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Summary: Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein of interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split/link design, we applied it to identify protein interactions of the glucocorticoid receptor and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (NSP7) proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control that builds upon previously established control requirements in the field. Motivation: In proximity labeling proteomics, protein-protein interactions are identified by in vivo biotinylation. However, the current lack of a universally applicable negative control for differential analysis affects accurate mapping of the interactome. To bridge this gap, we conceptualized a system based on the T2A self-cleaving peptide to match expression levels between control and bait protein setups while using the same bait protein. In addition, we implemented a versatile modular cloning system to build mammalian expression vectors for, but not limited to, proximity labeling assays.

Introduction: Multiparametric MRI (mpMRI) parameters of pT3a prostate cancer have not been examined in large cohort studies. Therefore, we aimed to identify factors associated with up-staging of mpMRI cT3a in post-operative histopathological confirmation., Methods: Retrospective analysis of a prospectively maintained database of a single UK cancer centre. Only cT3a cases who underwent robotic-assisted radical prostatectomy (RARP) were included (N = 383). MRI and specimen histopathology was reviewed independently by expert uro-radiologists and uro-histopathologists, respectively. Factors included age, BMI, prostate-specific antigen (PSA) level, biopsy international society of urological pathology (ISUP) grade, Prostate Imaging Reporting & Data System (PI-RADS ® ) score, tumour size, tumour coverage of gland (%), gland weight and surgical margins were analysed as predictors of pT3a prostate cancer., Results: N = 383. Mean age 66 years (58-71), mean BMI 27.1 kg/m 2 (25.0-30.0). 314 (82.0%) cases down- unchanged or down-staged, and 69 (18.0%) cases upstaged. PSA level (P = 0.002), PI-RADS score (P < 0.001) and ISUP grade (P < 0.001) are positively associated with upstage categories. ISUP grade ≥3 (OR 5.45, CI 1.88, 9.29, P < 0.002), PI-RADS score ≥4 (OR 3.92, CI 1.88-9.29, P < 0.001) and tumour coverage (OR 1.06, CI 1.05-1.08, P < 0.001) significantly positively associated with upstaging disease, with concurrent decreased probability of downstaging (OR 0.55, 0.14, 0.44, respectively, P < 0.05). Tumour coverage was positively correlated with increasing positive surgical margins (P < 0.05). Capsular contact > 15 mm was very unlikely to be upstaged (OR 0.36, CI 0.21-0.62, P < 0.001), aligning with published results past the widely accepted significant level for extracapsular disease on MRI., Conclusion: The study has identified PSA level, ISUP, PI-RADS score, tumour volume and percentage coverage are key predictive factors in cT3a upstaging. This study uniquely shows tumour coverage percentage as a predictor of cT3a upstaging on mpMRI. ISUP is the strongest predictor, followed by PI-RADS score and tumour coverage of gland. Multi-institutional studies are needed to confirm our findings., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: To report the incidence of malignancy in gynaecological organs removed during radical cystectomy (RC)., Patients and Methods: A retrospective multicentre study of 1600 RCs at three high-volume institutions between January 2009 and March 2022 was performed. Pathological findings in gynaecological organs in female RC specimens were reviewed. Multivariable logistic regression analyses were used to identify predictors of malignant gynaecological organ involvement (GOI) at time of RC., Results: Overall, 302 females with a median (interquartile range) age of 68 (61-75) years underwent RC for clinical (c)Ta-T4 bladder cancer. In all, 56 patients (18.5%) received neoadjuvant chemotherapy. Malignant GOI was seen in 20 patients (6.6%); the most common single sites of GOI were the uterus (five patients) and vaginal wall (four), followed by cervix (one), and ovaries (one). Nine patients had involvement of more than one gynaecological organ. No females had a primary gynaecological malignancy detected incidentally at RC. Patients with GOI were more likely to have cT3/T4 stage (P < 0.001), preoperative hydronephrosis (P = 0.004), lymphovascular invasion (P = 0.002), and squamous cell carcinoma (P = 0.005) than those without GOI. On multivariable analysis, cT4 stage was an independent predictor of malignant GOI (odds ratio 88.3, 95% confidence interval 10.1-1214; P < 0.001)., Conclusion: To our knowledge, we present the largest multi-institutional study examining malignant GOI in females with bladder cancer undergoing RC. The rate of GOI at the time of RC is low and associated with higher clinical stage. In the absence of clinical or radiological evidence of sexual organ involvement, our results do not support their routine removal at the time of RC., (© 2023 BJU International.)

