institution., Material and Methods: The clinical data of patients treated with ERASE for renal cell carcinoma at our Institution from January 2012 to September 2018 were retrospectively reviewed. Surface-Intermediate-Base (SIB) score was evaluated immediately after surgery. Patients with a ≥2 SIB score were excluded. The local and distant recurrence rates, the recurrence-free (RFS), cancer-specific (CSS) and overall (OS) survival were assessed. Kaplan-Meier and log-rank test were used to estimate survivals and to compare recurrence-free survival., Results: Overall, 259 patients were considered eligible for the present study. The median PADUA score was 7 (interquartile range [IQR] 6-9). Positive surgical margins (PSM) were registered in 7 (2.7%) cases. Median (IQR) follow-up time was 36 (27-51) months. Overall, 4 (1.5%) patients experienced systemic recurrence (SR) and 5 (1.9%) patients experienced local recurrence alone (LR) of whom 3 (1.1%) were on tumor resection bed, and 2 (0.8%) experienced recurrence elsewhere in the ipsilateral kidney. The 5-year RFS, CSS and OS were 94.2%, 98.9% and 93.6%, respectively. Higher nucleolar grade and PSM were the two factors significantly associated with worse RFS., Conclusions: ERASE is a safe procedure, achieving negative surgical margins in the vast majority of patients and providing excellent mid-term local control and oncologic outcomes., (Copyright © 2019 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

The chiral purity of some molecules such as nutraceuticals is fundamental to ensure their beneficial activities and it must be checked during quality control analysis. Carnosine is a natural histidine dipeptide used as ingredient for food supplements, but only his L-enantiomer is absorbed and active. Despite of this feature, a method for the separation of carnosine enantiomers without derivatization has only recently been published. Herein, we validated a method based on a Chirobiotic T column and an UV detector for the direct quantification of carnosine enantiomers, following ICH guideline. Moreover, we demonstrated that elution with water containing 0.1% formic acid and 20-40% ensures stereo-, chemo- and regio-selectivity for the separation and the identification of carnosine enantiomers and natural analogues. Moreover, the method allows a direct hyphenation with electrospray mass spectrometry to increase detection selectivity and sensitivity. As far as we know, this is the first method allowing the simultaneous identification and quantification of natural analogues of carnosine, which can be important for application such as the identification of enantiomeric impurities or adulteration that can occur during the storage or the preparation of foods or food supplements containing histidine dipeptides., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The aim of the study was to investigate the feasibility of Epidermal Growth Factor Receptor (EGFR) measurement in bladder washings of patients affected by non-muscle-invasive bladder cancer (NMIBC) and its prognostic role in identifying risk subgroups and predicting disease recurrence and progression., Patients and Methods: Patients with NMIBC treated with transurethral resection of bladder tumor (TURBT) from 2012 to 2015 were enrolled. Samples of bladder washings were collected and stored at -80°C until RNA extraction. The cDNA obtained from RNA was used to perform a gene expression analysis by a real time polymerase chain reaction., Results: An adequate cellular pellet was obtained in 50 (86.2%) of 58 patients and in 18 (85.7%) of 21 controls. Patients had a median 2.5-, a 1.6- and a 2.8-fold EGFR expression compared with controls before, during, and after adjuvant treatment, respectively. Patients at higher risk had a significantly higher EGFR expression compared with patients at low and intermediate risk when EGFR was measured during (P = .04) and after (P = .001) adjuvant therapy. At a median follow-up of 35.5 months (interquartile range, 19.0-54.8 months), in the high-risk group, patients with overexpression had a significantly lower recurrence-free survival (27.9% vs. 58%), progression-free survival (75.9% vs. 90.2%), and cancer-specific survival (77.7% vs. 93.3%). At multivariable analysis, EGFR overexpression was an additional independent prognostic factor to the European Organisation for Research and Treatment of Cancer scoring system of disease recurrence (hazard ratio, 1.98; 95% confidence interval, 1.32-2.97) and progression (hazard ratio, 1.84; 95% confidence interval, 1.27-2.65)., Conclusions: EGFR overexpression might represent an additional parameter to the current clinical tools for an individualized risk stratification., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tannins are a heterogeneous class of polyphenols that are present in several plants and foods. Their ability to interact and precipitate proline-rich proteins leads to different effects such as astringency or antidiarrheal activity. Thus, evaluation of the tannin content in plant extracts plays a key role in understanding their potential use as pharmaceuticals and nutraceuticals. Several methods have been proposed to study tannin-protein interactions but few of them are focused on quantification. The purpose of the present work is to set up a suitable and time efficient method able to quantify the extent of tannin protein precipitation. Bradykinin, chosen as a model, was incubated with increasing concentrations of 1,2,3,4,6-penta-O-galloyl-β-d-glucose and tannic acid selected as reference of tannic compounds. Bradykinin not precipitated was determined by a mass spectrometer TSQ Quantum Ultra Triple Quadrupole (direct infusion analysis). The results were expressed as PC 50 , which is the concentration able to precipitate 50% of the protein. The type of tannin-protein interaction was evaluated also after precipitate solubilisation. The involvement of proline residues in tannin-protein interactions was confirmed by repeating the experiment using a synthesized peptide (RR-9) characterized by the same bradykinin sequence, but having proline residues replaced by glycine residues: no interaction occurred between the peptide and the tannins. Moreover, modelling studies on PGG-BK and PGG-RR-9 were performed to deeply investigate the involvement of prolines: a balance of hydrophobic and H-bond contacts stabilizes the PGG-BK cluster and the proline residues exert a crucial role thus allowing the PGG molecules to elicit a sticking effect., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: Partial nephrectomy (PN) is standard for small renal masses, improving renal function by preserving renal parenchyma compared with radical nephrectomy. Recent work demonstrated that postoperative surgeon assessment of volume preservation (SAVP) and 3D imaging measurements agree and correlate with postoperative function. We hypothesize preoperative assessment of volume preservation (PAVP) with PN based on preoperative imaging will reliably indicate postoperative renal function., Materials and Methods: Data were collected from 336 patients undergoing PN for suspected renal cancer by 40 surgeons at 12 centers in Europe and the United States within the Surface-Intermediate-Base International Consortium. Surgeons recorded PAVP and SAVP for individual patients; pre- and postoperative glomerular filtration rate (GFR) was estimated by Chronic Kidney Disease Epidemiology Collaboration equations. Correlations between PAVP, SAVP, and postoperative GFR were assessed with linear regression models. Bland-Altman analysis was used to assess agreement between PAVP and SAVP with a significant cutoff of 5%., Results: Median PAVP was 90% (interquartile range [IQR] 85%-100%) and SAVP was 90% (IQR: 80%-94%). PAVP and SAVP were moderately correlated (R 2  = 0.67, P < 0.0001) and deemed "interchangeable" by Bland-Altman analysis at a 5% acceptable rate of difference (95% CI: -5.4, -3.1). Median postoperative GFR was 77.3 (IQR: 56.2, 92.0). Both PAVP (R 2  = 0.82, P < 0.0001) and SAVP (R 2  = 0.83, P < 0.0001) were correlated with postoperative GFR. Multivariable models utilizing volume-adjusted GFR based on PAVP or SAVP significantly and similarly predicted postoperative GFR (R 2  = 0.72 for each)., Conclusion: Renal function is closely linked to the amount of parenchymal volume preservation, whether estimated prior to surgery (PAVP) or afterward (SAVP). PAVP provides reasonably accurate information for decision-making in patients considering PN., (Copyright © 2018. Published by Elsevier Inc.)

