CRISPR-Cas12a-Assisted Recombineering in Bacteria.

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a-assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria.IMPORTANCE The CRISPR-Cas9 system has been widely used to facilitate genome editing in many bacteria. CRISPR-Cas12a (Cpf1), a new type of CRISPR-Cas system, allows efficient genome editing in bacteria when combined with recombineering. Cas12a and Cas9 recognize different target sites, which allows for more precise selection of the cleavage target and introduction of the desired mutation. In addition, CRISPR-Cas12a-assisted recombineering can be used for genetic manipulation of plasmids and plasmid curing. Finally, Cas12a-assisted recombineering in the generation of point mutations, deletions, insertions, and replacements in bacteria has been systematically analyzed. Taken together, our findings will guide efficient Cas12a-mediated genome editing in bacteria.

[Retropubic radical prostatectomy: 10 years' experience with 100 cases].

OBJECTIVE: To summarize the experience and lessons from 100 cases retropubic radical prostatectomy performed in the past 10 years. METHODS: From July 1999 to July 2009, we performed 100 cases of retropubic radical prostatectomy, of which 84 were followed up for 3 - 120 months. We analyzed their preoperative age, PSA level, amount of intraoperative blood transfusion, operation time, urinary continence, penile erectile function, stricture of the anastomotic stoma and Qmax. RESULTS: The mean age, PSA level, amount of intraoperative blood transfusion, operation time were 66.8 yr, 20.1 ng/ml, 585.7 ml and 198.9 min; the recovery rates of bladder control at 3, 6 and 12 months postoperatively were 65.5%, 81.7% and 92.4%, respectively. At 12 months after surgery, penile erection was restored in 19 cases (42.2%), anastomotic stoma stricture developed in 5 (5.9%), Qmax averaged 20.5 ml/min, biochemical recurrence was found in 13, and 1 died from prostate cancer. CONCLUSION: Retropubic radical prostatectomy is a desirable procedure for the treatment of local prostate cancer, in which ligation of the puboprostatic ligament and prostatic venous plexus before cutting off the ligament helps improve urinary continence, protection of the neurovascular bundle and collateral pudendal artery contributes to the recovery of penile erectile function, and proper connection of urethral and bladder mucosa can reduce anastomotic stoma stricture. Postoperative external-beam radiotherapy for those with T3a or local lymph node metastasis could decrease biochemical recurrence.

Primary malignant teratoma of the kidney: a rare case report and literature review.

Teratomas originate from pluripotent cells and can differentiate along one or more embryonic germ lines. Renal teratoma is infrequent and malignant renal teratoma is even rarer. Experience in the diagnosis and treatment of this uncommon malignancy is seriously limited. In this report, we described the case of a 64-year-old female who complained of right flank pain for 4 months. Computed tomography (CT) revealed a hypodense mass (50 mm in maximum diameter) with slow contrast enhancement and obscure boundary located in the lower pole of the right kidney. CT also showed multiple retroperitoneal lymphadenectasis. Retroperitoneal laparoscopic right radical nephrectomy along with regional lymphadenectomy was successfully performed, and postoperative pathological examination confirmed malignant teratoma of the kidney. After surgery, the patient received adjuvant chemotherapy with BEP (bleomycin, etoposide, and cisplatin) protocol. At the 6-month follow-up, pulmonary and liver metastases were discovered by CT and the patient refused any further treatment. Unfortunately, she died at 16 months postoperatively. Although primary renal malignant teratoma is extremely rare, this kind of tumor should be taken into consideration. Currently, there is no therapeutic standard consensus for this disease and the prognosis remains unclear. Early detection and surgical intervention is critical, and more research on postoperative adjuvant therapy should be performed.

A circular RNA, circSMARCA5, inhibits prostate cancer proliferative, migrative, and invasive capabilities via the miR-181b-5p/miR-17-3p-TIMP3 axis.