Background and Aims: Ketamine use as a recreational drug is becoming more popular nowadays. Ketamine-induced uropathy (KIU) is a late finding observed with long-term use of ketamine. A systematic review of Ketamine-Induced Uropathy was performed to emphasise its key clinical manifestations, mechanism of action and establish an effective treatment pathway., Methods and Results: A literature search was conducted in MEDLINE via Pubmed and Cochrane using the keywords ketamine and bladder, ketamine and uropathy, and ketamine and epidemiology. The search strategy was limited to articles published from 2000 to 2023. Both animal and human studies were included. A total of 101 papers were reviewed based on topic relevance from the title and abstracts available. While ketamine is a controlled drug in the United Kingdom (UK) and other countries, 283 ketamine-related deaths have been reported in the UK. There is no definite pathogenesis but multiple potential mechanisms that cause KIU and its related symptoms. KIU involves chronic inflammation of the bladder, ureteral wall thickening, hydronephrosis and finally, chronic renal failure. A multidisciplinary approach is paramount when managing these patients to break the vicious cycle. The mainstay of medical and surgical treatment pathways is continued abstinence to prevent symptom relapse. This review included the pathophysiology, novel medical treatments and surgical management of KIU., Conclusion: KIU is a rare but significantly disabling condition often seen among ketamine abusers. With the rising trend in drug addiction, KIU is expected to be more common. Unfortunately, it is a late complication in chronic ketamine abusers and is only partially reversible even with abstinence. This review discusses this rare entity's newer medical treatments and surgical options., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Introduction: We evaluated risk factors for biochemical recurrence (BCR) after robot-assisted radical prostatectomy (RARP) based on our department database., Material and Methods: Patients who underwent RARP between 2018 and 2020 were identified and included in our retrospective study. Patients who received neoadjuvant treatment, patients with positive lymph nodes, salvage prostatectomies, and patients with missing data were excluded. BCR was defined as PSA ≥0.2 ng/ml. Parameters that were investigated were the International Society of Urological Pathologists (ISUP) score, stage, and positive surgical margins (PSM) as they were reported in the pathology report. A subgroup analysis based on the tumour stage was performed., Results: A total of 414 patients were included in the analysis. Seventy-seven of them experienced BCR. Based on multivariable analysis, ISUP grade was a strong predictor for BCR with odds ratio (OR): 2.86 (CI: 1.49-5.65; p = 0.002), OR: 5.90 (CI: 1.81-18.6; p = 0.003), OR: 4.63 (CI: 1.79-11.9; p = 0.001) for ISUP grade 3, 4, 5, respectively. Regarding tumour stage, pT2 and pT3a did not show any significant difference in predicting BCR (p = 0.11), whereas pT3b stage was a predictor for BCR with OR: 6.2 (CI: 2.25-17.7; p < 0.001). In the subgroup analysis for 206 patients with pT2 disease, ISUP group and PSM were predictors for BCR. On the other hand, when patients with pT3 disease were inspected, the only parameter that was predictive of BCR was pT3b disease (OR: 4.68, CI: 1.71-13.6; p = 0.003). ISUP grade, the extent of T3 disease, and the extent and ISUP grade of surgical margins were not predictors of BCR., Conclusions: The most important risk factors for BCR after RARP are ISUP grade and tumour stage. In pT2 disease, PSM is a significant predictor of BCR, along with high ISUP grade. The substage pT3b can be considered a predictor of BCR in pT3 cases., Competing Interests: The authors declare no conflicts of interest., (Copyright by Polish Urological Association.)