Objective: Tumor enucleation has been shown to be oncologically safe for elective treatment of renal cell carcinoma (RCC); yet, evidence on long-term oncologic outcomes after robotic tumor enucleation is lacking. In this study we provide a detailed histopathological analysis of tumor-parenchyma interface and the long-term oncologic outcomes after robotic tumor enucleation for sporadic RCC in a high-volume referral center., Materials and Methods: We selected consecutive patients undergoing robotic tumor enucleation for sporadic RCC by experienced surgeons with at least 4 years of follow-up. Pattern of pseudocapsule (PC) invasion, thickness of healthy renal margin removed with the tumor, margin status and recurrence rate were the main study endpoints. Multivariable models evaluated independent predictors of PC invasion., Results: Overall, 140 patients were eligible for the study. Of these, 127 (91%) had complete data available for analysis. Median thickness of healthy renal margin was 0.57 mm (interquartile range [IQR] 0.24-103). A distinct peritumoral PC was present in 121/127 (95%) tumors with a median thickness of 0.28 mm (IQR 0.14-0.45). In 24/121 (19.8%) cases, RCC showed complete PC invasion. At multivariable analysis, increasing tumor diameter, endophytic rate > 50% and papillary histology were significantly associated with complete PC invasion. Positive surgical margins were reported in 3/127 (2.4%) cases. At a median follow-up of 61 months (range 48-76), one patient died due to metastatic RCC. Among patients alive at follow-up, no cases of recurrence at the enucleation site were recorded, while three cases (2.4%) of renal recurrence (elsewhere in the ipsilateral kidney) and three cases (2.4%) of systemic recurrence were found., Conclusions: Distinct RCC-related features were associated with complete PC invasion. By providing a microscopic layer of healthy renal margin in almost all cases, robotic tumor enucleation achieved negative surgical margins in the vast majority of patients, even in case of complete PC invasion. At long-term follow-up, no recurrences were found at the enucleation site. Although our findings need to be confirmed by larger studies with longer follow-up, robotic tumor enucleation appears oncologically safe in experienced hands for the treatment of sporadic RCC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Idebenone is a hydrophilic short-chain coenzyme (Co) Q analogue, which has been used as a potential bypass of defective complex I in both Leber Hereditary Optic Neuropathy and OPA1-dependent Dominant Optic Atrophy. Based on its potential antioxidant effects, it has also been tested in degenerative disorders such as Friedreich's ataxia, Huntington's and Alzheimer's diseases. Idebenone is rapidly modified but the biological effects of its metabolites have been characterized only partially. Here we have studied the effects of quinones generated during in vivo metabolism of idebenone with specific emphasis on 6-(9-carboxynonyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (QS10). QS10 partially restored respiration in cells deficient of complex I or of CoQ without inducing the mitochondrial permeability transition, a detrimental effect of idebenone that may offset its potential benefits [Giorgio et al. (2012) Biochim. Biophys. Acta 1817: 363-369]. Remarkably, respiration was largely rotenone-insensitive in complex I deficient cells and rotenone-sensitive in CoQ deficient cells. These findings indicate that, like idebenone, QS10 can provide a bypass to defective complex I; and that, unlike idebenone, QS10 can partially replace endogenous CoQ. In zebrafish (Danio rerio) treated with rotenone, QS10 was more effective than idebenone in allowing partial recovery of respiration (to 40% and 20% of the basal respiration of untreated embryos, respectively) and allowing zebrafish survival (80% surviving embryos at 60 h post-fertilization, a time point at which all rotenone-treated embryos otherwise died). We conclude that QS10 is potentially more active than idebenone in the treatment of diseases caused by complex I defects, and that it could also be used in CoQ deficiencies of genetic and acquired origin., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: To evaluate the surgical and functional outcomes of a matched-paired series of on-clamp vs off-clamp endoscopic robot-assisted simple enucleation (ERASE) and standardized renorraphy in a tertiary referral institution, to search for predictors of functional drop after surgery and to investigate the influence of off-clamp technique in patients presenting these characteristics., Materials and Methods: A matched-pair comparison of 120 on-clamp vs 120 off-clamp over 491 patients treated with ERASE was performed. Perioperative and functional outcomes were compared between groups., Results: Patients treated with on-clamp and off-clamp technique had comparable complication and positive surgical margin rate. The off-clamp group had a significantly lower eGFR drop compared to the on-clamp group at 3rd postoperative day (POD) (1% vs 7%, p = 0.0001) and at 30th POD (2.5% vs 9%, p = 0.01) from baseline. This difference lost its statistical significance at 6th month and at last follow-up (median 40 months). At multivariable analysis the Charlson comorbidity index (OR 2.06, p < 0.0001), uncontrolled type 2 diabetes mellitus (OR 4.13, p < 0.001) were independent predictive factors of a >15% eGFR drop from baseline to last follow-up. In a subanalysis over 64 comorbid patients, those patients who underwent off-clamp ERASE had a significantly lower eGFR drop compared to the comorbid counterpart during the whole follow-up., Conclusions: The off-clamp ERASE is a safe surgical technique with a significantly lower renal function drop compared to on-clamp ERASE in the early perioperative time. Patients with comorbidity might represent a subgroup of patients having a functional benefit after off-clamp RAPN even in the long-term period., (Copyright © 2018 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