SMARCA5 (circSMARCA5) is involved in the occurrence of different cancers, but its role in prostate cancer carcinogenesis and metastatic transformation remains elusive. Thus, we evaluated the circSMARCA5 functional relevance in prostate cancer and its associated molecular mechanism. First, circSMARCA5 expression and function in this cancer were evaluated. To determine the miR-181b-5p/miR-17-3p target and clarify how circSMARCA5 regulates the miR-181b-5p-TIMP3 and miR-17-3p-TIMP3 axis, RNA immunoprecipitation, biotin-coupled microRNA capture, luciferase reporter, Western blot, and quantitative real-time PCR assays were employed. In primary and metastatic prostate cancer tissues, circSMARCA5 was significantly downregulated compared with normal controls. Functionally, circSMARCA5 exhibited a suppressive effect on prostate cancer cells' metastasis and growth. At the molecular level, circSMARCA5 could affect the tissue inhibitor of metalloproteinases 3 (TIMP3) expression through miR-181b-5p or miR-17-3p interactions. Moreover, lysine acetyltransferase 5 (KAT5) induced circSMARCA5 biogenesis and regulated the miR-181b-5p-TIMP3 and miR-17-3p-TIMP3 axis. These results suggested that targeting circSMARCA5-miR-181b-5p-TIMP3 and circSMARCA5-miR-17-3p-TIMP3 axis might be a novel therapeutic strategy for prostate cancer.

[The clinical observation of bipolar transurethral plasma kinetic resection of prostate for high risk benign prostate hyperplasia].

OBJECTIVE: To evaluate therapeutic effect and reliability of bipolar transurethral plasma kinetic prostatectomy (TUPKP) for high risk level benign prostatic hyperplasia (BPH). METHODS: A total of 230 cases of high risk of BPH were treated with TUPKP. Among them, 132 cases with the residual urine of 40 to 420 ml had accepted long term but inefficient medical therapy, 98 cases were suffered with repeating acute urinary retention. One hundred and seventy-three cases with the functional capacity>4 MET were performed the standard transurethral resection of the prostate (TURP), the other 57 cases with the functional capacity<4 MET were accepted the minimally invasive TURP. Among them 12 cases complicated with bladder stones accepted Ho: YAG lithotripsy priory. The international prostate symptom score (IPSS), The maximal urinary flow rate (Qmax) and residual urine of the 2 groups before and after operation were analyzed. RESULTS: There was no transurethral resection syndrome occurred in both groups. After 3 to 12 months of follow-up postoperatively, the IPSS of the two groups were reduced from (21.9+/-5.7) and (23.7+/-5.0) to (4.4+/-2.3) and (5.5+/-2.4), residual urine were reduced from (61.8+/-18.4) ml and (103.9+/-77.3) ml to (13.0+/-6.2) ml and (15.8+/-6.1) ml, respectively. The Qmax was increased from (5.7+/-3.0) ml/s and (4.8+/-2.8) ml/s to (20.9+/-6.3) ml/s and (16.8+/-3.9) ml/s, there existed significant differences (P<0.01). However the IPSS, Qmax and residual urine of the standard group had progressed more obviously than the minimally invasive TURP group (P<0.05). CONCLUSIONS: It is safe and effective to use TUPKP for treating high risk patients of BPH with classic TURP and minimally invasive TURP according to different functional capacity. When the functional capacity is more than 4 MET, the standard procedures is preferred.

AU-Rich Long 3' Untranslated Region Regulates Gene Expression in Bacteria.

3' untranslated regions (3' UTRs) and particularly long 3' UTRs have been shown to act as a new class of post-transcriptional regulatory element. We previously reported that hmsT mRNA stability is negatively regulated by the 3' UTR of hmsT in Yersinia pestis. To investigate more general effects of 3' UTRs in Y. pestis, we selected 15 genes potentially possessing long 3' UTRs with different AU content and constructed their 3' UTR deletion mutants. Deletion of AU-rich 3' UTRs increased mRNA levels, whereas deletion of 3' UTRs with normal AU content resulted in slight or no changes in the mRNA level. In addition, we found that PNPase was important for 3' UTR-mediated mRNA decay when the transcriptional terminator was Rho-dependent. Finally, we showed that ribosomes promote mRNA stability when bound to a 3' UTR. Our findings suggest that functional 3' UTRs might be broadly distributed in bacteria and their novel regulatory mechanisms require further investigation.

