Immunoprecipitation of Cullin-Ring Ligases (CRLs) in Arabidopsis thaliana Seedlings.

CRL (Cullin-Ring ubiquitin ligases) are the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-Ring Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: (1) a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure.

Ruptured Proximal Anterior Cerebral Artery Aneurysm Treated with Flow Diverter.

AIM: To discuss the use of flow modulation in treating ruptured aneurysms of the proximal segment of the anterior cerebral artery (A1 aneurysms). A1 aneurysms are rare, constituting approximately 1% of all intracranial aneurysms. CASE REPORT: We report a left A1 aneurysm with a wide neck and small sac (3 × 1.8 mm). In order to treat the lesion, a flow diverter (4 × 12?18 mm, FRED, Microvention) was placed from M1 to the proximal end of the paraophthalmic internal carotid artery, without directly covering the neck of the aneurysm. No procedural bleeding occurred. During stent deployment, abciximab was infused. A day after the procedure, double antiplatelet therapy was initiated for 1 month, followed by single antiplatelet therapy for another 3 months. Due to the aneurysm morphology, we opted for a competitive flow diversion, covering the parent artery origin and leaving the A1A neck uncovered. A decreased flow into the aneurysmal parent artery gradually promoted aneurysm sac thrombosis. Both digital subtraction angiography at a 12-month follow-up and computed tomography angiography 24-month follow-up confirmed the regular patency of the stent and resolution of the aneurysm. In addition, the competitive modulation of flow in the ipsilateral anterior cerebral artery results in the narrowing of the vessel. CONCLUSION: A1 aneurysm endovascular treatment is often challenging. Coiling or assisted coiling is the most frequently employed. Although flow diverter stent (FDS) is a consolidated technique for treating ruptured intracranial blister-like and dissecting aneurysms, its role in treating intracranial saccular ruptured aneurysms has to be elucidated. However, more number of case studies is needed to confirm the efficacy and safety of an FDS in treating ruptured A1 aneurysms.

The value of small bowel wall contrast enhancement after sulfur hexafluoride-filled microbubble injection to differentiate inflammatory from fibrotic strictures in patients with Crohn's disease.

We analysed the value of contrast-enhanced ultrasound to differentiate inflammatory from fibrotic ileal strictures in patients with Crohn's disease (CD). Twenty-eight patients (17 male and 11 female; mean age ± SD, 48.5 ± 17.17 years) with a biopsy-proven diagnosis of CD were included. In each patient, the terminal ileal loop was scanned by a convex-array probe (2-5 MHz) before and after sulphur hexafluoride-filled microbubble injection. The digital cine-clip registered after microbubble injection during the first-pass dynamic enhancement was quantified in gray-scale levels by a dedicated software through manually drawn regions-of-interest (ROIs) encompassing the anterior bowel wall. Time-intensity curves from patients with inflammatory and fibrotic ileal strictures, fitted according to the theoretical gamma variate curve, were compared. Inflammatory vs. fibrotic ileal strictures differed in the percentage of maximal enhancement (45.86 ± 5.32 vs. 37.33 ± 16.24%; p < 0.05) and area under the enhancement curve (1168.25 ± 437.65 vs. 570.47 ± 323.08; p < 0.05), whereas the difference in time to peak enhancement was found not significant (9.25 ± 4.21 vs. 12.01 ± 7.34 s; p > 0.05). The quantitative analysis of small bowel wall contrast enhancement after microbubble contrast agent injection may differentiate inflammatory from fibrotic ileal strictures in patients with CD.

Analysis of the impact of digital tomosynthesis on the radiological investigation of patients with suspected pulmonary lesions on chest radiography.

OBJECTIVE: To assess the impact of digital tomosynthesis (DTS) on the radiological investigation of patients with suspected pulmonary lesions on chest radiography (CXR). METHODS: Three hundred thirty-nine patients (200 male; age, 71.19 ± 11.9 years) with suspected pulmonary lesion(s) on CXR underwent DTS. Two readers prospectively analysed CXR and DTS images, and recorded their diagnostic confidence: 1 or 2 = definite or probable benign lesion or pseudolesion deserving no further diagnostic workup; 3 = indeterminate; 4 or 5 = probable or definite pulmonary lesion deserving further diagnostic workup by computed tomography (CT). Imaging follow-up by CT (n = 76 patients), CXR (n = 256) or histology (n = 7) was the reference standard. RESULTS: DTS resolved doubtful CXR findings in 256/339 (76 %) patients, while 83/339 (24 %) patients proceeded to CT. The mean interpretation time for DTS (mean ± SD, 220 ± 40 s) was higher (P < 0.05; Wilcoxon test) than for CXR (110 ± 30 s), but lower than CT (600 ± 150 s). Mean effective dose was 0.06 mSv (range 0.03-0.1 mSv) for CXR, 0.107 mSv (range 0.094-0.12 mSv) for DTS, and 3 mSv (range 2-4 mSv) for CT. CONCLUSIONS: DTS avoided the need for CT in about three-quarters of patients with a slight increase in the interpretation time and effective dose compared to CXR.

A multicenter study of the prognosis and treatment of adult brain ependymal tumors.

BACKGROUND: The current analysis of outcomes in a large series of adult patients with intracranial ependymal tumors contributes to the characterization of the primary prognostic factors and to the therapeutic management of this rare disease, for which limited information is available in the literature. METHODS: The authors analyzed data on patient and tumor characteristics, treatment, and survival in a series of 70 patients age > 17 years with pathologic diagnoses of brain ependymal tumors from 4 institutions. RESULTS: The 5- and 10-year overall survival (OS) rates (+/- standard errors) were 67% +/- 6% and 50% +/- 8%, respectively. The 5- and 10-year failure-free survival (FFS) rates were 43% +/- 7% and 24% +/- 6%, respectively. Younger age and infratentorial tumor location were associated with longer survival. Among patients with Grade 2 ependymoma (n = 51), 21 (41%) received no postsurgical treatment. These 21 patients had a 5-year OS rate of 78% +/- 10% and a 10-year OS rate of 68% +/- 13%; the 5- and 10-year FFS rates for these patients were 47% +/- 12% and 12% +/- 11%, respectively. Twenty-six patients with Grade 2 ependymoma (51%) received postoperative radiotherapy (RT). These 26 patients had a 5-year OS rate of 71% +/- 9% and a 10-year OS rate of 59% +/- 11%; the 5- and 10-year FFS rates for these patients were 54% +/- 10% and 34% +/- 10%, respectively. Among patients with Grade 2 ependymoma, neither OS nor FFS differed significantly between those who did not receive postoperative RT and those who did; however, these two groups were heterogeneous with respect to prognostic factors. On multivariate analysis, RT use exhibited a trend toward improved OS and was significantly predictive of improved FFS. CONCLUSIONS: The current analysis does not rule out the possibility that deferral of RT at the time of recurrence could have a detrimental effect on FFS or OS in patients with Grade 2 ependymoma, regardless of the degree of ablation. The role of postoperative RT for patients who undergo imaging-based macroscopic total resection remains to be addressed.