The study combines HPLC-based with MS-based competitive analyses to evaluate the quenching activity of a set of carnosine derivatives towards methylglyoxal (MGO) and malondialdehyde (MDA) chosen as representative of α- and β-dicarbonyls, respectively. The obtained results underline that these derivatives are moderately reactive towards MDA with which they form the corresponding N-propenal adduct via Michael addition. In contrast they proved a rather poor quenching activity towards MGO with which they can condense to give MOLD-like adducts through a concerted mechanism involving more quenchers molecules. Even though both quenching mechanisms involve the amino group in its neutral form, in silico studies revealed that the reported reactivity values depend on different stereo-electronic parameters which are reflected in the different observed quenching mechanism. Finally, the MGO quenching reactivity and the unselective (and unwanted) pyridoxal quenching are found to be influenced by the same parameters thus rationalizing the known difficulty in the design of potent and selective quenchers towards β-dicarbonyls., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Introduction and Objectives: To analyze postoperative complications and to assess for significant predictive factors during partial nephrectomy (PN) using a large multicenter dataset., Methods: Patients who underwent PN for clinical T1 renal tumors at 19 urological Italian centers (Registry of Conservative Renal Surgery [RECORd] project) were evaluated between 2009 and 2012. Anthropometric data, comorbidities and perioperative outcomes were analyzed. Complications were divided as intra- and postoperative, medical and surgical, as appropriate. The severity of postoperative complications was graded according to the modified Clavien classification system. Patients who experienced intraoperative complications were excluded from the analyses for the potential confounding effect in the evaluation of predicting factors for postoperative complications., Results: Overall, 979 patients were analyzed: open, laparoscopic and robot-assisted (available since 2011) surgical approaches were used in 522 (56.4%), 286 (30.9%) and 117 (12.6%) cases, respectively. Surgical postoperative complications were reported in 121 (13.1%) cases (32 (3.5%) were Clavien 3), medical were reported in 52 (5.6%) cases (3 (0.3%) were Clavien 3). No Clavien 4 complications were reported. At multivariable analysis, ECOG score ≥1 (OR 1.98; p = 0.002), lower preoperative hemoglobin (OR 0.71; p < 0.0001) and open surgical approach (2.91; p = 0.02) were significant predictive factors of overall surgical postoperative complications, ECOG score ≥1 (OR 1.93; p = 0.04) and surgical approach (p = 0.05) were significant predictive factors of Clavien 3 either surgical or medical postoperative complications., Conclusions: Comorbidities and surgical approach should be considered in preoperative evaluation of patients undergoing PN, as they resulted to play a significant role in the occurrence of postoperative complications., (Copyright © 2016 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Background: After radiotherapy (RT) for prostate cancer (PCa), several patients reported lower urinary tract symptoms (LUTS) due to damage and discontinuation of the glycosaminoglycan layer of the bladder. Instillation of hyaluronic acid and chondroitin sulfate (HA-CS) represents replenishment therapy of the glycosaminoglycan layer. The aim of the study is to evaluate the efficacy and safety of HA-CS in men with symptomatic cystitis after RT for PCa., Materials and Methods: Eighty consecutive men were treated with RT for PCa; 30 of these (37.5%) reported clinically relevant LUTS and associated bother as measured by the Interstitial Cystitis Symptom Index and Problem Index (ICSI/ICPI) Questionnaire 3 months after RT. Symptomatic patients received instillation therapy with HA-CS weekly for the first month and then at weeks 6, 8, and 12. All patients completed the ICSI/ICPI questionnaire before and after RT and at the end of HA-CS treatment., Results: HA-CS significantly reduced postradiation LUTS (P < .001) and bother (P = .006). Age, Gleason score, and radiation dose were the main determinants of worsening of LUTS after radiation (ICSI score baseline vs. postradiation: P = .047, .043, and .023). In multivariate analysis, only age influenced LUTS worsening after RT (P = .01). Age, radiation dose, and radiation toxicity were related to recovery of LUTS (ICSI score postradiation vs. post-HA-CS P = .041, P = .050, and P = .046). In multivariate analysis, no factor was statistically significant., Conclusions: A remarkable worsening of symptoms and bother was observed after RT. HA-CS instillation is a safe treatment and resulted in an improvement of LUTS irrespective of age and clinical features, with full recovery of urinary bother., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A validated analytical procedure is here described for the quality control of the protein fraction of purified bovine colostrum used in food supplements. The proposed procedure starts with 1D and 2D-gel electrophoresis. The sample is then separated into two fractions by protein G affinity chromatography: the IgG enriched and the IgG depleted fraction (IgG-d). A size exclusion chromatography coupled to UV is then applied to the IgG and IgG-d fractions for the quantitative analysis of IgG and IgM, respectively. The IgG-d fraction is then analysed by HPLC-MS analysis for the quantitative analysis of β-lactoglobulins and α-lactoalbumin. The next step is to quantitatively measure a set of bioactive proteins selected from the bovine colostrum data bank on the basis of their claimed health benefits. The enzymatic activities of lactoperoxidase and xanthine dehydrogenase/oxidase are then tested as an index of protein functionality., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Four natural N(6)-substituted adenine derivatives (cytokinins) were evaluated for the first time in vitro for they antioxidant capacity by using fluorimetric and spectrophotometric assays, i.e., the oxygen radical absorbance capacity (ORAC), trolox equivalence antioxidant capacity (TEAC) and the 2-deoxyribose degradation (2-DRA) assays. The results from the TEAC assay show that only N(6)-(4-hydroxybenzyl)adenine (p-topolin) shows an electron transfer capacity due to the presence of a phenolic moiety in the N(6)-position. The results from the ORAC test show that the antioxidant activity of N(6)-furfuryladenine (kinetin, K) is the highest up to a concentration of 1 μM, whereas at concentrations higher than 1 μM p-topolin is the most efficient antioxidant. Analysis of the kinetic data suggests that, compared to the other cytokinins, more sites of the molecular structure of p-topolin are available for the quenching of peroxyl radicals. The hydroxyl radical scavenger ability, as measured by the 2-DRA assay, showed that all tested cytokinins react in this test and that N(6)-(Δ(2)-isopentenyl)adenine is slightly more potent, probably because of the allylic methylene group present in the N(6)-isopentenyl moiety. Our data suggest that a part of the biological activity of the evaluated cytokinins is likely to be related to an intrinsic antioxidant capacity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aim: To assess the role of simple enucleation (SE) for the treatment of highly complex renal tumors., Methods: Overall, 96 Preoperative Aspects and Dimensions Used for an Anatomical (PADUA) classification score 10 to 13 renal tumors were treated with SE at our institution. All conventional perioperative variables, surgical, functional and oncological results were gathered in a prospectively maintained database. Survival curves were generated using a Kaplan-Meier method. Univariate analysis assessed the outcome differences., Results: Mean (± 1s.d.) clinical tumor diameter was 4.8 (± 1.6 cm). 