Use of pelvic model-based simulation for sacrospinous ligament fixation education in novice learners: a single-blinded randomized controlled trial.

INTRODUCTION AND HYPOTHESIS: We hypothesize that there will be improvement in a novice learners' confidence and skill level with sacrospinous ligament fixation (SSLF) following a pelvic model-based simulation. METHODS: We performed a single-blinded randomized controlled trial with obstetrics and gynecology residents who were novices at SSLF. The residents were randomly assigned to two groups. The control group received a lecture on the SSLF procedure and anatomy, whereas the intervention group received the same lecture in addition to a pelvic model-based simulation session taught by urogynecologists. The residents' knowledge of SSLF anatomy and confidence level with the procedure were measured via assessments administered before and after the educational interventions. Their technical skills were objectively assessed by one of two fellowship-trained urogynecologists who were blinded to their group allocation. RESULTS: A total of 28 residents were recruited with 14 residents in each group and equal distribution of junior and senior trainees. None of the residents had previously performed the SSLF procedure. There was no difference in anatomical knowledge between the two groups. The intervention group showed a greater increase in their average confidence score compared with the control group: 4.0 ± 1.4 (95% CI 3.1-4.8) versus 2.6 ± 1.6 (95% CI 1.7-3.4) respectively, with p = 0.02. The intervention group also showed better objective scores in specific technical skills, such as instrument handling (p < 0.001), instrument movement/motion (p < 0.001), and speed (p = 0.01). CONCLUSION: Our results demonstrate that inclusion of a pelvic model simulation significantly improves confidence and certain technical skills of novice trainees in performing SSLF.

Somatic coding mutations in human induced pluripotent stem cells.

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.

Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.

Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface.

Adaptor protein complex-2 (AP-2) and epsin-1 mediate protease-activated receptor-1 internalization via phosphorylation- and ubiquitination-dependent sorting signals.

Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by ß-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of ß-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the µ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs.

The Anatomical Distribution of the Pudendal Nerve Block Injection: A Cadaveric Study.

OBJECTIVE: The objective of this study was to assess the accuracy of commonly used injection locations of the pudendal nerve block by examining the proximity of the injected dye to the pudendal nerve in a cadaveric model. METHODS: Pudendal block injections at 4 sites were placed transvaginally on 5 cadaveric pelvises. These sites were 1 cm proximal to the ischial spine (black dye), at the ischial spine (red dye), 1 cm distal to the ischial spine (blue dye), and 2 cm lateral and 2 cm distal to the ischial spine (green dye). The cadavers were dissected via a posterior approach. RESULTS: We measured the shortest distance from the center of the dye-stained tissue to the pudendal nerve. As expected, the injections at the ischial spine (red) resulted in a distribution of dye closest to the pudendal nerve, averaging 3.0 ± 0.95 mm. Dyes at other sites were close to the nerve: 3.1 ± 1.00 mm (black), 3.6 ± 1.14 mm (blue), and 4.05 ± 1.28 mm (green). CONCLUSIONS: Regardless of the injection site, all dyes were close the pudendal nerve, indicating accuracy. We observed wide variation in the dye distribution even though all injections were performed by the same provider, implicating lack of precision. Based on our findings, we propose that the most effective injection location is at the ischial spine because it is the closest to the pudendal nerve; however, all injections were within 4 mm of the pudendal nerve, suggesting that only 1 to 2 injections may be sufficient.

Examination of potential mechanisms of amyloid-induced defects in neuronal transport.

Microtubule-based neuronal transport pathways are impaired during the progression of Alzheimer's disease and other neurodegenerative conditions. However, mechanisms leading to defects in transport remain to be determined. We quantified morphological changes in neuronal cells following treatment with fibrils and unaggregated peptides of beta-amyloid (Abeta). Abeta fibrils induce axonal and dendritic swellings indicative of impaired transport. In contrast, Abeta peptides induce a necrotic phenotype in both neurons and non-neuronal cells. We tested several popular hypotheses by which aggregated Abeta could disrupt transport. Using fluorescent polystyrene beads, we developed experimental models of physical blockage and localized release of reactive oxygen species (ROS) that reliably induce swellings. Like the beads, Abeta fibrils localize in close proximity to swellings; however, fibril internalization is not required for disrupting transport. ROS and membrane permeability are also unlikely to be responsible for fibril-mediated toxicity. Collectively, our results indicate that multiple initiating factors converge upon pathways of defective transport.

Conformation dependent monoclonal antibodies distinguish different replicating strains or conformers of prefibrillar Aß oligomers.

BACKGROUND: Age-related neurodegenerative diseases share a number of important pathological features, such as accumulation of misfolded proteins as amyloid oligomers and fibrils. Recent evidence suggests that soluble amyloid oligomers and not the insoluble amyloid fibrils may represent the primary pathological species of protein aggregates. RESULTS: We have produced several monoclonal antibodies that specifically recognize prefibrillar oligomers and do not recognize amyloid fibrils, monomer or natively folded proteins. Like the polyclonal antisera, the individual monoclonals recognize generic epitopes that do not depend on a specific linear amino acid sequence, but they display distinct preferences for different subsets of prefibrillar oligomers. Immunological analysis of a number of different prefibrillar Aß oligomer preparations show that structural polymorphisms exist in Aß prefibrillar oligomers that can be distinguished on the basis of their reactivity with monoclonal antibodies. Western blot analysis demonstrates that the conformers defined by the monoclonal antibodies have distinct size distributions, indicating that oligomer structure varies with size. The different conformational types of Aß prefibrillar oligomers can serve as they serve as templates for monomer addition, indicating that they seed the conversion of Aß monomer into more prefibrillar oligomers of the same type. CONCLUSIONS: These results indicate that distinct structural variants or conformers of prefibrillar Aß oligomers exist that are capable of seeding their own replication. These conformers may be analogous to different strains of prions.