Methods to account for movement and flexibility in cryo-EM data processing.

Recent advances in direct electron detectors and improved CMOS cameras have been accompanied by the development of a range of software to take advantage of the data they produce. In particular they allow for the correction of two types of motion in cryo electron microscopy samples: motion correction for movements of the sample particles in the ice, and differential masking to account for heterogeneity caused by flexibility within protein complexes. Here we provide several scripts that allow users to move between RELION and standalone motion correction and centring programs. We then compare the computational cost and improvements in data quality with each program. We also describe our masking procedures to account for conformational flexibility. For the different elements of this study we have used three samples; a high symmetry virus, flexible protein complex (∼1MDa) and a relatively small protein complex (∼550kDa), to benchmark four widely available motion correction packages. Using these as test cases we demonstrate how motion correction and differential masking, as well as an additional particle re-centring protocol can improve final reconstructions when used within the RELION image-processing package.

New benzomorphan derivatives of MPCB as MOP and KOP receptor ligands.

There is considerable interest in developing KOP Opioid receptor ligands as clinically useful analgesics. Moreover, compounds with mixed KOP receptor and mu-opioid peptide (MOP) receptor agonist/antagonist properties could have a better therapeutic potential. The benzomorphan-based synthetic ligands MPCB and CCB have been shown to bind KOP receptors with high affinity and selectivity. We report here a series of compounds synthesized to perform structure-affinity relationship (SAR) studies on MPCB. The aim of this study was to optimise KOP receptor-ligand interaction and to modulate MOP receptor selectivity. In the benzylamide analogue of MPCB (compound 9) the presence of a third aromatic nucleus, at an appropriate distance and conformation with respect to aromatic pharmacophoric residues, increased KOP receptor affinity by about 6-fold compared to MPCB (Ki = 35 nM and Ki = 240 nM, respectively). Instead, compound 28 with a tertiary amino group in the nitrogen substituent displayed a comparable KOP receptor affinity (Ki = 179 nM) but also high MOP receptor affinity (Ki = 45 nM). Thus, the present study shows that in benzomorphan-based ligands the presence of different functional groups in the nitrogen substituent, ranging from a positive charged amine to an additional aromatic ring, is able to promote the correct aligment of aromatic pharmacophoric residues with MOP and KOP receptor types. Evaluation of docking simulations of compounds 9 and 28 into the KOP and MOP receptor displayed selective ligand interactions with the important amino acid residues Tyr320 (TMVII) and Trp318 (TMVII), respectively.

[Magnetic resonance with endorectal coil in the local staging of prostatic carcinoma. Comparison with histologic macrosections in 40 cases]. / La Risonanza Magnetica con bobina endorettale nella stadiazione locale del carcinoma prostatico. Confronto con macrosezioni istologiche in 40 casi.

PURPOSE: Endorectal coil MRI is widely used in the diagnostic workup of prostate cancer, but diagnostic accuracy rates reported in the literature are quite variable. We report our personal experience with endorectal coil MRI in the local staging of prostate carcinoma. MATERIAL AND METHODS: Forty consecutive patients with histologically proved prostate carcinoma were examined with endorectal coil MRI at high field strength (1.5 T). All patients underwent a sagittal T1-weighted SE location sequence (TR 400, TE 20), an axial T1-weighted SE (TR 400, TE 20), two axial T2-weighted FSE sequences (TR 3000, TE 102, ETL 8) with and without fat suppression, and a coronal T2-weighted FSE sequence (TR 3000, TE 102, ETL 8); an axial Fast Multiplanar Spoiled Gradient Recalled (FMSPGR) dynamic sequence after Gd-DTPA injection was also performed in 18 patients. MR staging of local tumor spread was done according to the current literature criteria. All patients were submitted to radical prostatectomy, and histologic macrosections on the same plane as MR images were obtained from surgical specimens. MR and histologic staging were compared to assess MR accuracy in detecting capsular infiltration, seminal vesicles and apex involvement. The diagnostic yield of Gd-DTPA was also investigated. RESULTS: MRI correctly staged 31 of 40 cases (77.5%). MR accuracy was 80% in detecting capsular infiltration (85.7% sensitivity and 73.6% specificity), 90% in seminal vesicle involvement (91.6% sensitivity, 89.2% specificity) and 72.5% in apex involvement (79.1% sensitivity, 62.5% specificity). Dynamic studies with Gd-DTPA did not improve staging accuracy in any case. DISCUSSION AND CONCLUSIONS: In agreement with most of the current literature, MRI showed moderate overall accuracy in the local staging of prostate carcinoma. Particularly, MRI had good accuracy in detecting seminal vesicle involvement but moderate sensitivity and specificity in demonstrating capsular infiltration and apex involvement. Due to its high cost, MRI should not be routinely used in prostate cancer staging but should be reserved to the patients whose clinical and serological data suggest extraprostatic tumor spread, whose preoperative demonstration could avoid noncurative surgery.