70.8% of patients had ≥ cT1b stage. The PADUA score was recorded as 10, 11, 12 and 13 in 57.3%, 29.2%, 11.5%, and 2.1% of tumors respectively. Overall, 76 patients were treated with an open approach and 20 robotically. Mean warm ischemia time (WIT) was 19.2 min, and WIT greater than 25 min occurred in 14.6% of cases. Positive surgical margin (PSM) rate was 3.6% and trifecta was achieved in 64.3% of patients. Postoperative surgical complications occurred in 24% of patients, with 14.6% Clavien-Dindo grade 1-2, 8.3% grade 3, and 1% grade 4. Five-year cancer specific survival (CSS), recurrent free survival (RFS), and overall survival (OS) rates resulted 96.1%, 90.8% and 88.0%, respectively. Overall, 4.2% of patients experienced progressive disease. At follow-up, the mean decrease of eGFR from preoperative value was 13.9 ml/min. This was not significantly correlated with PADUA score (p = 0.69). The surgical approach was neither a predictor of Trifecta outcome, nor of postoperative complications, WIT > 25 min or PSM rate., Conclusions: SE is an effective treatment for highly-complex renal tumors, with a potential key role to widen the NSS (nephron sparing surgery) indications according to guidelines., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The purpose of this study was to evaluate the predictors of positive margins in one of the largest available prospective multi-institutional studies., Patients and Methods: We evaluated all patients who underwent NSS for radiologically diagnosed kidney tumors between January 2009 and December 2012 at 19 urological Italian centers (Registry of Conservative Renal Surgery [RECORd] project). Preoperative and anthropometric data, comorbidities, intraoperative and postoperative outcomes, and histological findings were analyzed. The negative and PSMs were compared according to the clinical and surgical variables. Multivariable logistic regression models were applied to analyze predictors of PSMs., Results: Eight hundred consecutive patients were evaluated. Seven hundred sixty-one (95.1%) and 39 patients (4.9%) achieved negative and PSMs, respectively. Patients with PSMs were significantly older compared with those with negative margins (median age: 66.6 vs. 61.8 years, respectively; P = .001). A higher incidence of PSMs was observed when NSS was performed for renal masses located in the upper pole (P = .001). A lower rate of PSMs was found in patients treated with simple enucleation rather than standard PN (1.6% vs. 7.4%, respectively; P < .0001). A greater incidence of PSMs was found in Fuhrman 3/4 tumors (11.3%; P < .0001). At multivariable analysis, age (odds ratio [OR], 1.04; P = .01), upper pole tumor location (OR, 2.85; P = .005), standard PN (OR, 3.45; P = .004), and Fuhrman 3-4 nuclear grade (OR, 4.81; P = .001) were found to be independent predictors of PSMs., Conclusion: In our multi-institutional report, young age, simple enucleation, middle or lower tumor location, and low-grade tumor were demonstrated to be independent predictors of negative SMs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: In recent years, new-onset hypothyroidism was extensively reported in patients receiving sunitinib for malignancy. Effects of sunitinib on serum lipids are not described, however a hyperlipidemic state is commonly observed in hypothyroid patients. Here we report about the incidence and severity of hypercholesterolemia and hypertriglyceridemia in a cohort of patients receiving sunitinib for metastatic renal cell carcinoma., Patients and Methods: Thyroid function tests, serum triglycerides, and cholesterol were prospectively evaluated in 39 consecutive metastatic renal cell carcinoma patients, who were receiving sunitinib as a first-line treatment. Incidence of hyperlipidemia, thyroid function impairment, and their possible relationship were investigated., Results: Thyroid function tests, serum cholesterol, and triglycerides were assessed at baseline and before the beginning of each sunitinib cycle. During treatment, median triglyceride levels increased up to 271.3 mg/dL, and median cholesterol increased up to 234.7 mg/dL (+113% and +22%, respectively). A hyperlipidemic state developed in 27 patients (69.2%) within a mean time of 1.8 six-week cycles (range, 1-5 cycles) and persisted during treatment. Hypothyroidism was observed in 20 patients (51.2%) and usually developed within 2.3 cycles. Because hypothyroidism and hyperlipidemia developed at different time points of treatment and among different patients, our results failed to demonstrate a correlation between these adverse events., Conclusion: New-onset hyperlipidemia was observed in an increased percentage of patients taking sunitinib. The mechanism of this side effect is still unclear. We recommend careful monitoring of serum lipid levels during sunitinib administration to recognize possible consequences, especially on cardiovascular health., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Nephron-sparing surgery (NSS) has become the standard of care for the surgical management of small and clinically localized renal cell carcinoma (RCC). The conservative management of those RCCs is increasing over time. Aim of this study was to report a snapshot of the clinical, perioperative and oncological results after NSS for RCC in Italy., Material and Methods: We evaluated all patients who underwent conservative surgical treatment for renal tumours between January 2009 and December 2012 at 19 urological Italian Centers (RECORd project). Perioperative, radiological and histopathological data were recorded. Surgical eras (2009 vs 2012 and year periods 2009-2010 vs 2011-2012) were compared., Results: Globally, 983 patients were evaluated. More recently, patients undergoing NSS were found to be significantly younger (p = 0.05) than those surgically treated in the first study period, with a significantly higher rate of NSS with relative and imperative indication (p < 0.001). More recently, a higher percentage of procedures for cT1b or cT2 renal tumours was observed (p = 0.02). Utilization rate of open partial nephrectomy (OPN) constantly decreased during years, laparoscopic partial nephrectomy (LPN) remained almost constant while robot-assisted partial nephrectomy (RAPN) increased. The rate of clampless NSS constantly increased over time. The use of at least one haemostatic agent has been significantly more adopted in the most recent surgical era (p < 0.001)., Conclusions: The utilization rate of NSS in Italy is increasing, even in elective and more complex cases. RAPN has been progressively adopted, as well as the intraoperative utilization of haemostatic agents and the