CRISPR-Cas12a-mediated DNA clamping triggers target-strand cleavage

Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas12a is widely used for genome editing and diagnostics, so it is important to understand how RNA-guided DNA recognition activates the cleavage of the target strand (TS) following non-target-strand (NTS) cleavage. Here we used single-molecule magnetic tweezers, gel-based assays and nanopore sequencing to explore DNA unwinding and cleavage. In addition to dynamic and heterogenous R-loop formation, we also directly observed transient double-stranded DNA unwinding downstream of the 20-bp heteroduplex and, following NTS cleavage, formation of a hyperstable ‘clamped’ Cas12a–DNA intermediate necessary for TS cleavage. Annealing of a 4-nucleotide 3′ CRISPR RNA overhang to the unwound TS downstream of the heteroduplex inhibited clamping and slowed TS cleavage by ~16-fold. Alanine substitution of a conserved aromatic amino acid in the REC2 subdomain that normally caps the R-loop relieved this inhibition but favoured stabilisation of unwound states, suggesting that the REC2 subdomain regulates access of the 3′ CRISPR RNA to downstream DNA. [Image: see text

Impact of radical nephrectomy on renal functional outcome in patients with no other co-morbidity as determined by 24-h urinary creatinine clearance

Introduction: Renal cell carcinoma (RCC) accounts for 3% of all adult tumors. The mainstay of treatment of RCCs in the past has remained radical nephrectomy (RN). Studies have found a higher cumulative incidence of development of chronic renal insufficiency in patients undergoing RN for RCC. Objectives: To determine the frequency of decrease in 24-h urinary creatinine clearance (CrCl) as an indicator of functional decline after nephrectomy for RCC. Subjects and methods: A total of 103 patients of RCC undergoing RN were included in the study. Patients’ 24-h urinary CrCls were measured pre-nephrectomy and 3 months post-nephrectomy. The patients’ demographic and tumor characteristics were noted from case files. Data was analyzed by using SPSS version 15.0. Results: There were 61 (59.2%) males and 42 (40.8%) females with a mean age of 60.12 ± 8.88 years. The mean maximum tumor diameter was 8.5 ± 2.6 cm. The mean preoperative serum creatinine in the study group was 1.01 ± 0.24 mg/dl, while the mean 3-month postoperative serum creatinine was 1.29 ± 0.46 mg/dl. The mean preoperative CrCl measured in this study was 112.02 ± 6.04 ml/min/1.73 m2, while the 3-month postoperative value was 102.94 ± 14.10 ml/min/1.73 m2, a mean decrease of 9.08 ml/min/1.73 m2. The decrease in CrCl was identified in 34 (33%) patients. No association was found between the measured functional decline and the patients’ age and gender and stage of the disease. Conclusion: The results from this study show that RN is associated with a decrease in CrCl in one third of the study population. Therefore it is recommended that patients undergoing RN should be strictly monitored for occult renal failure and managed promptly to prevent serious morbidity of frank renal failure

Renal Tumors in Young Adults A Single-Center Experience From a Developing Country

Purpose: To determine the pattern and outcome of renal tumors in young adults in a large surgical series in Pakistan. Materials and Methods: We retrospectively analyzed 133 young adults (age: ≥ 16 to ≤ 40 years) with 136 renal tumors, who underwent surgical treatment for suspected renal cancer from 1994 till 2010. The clinical and pathological parameters were determined and their impact on final outcome was analyzed. Results: The mean age of the patients was 33.3 ± 6.2 years. Of 136, 121 (88.9%) renal tumors were malignant and 15 (11%) were benign. Among malignancies, 76 (62.7%) patients had stage I or II tumors, 22 (18.1%) stage III, and 23 (19%) stage IV at surgery. The overall cancer-specific survival for malignant tumors at 1, 5, and 10 years was 97%, 83%, and 83%, whereas the cancer-free survival (CFS) was 80%, 63%, and 37%, respectively. Patients with age ≤ 35 years had 1 and 5-year CFS of 83% and 71%, respectively, as compared with 76% and 49% for patients > 35 years (P = .02; odds ratio = 2.3; P = .03). Regarding tumor size, 1 and 5-year CFS for tumors ≤ 10 cm was 93% and 75%, while tumors > 10 cm showed CFS of 56% and 41%, respectively (P = .0001; odds ratio = 4.2; P = .0001). For stage I tumors, CFS at 1 and 5 years was 98% and 84%; for stage II, 82% and 63%; and for stage III, 62% and 50%, respectively. One-year survival for stage IV was 48% only (P = .0001). Conclusion: A wide heterogeneity of renal tumors is seen in young adults with delayed presentation

Primitive neuroectodermal tumor/Ewing's sarcoma in adult uro-oncology: A case series from a developing country

Peripheral primitive neuroectodermal tumor/Ewing's sarcoma (PNET/EWS) is primarily a tumor of soft tissues and bones. Primary localization of PNET/EWS in genitourinary organs is rare. No data on this localization of PNET/EWS are available in literature from Pakistan. We searched our adult uro-oncology records from 1994 till date and identified all cases of adult genitourinary and adrenal PNET/EWS diagnosed on histology and immunohistochemistry. Their case records were reviewed to obtain data on demographics, presentation, pathologic features, management and outcome. Six cases were found; all were young and had aggressive disease at presentation. Four had renal PNET/EWS. One case each of prostate and adrenal PNET/EWS was seen. Surgery and chemotherapy formed the mainstay of management. Three patients (50%) died during treatment, two were lost to follow-up and one case with renal PNET/EWS showed good initial response to chemotherapy but was later on lost to follow-up. In conclusion, PNET/EWS should be considered in the differential diagnosis of genitourinary malignant tumors in young patients. These tumors are aggressive with poor outcome