Pharmacokinetics of mycophenolic acid and estimation of exposure using multiple linear regression equations in Chinese renal allograft recipients.

OBJECTIVES: To investigate the pharmacokinetics of mycophenolic acid (MPA) in Chinese adult renal allograft recipients, and to generate the validated model equations for estimation of the MPA area under the plasma concentration-time curve from 0 to 12 hours (AUC(12)) with a limited sampling strategy. PATIENTS AND METHODS: The pharmacokinetics in 75 Chinese renal allograft recipients treated with mycophenolate mofetil 2 g/day in combination with cyclosporin and corticosteroids were determined. The MPA concentration was assayed by high-performance liquid chromatography at pre-dose (C(0)) and at 0.5 (C(0.5)), 1 (C(1)), 1.5 (C(1.5)), 2 (C(2)), 4 (C(4)), 6 (C(6)), 8 (C(8)), 10 (C(10)) and 12 (C(12)) hours after dosing on day 14 post-transplant. Patients were randomly divided into: (i) a model group (n = 50) to generate the model equations by multiple stepwise regression analysis for estimation of the MPA AUC by a limited sampling strategy; and (ii) a validation group (n = 25) to evaluate the predictive performance of the model equations. RESULTS: The mean MPA AUC(12) was 52.97 +/- 15.09 mg . h/L, ranging from 24.0 to 102.3 mg . h/L. The patient's age and serum albumin level had a significant impact on the MPA AUC(12). The correlation between the pre-dose MPA trough level (C(0)) and the MPA AUC(12) was poor (r(2) = 0.02, p = 0.33). Model equations 7 (MPA AUC(12) = 14.81 + 0.80 . C(0.5) + 1.56 . C(2) + 4.80 . C(4), r(2) = 0.70) and 11 (MPA AUC(12) = 11.29 + 0.51 . C(0.5) + 2.13 . C(2) + 8.15 . C(8), r(2) = 0.88) were selected for MPA AUC calculation in Chinese patients, resulting in good agreements between the estimated MPA AUC and the full MPA AUC(12), with a mean prediction error of +/-10.1 and +/-6.9 mg . h/L, respectively. CONCLUSION: In Chinese renal allograft recipients, MPA pharmacokinetics manifest substantial interindividual variability, and the MPA AUC(12) tends to be higher than that in Caucasian patients receiving the same dose of mycophenolate mofetil. Two validated model equations with three sampling timepoints are recommended for MPA AUC estimation in Chinese patients.

Life-threatening meningitis resulting from transrectal prostate biopsy.

After antibiotic prophylaxis with metronidazole and levofloxacin, a transrectal sextant biopsy was performed under the guide of transrectal ultrasonography (TRUS) for a 75-year-old suspicious patient with prostate adenocarcinoma. Although antibiotics were also given after this procedure, the patient still developed fever, anxious, agrypnia and headache. Blood cultures remained negative. Lumbar puncture was performed and was consistent with Escherichia coli bacterial meningitis.

Myxoid adrenal cortical tumor: report of four cases.

Myxoid adrenocortical neoplasms are rare. Surgical resection of the mass is the first-line therapy. Here we reported a total of four patients, aged 44­66 years, diagnosed with myxoid adrenocortical tumor. The clinical characteristics and immunohistochemical features of the tumor are discussed in the current literature.

Significance of heparanase-1 and vascular endothelial growth factor in adrenocortical carcinoma angiogenesis: potential for therapy.