rate of clampless procedures., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction/background: The prediction of histology of SRM could be essential for their management. The RNN is a statistical tool designed to predict malignancy or high grading of enhancing renal masses. In this study we aimed to perform an external validation of the RNN in a cohort of patients who received a PN for SRM., Materials and Methods: This was a multicentric study in which the data of 506 consecutive patients who received a PN for cT1a SRM between January 2010 and January 2013 were analyzed. For each patient, the probabilities of malignancy and aggressiveness were estimated preoperatively using the RNN. The performance of the RNN was evaluated according to receiver operating characteristic (ROC) curve, calibration plot, and decision curve analyses., Results: The area under the ROC curve for malignancy was 0.57 (95% confidence interval [CI], 0.51-0.63; P = .031). The calibration plot showed that the predicted probability of malignancy had a bad concordance with observed frequency (Brier score = 0.17; 95% CI, 0.15-0.19). Decision curve analysis confirmed a poor clinical benefit from use of the system. The estimated area under the ROC curve for high-grade prediction was 0.57 (95% CI, 0.49-0.66; P = .064). The calibration plot evidenced a bad concordance (Brier score = 0.15; 95% CI, 0.13-0.17). Decision curve analysis showed the lack of a remarkable clinical usefulness of the RNN when predicting aggressiveness., Conclusions: The RNN cannot accurately predict histology in the setting of cT1a SRM amenable to PN., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objectives: To compare simple enucleation (SE) and standard partial nephrectomy (SPN) in terms of surgical results in a multicenter dataset (RECORd Project)., Materials and Methods: patients treated with nephron sparing surgery (NSS) for clinical T1 renal tumors between January 2009 and January 2011 were evaluated. Overall, 198 patients who underwent SE were retrospectively matched to 198 patients who underwent SPN. The SPN and SE groups were compared regarding intraoperative, early post-operative and pathologic outcome variables. Multivariable analysis was applied to analyze predictors of positive surgical margin (PSM) status., Results: SE was associated with similar WIT (18 vs 17.8 min), lower intraoperative blood loss (177 vs 221 cc, p = 0.02) and shorter operative time (121 vs 147 min; p < 0.0001). Surgical approach (laparoscopic vs. open), tumor size and type of indication (elective/relative vs absolute) were associated with WIT >20 min. The incidence of PSM was significantly lower in patients treated with SE (1.4% vs 6.9%; p = 0.02). At multivariable analysis, PSM was related to the surgical technique, with a 4.7-fold increased risk of PSM for SPN compared to SE. The incidence of overall, medical and surgical complications was similar between SE and SPN., Conclusions: Type of NSS technique (SE vs SPN) adopted has a negligible impact on WIT and postoperative morbidity but SE seems protective against PSM occurrence., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: To evaluate the pathological characteristics of peritumoral capsule (PC) and the prognostic effect of capsule penetration on tumor recurrence in patients treated with tumor enucleation for clinically intracapsular renal cell carcinomas (RCCs)., Methods and Materials: PC status was analyzed in 304 consecutive patients with single intracapsular RCC. Degree and side of capsule penetration if present were evaluated. Mean (median, range) follow-up was 49 months (46, 25-69). Local recurrence rate, progression-free survival (PFS), and cancer-specific survival were the main outcomes. Statistical analyses included the Kaplan-Meier method, log-rank test, and univariate and multivariate Cox regression models., Results: Overall, 51% of RCCs had intact PC and free from neoplastic invasion (PC-), 34.9% had capsular penetration on the parenchymal side (PCK), and 14.1% had tumor invasion on the perirenal fat tissue side (PCF). None of the patients had positive surgical margins. The 5-year PFS rates for tumors PC-, PCK, and PCF were 97.5%, 96.7%, and 77.1%, respectively (P<0.0001). The multivariate Cox model showed PCF to be the sole significant independent predictor of PFS, whereas patients who had PCK did not present a significant increased risk in developing recurrence., Conclusions: Tumor enucleation is an oncologically safe nephron-sparing surgery technique. PCF is a significant and independent predictor of tumor recurrence in patients with clinically intracapsular RCCs scheduled for nephron-sparing surgery. PCK does not predict the risk of recurrence., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Our current knowledge of the occurrence of proteins covalently modified by reactive carbonyl species (RCS) generated by lipid peroxidation indicates their involvement as pathogenic factors associated with several chronic degenerative diseases. Proteomics and mass spectrometry (MS) in the last decade have played a fundamental role in this context, allowing the demonstration of the formation of RCS-protein adducts in vitro and in vivo under different experimental conditions. In conjunction with functional and computational studies, MS has been widely applied in vitro to study the stoichiometry of the protein-RCS adduct formation, and, by identifying the site(s) of modification, to elucidate the molecular mechanisms of protein carbonylation and the physiologic impact of such modification on protein function. This review will provide an update of the MS methods commonly used in detecting and characterizing protein modification by RCS generated by lipid peroxidation, among which 4-hydroxy-trans-2-nonenal and acrolein represent the most studied and cytotoxic compounds. Research in this field, employing state-of-the-art MS, is rapidly and continuously evolving, owing also to the development of suitable derivatization and enrichment procedures enabling the improve MS detectability of RCS-protein adducts in complex biological matrices. By considering the emerging role of RCS in several human diseases, unequivocal analytical approaches by MS are needed to provide levels of intermediate diagnostic biomarkers for human diseases. This review focuses also on the different MS-based approaches so far developed for RCS-protein adduct quantification. