Technical Description and Microsurgical Outcomes in Phalloplasty Using the Deep Inferior Epigastric Artery and Locoregional Veins.

BACKGROUND: Phalloplasty often requires free tissue transfer. There is ample literature describing flap-related outcomes, but the microsurgical technique used, including choice of recipient vessels, has been an overlooked yet important topic. In this study, the authors review the outcomes of their experience with the deep inferior epigastric artery and locoregional veins and outline technical modifications that occurred during the study period. METHODS: A retrospective chart analysis of patients who underwent microsurgical phalloplasty between September of 2016 and July of 2019 was performed. Variables included flap design, donor site, and recipient vessels. The outcome measures were return to the operating room for flap compromise and partial or complete flap loss. RESULTS: Forty-two phalloplasties using the deep inferior epigastric artery were identified. There were six take-backs for flap compromise, and four patients required venous revision, one of whom lost his urethral flap on postoperative day 9. There was a decrease in take-back rate from 30 percent in the first 20 patients to 0 percent in the second 22 patients in the study period. A total of 11.9 percent of patients had partial flap loss. This decreased from 15 percent to 9 percent in the two groups. CONCLUSION: After an initial learning curve, the combination of deep inferior epigastric artery, deep inferior epigastric vein, and great saphenous vein combined with specific technical modifications such as targeted coagulation of the vasa nervorum of the clitoral nerve has proven to be a reliable technique. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Masculinizing Genital Surgery: An Imaging Primer for the Radiologist.

OBJECTIVE. Masculinizing genital surgeries for transgender individuals are currently performed at only a select few centers; however, radiologists in any geographic region may be confronted with imaging studies of transgender patients. The imaging findings of internal and external genital anatomy of a transgender patient may differ substantially from the imaging findings of a cisgender patient. This article provides the surgical and anatomic basis to allow appropriate interpretation of preoperative and postoperative imaging findings. We also expand on the most common complications and associated imaging findings. CONCLUSION. As these procedures become more commonplace, radiologists will have a growing role in the care of transgender patients and will be faced with new anatomic variants and differential diagnoses. Familiarity with these anatomic variations and postoperative complications is crucial for the radiologist to provide an accurate and useful report.

Probing the Effects of Gating on the Ion Occupancy of the K+ Channel Selectivity Filter Using Two-Dimensional Infrared Spectroscopy.

The interplay between the intracellular gate and the selectivity filter underlies the structural basis for gating in potassium ion channels. Using a combination of protein semisynthesis, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we probe the ion occupancy at the S1 binding site in the constricted state of the selectivity filter of the KcsA channel when the intracellular gate is open and closed. The 2D IR spectra resolve two features, whose relative intensities depend on the state of the intracellular gate. By matching the experiment to calculated 2D IR spectra of structures predicted by MD simulations, we identify the two features as corresponding to states with S1 occupied or unoccupied by K+. We learn that S1 is >70% occupied when the intracellular gate is closed and <15% occupied when the gate is open. Comparison of MD trajectories show that opening of the intracellular gate causes a structural change in the selectivity filter, which leads to a change in the ion occupancy. This work reveals the complexity of the conformational landscape of the K+ channel selectivity filter and its dependence on the state of the intracellular gate.

Instantaneous ion configurations in the K+ ion channel selectivity filter revealed by 2D IR spectroscopy.

Potassium channels are responsible for the selective permeation of K+ ions across cell membranes. K+ ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose four K+ binding sites. Here, we report on the two-dimensional infrared (2D IR) spectra of a semisynthetic KcsA channel with site-specific heavy (13C18O) isotope labels in the selectivity filter. The ultrafast time resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by molecular dynamics simulations of structures with water separating two K+ ions in the binding sites, ruling out configurations with ions occupying adjacent sites.

Individual Ion Binding Sites in the K(+) Channel Play Distinct Roles in C-type Inactivation and in Recovery from Inactivation.

The selectivity filter of K(+) channels contains four ion binding sites (S1-S4) and serves dual functions of discriminating K(+) from Na(+) and acting as a gate during C-type inactivation. C-type inactivation is modulated by ion binding to the selectivity filter sites, but the underlying mechanism is not known. Here we evaluate how the ion binding sites in the selectivity filter of the KcsA channel participate in C-type inactivation and in recovery from inactivation. We use unnatural amide-to-ester substitutions in the protein backbone to manipulate the S1-S3 sites and a side-chain substitution to perturb the S4 site. We develop an improved semisynthetic approach for generating these amide-to-ester substitutions in the selectivity filter. Our combined electrophysiological and X-ray crystallographic analysis of the selectivity filter mutants show that the ion binding sites play specific roles during inactivation and provide insights into the structural changes at the selectivity filter during C-type inactivation.

Engineering the glutamate transporter homologue GltPh using protein semisynthesis.

Glutamate transporters catalyze the concentrative uptake of glutamate from synapses and are essential for normal synaptic function. Despite extensive investigations of glutamate transporters, the mechanisms underlying substrate recognition, ion selectivity, and the coupling of substrate and ion transport are not well-understood. Deciphering these mechanisms requires the ability to precisely engineer the transporter. In this study, we describe the semisynthesis of GltPh, an archaeal homologue of glutamate transporters. Semisynthesis allows the precise engineering of GltPh through the incorporation of unnatural amino acids and peptide backbone modifications. In the semisynthesis, the GltPh polypeptide is initially assembled from a recombinantly expressed thioester peptide and a chemically synthesized peptide using the native chemical ligation reaction followed by in vitro folding to the native state. We have developed a robust procedure for the in vitro folding of GltPh. Biochemical characterization of the semisynthetic GltPh indicates that it is similar to the native transporter. We used semisynthesis to substitute Arg397, a highly conserved residue in the substrate binding site, with the unnatural analogue, citrulline. Our studies demonstrate that Arg397 is required for high-affinity substrate binding, and on the basis of our results, we propose that Arg397 is involved in a Na+-dependent remodeling of the substrate binding site required for high-affinity Asp binding. We anticipate that the semisynthetic approach developed in this study will be extremely useful in investigating functional mechanisms in GltPh. Further, the approach developed in this study should also be applicable to other membrane transport proteins.