The purpose of this study was to determine the correlation between human adrenocortical carcinoma (ACC) and the proteins involved in tumor angiogenesis, and to evaluate the angiogenic status of ACC. The expression of heparanase-1 (HPA-1), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor-2 (VEGFR-2) as well as microvessel density (MVD) were measured in a series of tissue samples from 44 human sporadic adrenocortical tumors by immunohistochemistry. These specimens were classified as adenomas (n = 20) and carcinomas (n = 24) according to the histological criteria defined by Weiss. A total of 22 of 24 (91.67%) malignant cases showed positive staining for HPA-1 and 3 of 20 (15%) benign cases showed positive, the difference of HPA-1 expression between ACA and ACC was statistically significant (P < 0.001). Similarly, VEGF staining was seen in 70.83% (17/24) of the malignant cases versus 25% (5/20) of the benign, the difference of VEGF expression among two groups was statistically significant (P = 0.002). VEGFR-2 expressed highly in the ACC group (79.17%, 19/24) and lowly in the benign group (25%, 5/20), the two groups had extremely significant difference (P < 0.001). Malignant cases showed higher MVD compared to benign tumors (84.70 ± 12.44 vs. 21.05 ± 8.07, P < 0.001). HPA-1 and VEGF expression were positively correlated with MVD in all specimens (r_s = 0.812, P = 0.001; r_s = 0.834, P < 0.001). In conclusion, these results suggest that angiogenesis of human ACC maybe mediated by these proteins and they could represent selective targets for the molecularly targeted treatments of ACC.

Heparanase-1 and Cyclooxygenase-2: prognostic indicators of malignancy in pheochromocytomas.

The objective of this article is to evaluate Heparanase-1 and Cyclooxygenase-2 as tissue-based markers of pheochromocytoma prognosis. Ninety-two sporadic pheochromocytoma patients with a minimum of 8-year follow-up post-diagnosis were enrolled. Slides of normal adrenal glands in nephrectomy specimens from 20 patients with benign renal tumors were as control. Heparanase-1 and Cyclooxygenase-2 expression as well as microvessel density were examined using immunohistochemistry in tissues from these patients. Positive staining for Heparanase-1 was observed in 23.68% of the benign and 77.78% of the malignant cases, whereas none of the normal adrenal controls showed positive staining. Similarly, Cyclooxygenase-2 staining was seen in 23.68% of the benign versus 83.33% of the malignant cases, and none of the normal controls appeared positive for Cyclooxygenase-2. Using both HPA-1 and Cox-2 combined, the positive predictive value of malignancy was significantly increased to 0.72, compared to about 0.45 by their own. Malignant cases showed higher microvessel density compared to benign tumors and normal controls (36.41, 21.43, and 13.36%, respectively). Heparanase-1 and Cyclooxygenase-2 may contribute to the invasive characteristics of malignant pheochromocytomas. Heparanase-1 and Cyclooxygenase-2 combined is better than their own to be used as a marker to distinguish malignant from benign pheochromocytoma.

Selective α1-adrenoceptor antagonist (controlled release tablets) in preoperative management of pheochromocytoma.

The objective of this article is to evaluate the efficacy of Doxazosin Mesylate Controlled Release Tablets for preoperative treatment of patients with pheochromocytoma. Between 2003 and 2008, 67 patients with confirmed diagnoses of pheochromocytoma were enrolled in this study. According to the drug used in preoperative management, patients were divided into two groups: Doxazosin Mesylate pretreatment group (n=36) and Phenoxybenzamine pretreatment group (n=31). Surgery was performed only in patients who met the optimal preoperative condition. The hematocrit decreased significantly (P<0.001) after antiadrenergic therapy in patients pretreated with phenoxybenzamine or doxazosin. There was no significant difference between the fluid intakes during operation in both groups. The systolic arterial pressures both before and after induction of anesthesia were all significantly higher in the doxazosin patients than in the phenoxybenzamine group (P<0.05). After tumor removed, the lowest systolic arterial pressure was significantly higher in doxazosin group than in phenoxybenzamine group (P<0.05). The fluctuation of systolic arterial pressure during operation was more stable in doxazosin group than in phenoxybenzamine group (P<0.05). Doxazosin mesylate controlled release tablet was as effective as phenoxybenzamine in preoperative volume expansion. Although phenoxybenzamine provided better arterial pressure control, patients pretreated with DOX experienced more stable perioperative hemodynamic changes, shorter preoperative management periods and more simple medication.