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: To evaluate the correlation between the side of positive biopsy (Bx) and the risk of lymph-node metastases (LNMs) on each side and to quantify the risk of contralateral LNMs in patients with unilateral positive biopsy., Methods: We analyzed the outcomes of 1599 patients with complete data regarding the sides of positive Bx and LN (lymph-node). By dividing each prostate into two separate sides, we assessed the accuracy of the side-specific Bx details in determining the side of positive nodes; the area under the receiver-operating characteristic (ROC) (AUCs) was used. For patients with unilateral positive Bx, we assessed the risk of homolateral and contralateral LNMs according to the number of total Bx taken and the preoperative risk of LN invasion., Results: Considering the 3198 prostate sides, there was a strict correlation between the side of positive Bx and the side of LNMs. The ratio of positive/total Bx was more informative than the number of positive core. The AUC for ipsilateral LNMs was significantly higher than that for contralateral LNMs (P = 0.039). In the 805 patients with unilateral positive Bx, the percentage of contralateral LNMs was >30% even considering a more meticulous biopsy scheme and increased in the patients at a higher clinical risk for LN invasion., Conclusion: PCa preferentially metastasizes to ipsilateral LNs but >30% of contralateral LNMs are present. A unilateral LN dissection that is limited to the tumor-bearing side of the gland should not be recommended because of the substantial risk of missing contralateral metastases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Allergy towards wide spectrum antibiotics such as amoxicillin (AX) is a major health problem. Protein haptenation by covalent conjugation of AX is considered a key process for the allergic response. However, the nature of the proteins involved has not been completely elucidated. Human serum albumin (HSA) is the most abundant protein in plasma and is considered a major target for haptenation by drugs, including β-lactam antibiotics. Here we report a procedure for immunological detection of AX-protein adducts with antibodies recognizing the lateral chain of the AX molecule. With this approach we detected human serum proteins modified by AX in vitro and identified HSA, transferrin and immunoglobulins heavy and light chains as prominent AX-modified proteins. Since HSA was the major AX target, we characterized AX-HSA interaction using high resolution LTQ orbitrap MS. At 0.5mg/mL AX, we detected one main AX-HSA adduct involving residues Lys 190, 199 or 541, whereas higher AX concentrations elicited a more extensive modification. In molecular modeling studies Lys190 and Lys 199 were found the most reactive residues towards AX, with surrounding residues favoring adduct formation. These findings provide novel tools and insight for the study of protein haptenation and the mechanisms involved in AX-elicited allergic reactions., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Several studies have reported unexpected rates of hypothyroidism in patients treated with sunitinib. The biochemical basis of this impairment is unknown. A relationship between hypothyroidism and improved outcome has been suggested in some cancer patients. Here we describe the incidence of newly onset hypothyroidism and its relationship with progression-free survival in metastatic renal cell carcinoma patients undergoing sunitinib treatment at our institution., Patients and Methods: Between July 2007 and June 2009, 22 patients received a first line sunitinib for metastatic renal cell carcinoma. Thyroid function tests were prospectively evaluated as routine laboratory assessment in every patient, at baseline and on the first and last day of every ON and OFF sunitinib period., Results: The median duration of treatment was 39.5 weeks. During sunitinib therapy, 13 patients (59.1%) showed at least one elevated TSH level. No reductions of TSH below normal ranges were observed. L-thyroxine replacement therapy was required in 2 patients. Based on thyroid function, median progression-free survival was 8.55 months for hypothyroid compared with 7.03 months for euthyroid patients (P < 0.05)., Conclusion: Patients administered sunitinib have an high incidence of hypothyroidism. The improved outcome of hypothyroid patients suggests an important relationship between sunitinib and this uncommon side effect., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A fully automated, non invasive, rapid and high-throughput method for the direct determination of sarcosine and N-ethylglycine in urine and urinary sediments using hexyl chloroformate derivatization followed by direct immersion solid-phase micro extraction and fast gas chromatography-mass spectrometric analysis was developed and validated. The use of a new ionic liquid narrow bore column, as well as the automation and miniaturization of the preparation procedure by a customized configuration of the utilized XYZ robotic system, allowed a friendly use of the GC apparatus achieving a quantitation limit of 0.06 μg L(-1) for sarcosine, good repeatability with CV always lower than 7% and reduced analysis times useful for point-of-care testing. The method was then applied for the analysis of 56 samples of urine and urinary sediments in healthy subjects, in those with benign prostatic hypertrophy and in patients with clinically localized prostate cancer. The results obtained showed that the medians of sarcosine/creatinine in urine were 103, 137 and 267 μg g(-1) respectively, thus assessing the potential use of sarcosine as urinary biomarker for prostate cancer detection. The highest values of sensitivity (79%) and specificity (87%) were obtained in correspondence of a cut-off value of 179 μg sarcosine(g creatinine)(-1), thus by using this cut-off threshold, sarcosine was significantly associated with the presence of cancer (p<0.0001). Finally, ROC analyses proved that the discrimination between clinically localized prostate cancer and patients without evidence of tumor is significantly correlated with sarcosine., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Methylprednisolone (MP) released by poly(d,l-lactide-co-glycolide) microspheres (PLGA MS) was monitored in plasma after intra-articular (i.a.) administration into rat joint. A validated LC-ESI-MS/MS method was used to quantify the plasmatic concentrations of MP. The calculated pharmacokinetic parameters were compared to those obtained after the i.a. administration of a commercially available suspension of MP acetate (MPA). Different pharmacokinetic profiles were observed in the two formulations, and a lower peak level (C(max) = 13.7 ± 4.3 ng · mL(-1)) and AUC(0-72 h) (198 ± 45 ng · mL(-1) · h) were observed for MP-PLGA MS than MPA (C(max) = 18.4 ± 2.7 ng · mL(-1)) and AUC(0-72 h) (943 ± 249 ng · mL(-1) · h). The administration of MP-PLGA MS resulted in a rapid increase in the MP concentration at 30 min, with a t(max) at 0.8 ± 0.3 h. Instead, for the MPA suspension the t(max) was 32.0 ± 13.9 h. These differences were indirectly confirmed by the evaluation of the extra-articular effects, namely, carrageenan-induced paw edema, since MP-PLGA MS showed a lower anti-inflammatory activity than MPA., (Copyright © 2011 Wiley-Liss, Inc.)

