Blinded assessment of operative performance after fundamentals of laparoscopic surgery in gynecology training.

STUDY OBJECTIVE: To determine the pass rate for the Fundamentals of Laparoscopic Surgery (FLS) examination among senior gynecology residents and fellows and to find whether there is an association between FLS scores and previous laparoscopic experience as well as laparoscopic intraoperative (OR) skills assessment. DESIGN: Prospective cohort study (Canadian Task Force classification II-2). SETTING: Three gynecology residency training programs. PARTICIPANTS: Third- and fourth-year gynecology residents and urogynecology fellows. INTERVENTIONS: All participants participated in the FLS curriculum, written and manual skills examination, and completed a survey reporting baseline characteristics and opinions. Fourth-year residents and fellows underwent unblinded and blinded pre- and post-FLS OR assessments. Objective OR assessments of fourth-year residents after FLS were compared with those of fourth-year resident controls who were not FLS trained. MEASUREMENTS AND MAIN RESULTS: Twenty-nine participants were included. The overall pass rate was 76%. The pass rate for third- and fourth-year residents and fellows were 62%, 85%, and 100%, respectively. A trend toward improvement in OR assessments was observed for fourth-year residents and fellows for pre-FLS curriculum compared with post-FLS testing, and FLS-trained fourth-year residents compared with fourth-year resident controls; however, this did not reach statistical significance. Self-report of laparoscopic case load experience of >20 cases was the only baseline factor significantly associated with passing the FLS examination (p = .03). CONCLUSION: The FLS pass rate for senior residents and fellows was 76%, with higher pass rates associated with increasing levels of training and laparoscopic case experience.

Cyclic peptide inhibitors of staphylococcal virulence prepared by Fmoc-based thiolactone peptide synthesis.

Virulence factor production in Staphylococcus aureus is largely under the control of the accessory gene regulator (agr) quorum sensing system. There are four agr groups, all of which exhibit bacterial interference: each agr type synthesizes a cyclic autoinducing peptide (AIP) with a distinct sequence that activates its cognate AgrC receptor and inhibits activation of others. To better understand inhibitory AIP-AgrC interactions, we aimed to identify the minimal molecular determinants required to inhibit both non-cognate and cognate receptors. This minimization of the AIP pharmacophore also may have therapeutic relevance as the use of native AIPs to block virulence of non-cognate agr strains can prevent the establishment of an infection in vivo. We synthesized and evaluated the inhibitory activities of 10 AIP derivatives based on a truncated AIP analogue that inhibits all four agr types. To carry out the rapid, parallel synthesis of these peptides, we employed a new linker for Fmoc-based thioester peptide synthesis. Our results identify key structural elements that are necessary for AgrC inhibition and reveal key differences between non-cognate and cognate inhibitory requirements.

Identification of ligand specificity determinants in AgrC, the Staphylococcus aureus quorum-sensing receptor.

Activation of the agr system, a major regulator of staphylococcal virulence, is initiated by the binding of a specific autoinducing peptide (AIP) to the extracellular domain of AgrC, a classical receptor histidine protein kinase. There are four known agr specificity groups in Staphylococcus aureus, and we have previously localized the determinant of AIP receptor specificity to the C-terminal half of the AgrC sensor domain. We have now identified the specific amino acid residues that determine ligand activation specificity for agr groups I and IV, the two most closely related. Comparison of the AgrC-I and AgrC-IV sequences revealed a set of five divergent residues in the region of the second extracellular loop of the receptor that could be responsible. Accordingly, we exchanged these residues between AgrC-I and AgrC-IV and tested the resulting constructs for activation by the respective AIPs, measuring activation kinetics with a transcriptional fusion of blaZ to the principal agr promoter, P3. Exchange of all five residues caused a complete switch in receptor specificity. Replacement of two of the AgrC-IV residues by the corresponding residues in AgrC-I caused the receptor to be activated by AIP-I nearly as well as the wild type AgrC-I receptor. Replacement of two different AgrC-I residues by the corresponding AgrC-IV residues broadened receptor recognition specificity to include both AIPs. Various types of intermediate activity were observed with other replacement mutations. Preliminary characterization of the AgrC-I-AIP-I interaction suggests that ligand specificity may be sterically determined.

Hydrophobic interactions drive ligand-receptor recognition for activation and inhibition of staphylococcal quorum sensing.

Two-component systems represent the most widely used signaling paradigm in living organisms. Encoding the prototypical two-component system in Gram-positive bacteria, the staphylococcal agr (accessory gene regulator) operon uses a polytopic receptor, AgrC, activated by an autoinducing peptide (AIP), to coordinate quorum sensing with the global synthesis of virulence factors. The agr locus has undergone evolutionary divergence, resulting in the formation of several distinct inter- and intraspecies specificity groups, such that most cross-group AIP-receptor interactions are mutually inhibitory. We have exploited this natural diversity by constructing and analyzing AgrC chimeras generated by exchange of intradomain segments between receptors of different agr groups. Functional chimeras fell into three general classes: receptors with broadened specificity, receptors with tightened specificity, and receptors that lack activation specificity. Testing of these chimeric receptors against a battery of AIP analogs localized the primary ligand recognition site to the receptor distal subdomain and revealed that the AIPs bind primarily to a putative hydrophobic pocket in the receptor. This binding is mediated by a highly conserved hydrophobic patch on the AIPs and is an absolute requirement for interactions in self-activation and cross-inhibition of the receptors. It is suggested that this recognition scheme provides the fundamental basis for agr activation and interference.