An integrated approach based on high resolution MS analysis (orbitrap), database (db) searching and MS/MS fragmentation prediction for the rapid identification of plant phenols is reported. The approach was firstly validated by using a mixture of phenolic standards (phenolic acids, flavones, flavonols, flavanones, flavanols, isoflavones). In particular, the integrated approach consists of the following steps: (1) LC-ESI-MS/MS analysis in data dependent scan mode using an orbitrap mass analyzer (resolution 60,000; positive ion-mode, ESI source); (2) searching the experimental monoisotopic masses (tolerance 1 ppm) in plant phenols databases; (3) filtering the entries on the basis of the phenol class to which the unknown belongs, as determined on the basis of the UV spectrum. Final identification is achieved by matching the isotopic pattern and by MS/MS fragmentation studies. In particular, experimental MS/MS fragments are matched with those predicted by a commercially available software. The method was then successfully applied for the rapid identification of phenolics contained in an EtOH extract of Angelica keiskei., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The challenging search of ligands for the amyloidogenic protein β(2)-microglobulin led us to set up an integrated strategy that combines analytical techniques and molecular modelling. Using a chemical library composed of 90 sulphonated molecules and a novel MS screening approach, we initially single out a few new binders. To check for anti-amyloid activity, the best hit obtained was thoroughly studied by docking analysis, affinity and refolding experiments by capillary electrophoresis and in vitro fibrillogenesis Thioflavin T test. Correlative analysis of the overall results obtained from the MS screening led to develop an equation able to identify the key factors of the affinity for β(2)-microglobulin and to predict the affinity for novel derivatives. The proposed equation was then used for a virtual screening of a large compound database. Studies on the new hit thus retrieved confirm the predictive potential of both the equation on affinity and of docking analysis on anti-amyloid activity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Glutathionylated hemoglobin (Hb-SSG) is now recognized as a promising biomarker of systemic oxidative stress. Aim of this study is to gain a mechanistic insight into its formation. The ability of GSSG to form Hb-SSG through a thiol-disulfide exchange mechanism was firstly examined. For this purpose, GSSG (ranging from 0.23 to 230micromol/g Hb, 15microM-15mM final concentrations) was incubated with 1mM Hb and the relative content of Hb-SSG determined by direct infusion mass spectrometry (Orbitrap as analyzer). No detectable Hb-SSG was observed at a GSSG concentration range found in physiopathological conditions (0.13-0.23micromol/g Hb). To reach a detectable Hb-SSG signal, the GSSG concentration was raised to 2.3micromol/g Hb (0.5% relative abundance). The relative content of Hb-GSSG dose-dependently increased to 6% and 11% at 77 and 153micromol/g Hb, respectively. The second step was to demonstrate whether Hb-SSG is formed through a sulfenic acid intermediate, a well-recognized mechanism of S-protein glutathionylation. Cys beta93 sulfenic acid was found to be formed by oxidizing Hb with 1mM H(2)O(2), as demonstrated by direct infusion and LC-ESI-MS/MS experiments and using dimedone as derivatazing agent. When H(2)O(2)-treated Hb was incubated with physiological concentrations of GSH (9micromol/g Hb), the corresponding Hb-SSG form was detected, reaching 15% of relative abundance. In summary, we here demonstrate that Hb glutathionylation can occur through a Cys sulfenic acid intermediate which is formed in oxidizing conditions. Hb glutathionylation is also mediated by a thiol-disulfide transfer mechanism, but this requires a concentration of GSSG which is far to be achieved in physiopathological conditions.

Aims: To evaluate the role of nephron-sparing surgery (NSS) compared to radical nephrectomy (RN) for treating multiple ipsilateral renal tumors., Methods: We retrospectively reviewed the clinical and pathological data of 960 patients who had surgery for pathologically confirmed RCC between 1986 and 2006. Thirty-four patients were diagnosed as having at least one ipsilateral smaller solid lesion associated with the primary RCC: 22 had RN while 12 had NSS for tumor enucleation., Results: All patients who had NSS had tumors confined within the kidney, as did 82% of patients treated with RN. The sole presence of concomitant accompanying benign histology to the primary RCC was diagnosed in 20% of patients. The mean (median, range) follow-up for patients treated with RN and NSS was 69 (58, 12-214) and 58 (44, 12-151) months. Tumor stage was significantly associated with tumor-specific survival (TSS) in the RN group (p<0.001). None of the patients who had tumor enucleation had positive surgical margins. Two patients recurred locally after NSS, elsewhere in the kidney, resulting in a crude ipsilateral recurrence rate of 17%. The analysis of TSS for patients with multiple ipsilateral tumors with a pT1 primary lesion showed no statistically significant differences between patients who had RN or NSS. Two patients had contralateral recurrence, resulting in a crude rate of 6%., Conclusions: For patients with multiple ipsilateral renal tumors, 20% of the satellite lesions are benign and 6% develop a contralateral metachronous recurrence. We also observed similar TSS for patients treated with NSS and RN.

Up to the present, only one major allergen had been univocally identified by chemical analysis (N-terminal sequencing) in the salt-extractable (cytoplasmic) protein fraction of maize kernels (Zea mays): the lipid transfer protein (Pastorello et al., Allergy Clin. Immunol. 2000;106:744-751). In the present study, two-dimensional maps of kernel flour have been set up, the proteins transferred to nitrocellulose membranes and confronted with sera of various patients allergic to maize proteins. Via spot excision and Orbitrap mass analysis, the following new allergens have been identified: vicilin, globulin-2, 50 kDa gamma-zein, endo-chitinase, thioredoxin and trypsin inhibitor. Vicilin was found to be composed of a string of six spots, all of them allergenic; also globulin-2 was composed of a string of five spots, exhibiting equivalent allergenicity. The 50 kDa gamma-zein, found here in the maize flour soluble fraction, is identical to the 50 kDa allergen reported by Pasini et al. (Allergy 2002; 57:98-106), but present in the insoluble fraction and solubilized via -S-S- reducing agents. However, the form here described might be a truncated species, since it exhibits an apparent Mr, in SDS-PAGE, of ca. 35 kDa. The homology of three of them (vicilin, globulin-2 and thioredoxin) with other vegetable systems has been investigated via BLAST analysis, the ones with highest homology belonging to rice, wheat and barley. The present data add a non-negligible amount of previously unreported allergens to the scanty panorama of maize proteins.

Because of a lack of specific clonality markers, information on lineage involvement and cell of origin of acute myeloid leukemia with normal karyotype (AML-NK), is missing. Because Nucleophosmin (NPM) gene is frequently mutated in AML-NK and causes aberrant NPM cytoplasmic localization (NPMc+), it was used as an AML lineage clonality marker. Clonal NPM exon 12 mutations were detected in myeloid, monocytic, erythroid, and megakaryocytic cells but not in fibroblasts or endothelia that were laser-microdissected from 3 patients with NPMc+ AML. Aberrant cytoplasmic expression of mutated NPM proteins was identified with anti-NPM antibodies in 2 or more myeloid hemopoietic cell lineages in 99 (61.5%) of 161 of NPMc+ AML paraffin-embedded bone marrow biopsies; lymphoid involvement was excluded in 3 investigated cases. These findings suggest that NPMc+ AML derives from either a common myeloid or earlier progenitor. Immunohistochemical studies show that varying combinations and ratios of NPMc+ leukemic cells from distinct lineages are responsible for heterogeneity within each French-American-British (FAB) classification type and for NPMc+ AML falling into different FAB categories. These findings question the value of FAB criteria in subdividing the WHO category of "AML not otherwise characterized" and suggest that, for clinical use, NPMc+ AML be provisionally regarded as a separate AML with prognostic significance.

A sensitive, selective, specific and rapid liquid chromatographic-electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination in skeletal muscle of the Michael adducts between 4-hydroxy-trans-2-nonenal (HNE), one of the most reactive lipid peroxidation-driven unsaturated aldehyde, and glutathione (GSH) and the endogenous histidine-containing dipeptides carnosine (CAR) and anserine (ANS), with the final aim to use conjugated adducts as specific and unequivocal markers of lipid peroxidation. Samples (skeletal muscle homogenates from male rats) were prepared by protein precipitation with 1 vol. of a HClO(4) solution (4.2%; w/v) containing H-Tyr-His-OH as internal standard. The supernatant, diluted (1:1, v/v) in mobile phase, was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9:1:0.01, v/v/v) at a flow rate of 0.2 ml/min, with a run time of 12 min. Detection was on a triple quadrupole mass spectrometer equipped with an ESI interface operating in positive ionization mode. The acquisitions were in multiple reaction monitoring (MRM) mode using the following precursor-->product ion combinations: H-Tyr-His-OH (IS): m/z 319.2--> 156.5+301.6; GS-HNE: m/z 464.3--> 179.1+308.0; CAR-HNE: m/z 383.1--> 110.1+266.6; ANS-HNE: m/z 397.2--> 109.1+126.1. The method was validated over the concentration ranges 1.5-90 (GS-HNE) and 0.4-40 (CAR-HNE, ANS-HNE) nmoles/g wet tissue, and the LLOQ were 1.25 and 0.33 pmoles injected respectively. The intra- and inter-day precisions (CV%) were <7.38% (

A saturated nitric oxide (NO) solution (1.88 mM) infused i.v. in the anesthetized pig at a dose of 68 nmol/kg/min for 24 min resulted in a time-dependent increase of nitrosylhemoglobin [HbFe(II)NO] as determined by electron spin resonance (ESR), reaching a C(max) of 7.99 +/- 0.42 microM at the end of the infusion, compared to 1.13 +/- 0.42 microM before (p < 0.01). This indicates that NO i.v. is efficiently bioconserved as HbFe(II)NO (approximately 34% of the NO dose) and to a greater extent than by the oxidative pathway (approximately 24% of the NO dose), as determined by measuring plasma nitrites/nitrates (chemiluminescence) and Met-Hb (ESR analysis). When the NO infusion was stopped, HbFe(II)NO declined with a t(1/2) of 15 min, indicating that it is a stable storage form of NO, able to deliver NO distally to the site of administration. No significant differences were observed in systemic and pulmonary vascular resistances during and after NO infusion, but PO(2) showed a significant decrease 15 and 30 min after the infusion. Thus, in normoxic/physiological conditions, HbFe(II)NO does not induce significant NO-dependent vasorelaxation.

The metabolic fate of nitric oxide (NO) released from nitroaspirin, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX 4016), the lead compound of a new class of NO-releasing non steroidal anti-inflammatory drugs (NO-NSAIDs), has been studied in the rat following p.o. and i.p. administration of 100 mg/kg, by monitoring in plasma the bioactive storage forms of NO (S-nitrosothiols, RS-NO) and its oxidation products (nitrites/nitrates, NOx) by a chemiluminescent assay. In parallel, plasma was analyzed for unchanged drug and metabolites by reverse-phase HPLC. In orally treated rats, no unchanged drug is observed in the 0-24 h interval post-dosing, but only salicylic acid (SA), NOx and RS-NO. The time-course of SA formation parallels that of plasma NOx (plateau after 6 h). Nitrosothiols in plasma are detectable at 1 h, peak at 4 h post-administration, and decline thereafter. The results relative to i.p. administration show a more pronounced and rapid NO delivery (peak of both NOx and RS-NO at 1 h and plateau between 1 and 2 h), still coincident with the peak of SA, and the presence in plasma of NCX 4015 (a metabolite of NCX 4016 which still bears the nitrate function). In myocardial tissue from p.o. treated rats, no drug or metabolites were ever detected, and the NOx levels were always in the range of the controls. Conversely, following i.p. treatment, we observed a rapid compartmentalization within the heart of the unchanged drug, which rapidly disappears in favour of its breakdown products NCX 4015 and SA, with a concomitant rise in myocardial NOx levels up to 2 h. To check the stability of NCX 4016 in the acidic gastric milieu and to explain the different distribution of the drug following p.o. or i.p. administration, the gastric content of the orally-treated animals at different post-dosing times was analysed by HPLC. The unchanged drug was detected up to 8 h post-dosing (levels slowly decreased with time), and the only metabolite to be detected was the O-deacetylated derivative (NCX 4023), which was present in low concentrations up to 4 h post-dosing. This indicates that NCX 4016 does not undergo biotransformation in the upper part of gastrointestinal tract (no direct release of NO in this district) and that the stomach acts as a reservoir for the drug.