Modeling Performance of Open Surgical Cases.

INTRODUCTION: Previous efforts used digital video to develop computer-generated assessments of surgical hand motion economy and fluidity of motion. This study tests how well previously trained assessment models match expert ratings of suturing and tying video clips recorded in a new operating room (OR) setting. METHODS: Enabled through computer vision of the hands, this study tests the applicability of assessments born out of benchtop simulations to in vivo suturing and tying tasks recorded in the OR. RESULTS: Compared with expert ratings, computer-generated assessments for fluidity of motion (slope = 0.83, intercept = 1.77, R2 = 0.55) performed better than motion economy (slope = 0.73, intercept = 2.04, R2 = 0.49), although 85% of ratings for both models were within ±2 of the expert response. Neither assessment performed as well in the OR as they did on the training data. Assessments were sensitive to changing hand postures, dropped ligatures, and poor tissue contact-features typically missing from training data. Computer-generated assessment of OR tasks was contingent on a clear, consistent view of both surgeon's hands. CONCLUSIONS: Computer-generated assessment may help provide formative feedback during deliberate practice, albeit with greater variability in the OR compared with benchtop simulations. Future work will benefit from expanded available bimanual video records.

Evidence That Female Urologists and Urology Trainees Tend to Underrate Surgical Skills on Self-Assessment.

BACKGROUND: Historically low, the proportion of female urology residents now exceeds 25% in recent years. Self-assessment is a widely used tool to track progress in medical education. However, the validity of its results and gender differences may influence interpretation. Simulation of surgical skills is increasingly common in modern residency training and standardizes certain objective tasks and skills. The objective of this study was to identify gender differences in self-assessment of surgeons and trainees when using simulation of surgical skills. METHODS: Medical students, residents, and attending and retired surgeons completed simple interrupted suturing. Assessment was self-rated using previously tested visual analog motion scales. Tasks were video recorded and rated by blinded expert surgeons using identical motion scales. Computer vision motion tracking software was used to objectively analyze the kinematics of surgical tasks. RESULTS: Proportion of female (n = 17) and male (n = 20) participants did not differ significantly by the level of training, P = 0.76. Five expert surgeons evaluated 84 video segments of simple interrupted suturing tasks (mean 3.0 segments per task per participant). Self-assessment correlated well overall with expert rating for motion economy (Pearson correlation coefficient 0.61, P < 0.001) and motion fluidity (0.55, P = 0.002). Women underrated their performance in accordance with mean individual difference of self-assessment and expert assessment scores (Δ SAS-EAS) for both economy of motion (mean ± SEM -1.1 ± 0.38, P = 0.01) and fluidity of motion (-1.3 ± 0.39, P < 0.01). On the same measures, men tended to rate themselves in accordance with experts (-0.16 ± 0.36, P = 0.63; -0.09 ± 0.41, P = 0.82, respectively). Δ SAS-EAS did not differ significantly on any rating scale across levels of training. Expert ratings did not differ significantly by gender for any domain. CONCLUSIONS: Female surgeons and trainees underrate some technical skills on self-assessment when compared with expert ratings, whereas male surgeon and trainee self-ratings and expert ratings were similar. Further work is needed to determine if these differences are accentuated across increasingly difficult tasks.

Quantifying surgeon maneuevers across experience levels through marker-less hand motion kinematics of simulated surgical tasks.

This paper compares clinician hand motion for common suturing tasks across a range of experience levels and tissue types. Medical students (32), residents (41), attending surgeons (10), and retirees (2) were recorded on digital video while suturing on one of: foam, pig feet, or porcine bowel. Depending on time in position, each medical student, resident, and attending participant was classified as junior or senior, yielding six experience categories. This work focuses on trends associated with increasing tenure observed from those medical students (10), residents (15), and attendings (10) who sutured on foam, and draws comparison across tissue types where pertinent. Utilizing custom software, the two-dimensional location of each of the participant's hands were automatically recorded in every video frame, producing a rich spatiotemporal feature set. While suturing on foam, increasing clinician experience was associated with conserved path length per cycle of the non-dominant hand, significantly reducing from junior medical students (mean = 73.63 cm, sd = 33.21 cm) to senior residents (mean = 46.16 cm, sd = 14.03 cm, p = 0.015), and again between senior residents and senior attendings (mean = 30.84 cm, sd = 14.51 cm, p = 0.045). Despite similar maneuver rates, attendings also accelerated less with their non-dominant hand (mean = 16.27 cm/s2, sd = 81.12 cm/s2, p = 0.002) than senior residents (mean = 24.84 cm/s2, sd = 68.29 cm/s2, p = 0.002). While tying, medical students moved their dominant hands slower (mean = 4.39 cm/s, sd = 1.73 cm/s, p = 0.033) than senior residents (mean = 6.53 cm/s, sd = 2.52 cm/s). These results suggest that increased psychomotor performance during early training manifest through faster dominant hand function, while later increases are characterized by conserving energy and efficiently distributing work between hands. Incorporating this scalable video-based motion analysis into regular formative assessment routines may enable greater quality and consistency of feedback throughout a surgical career.

Do I need to see a urologist for my vasectomy? A comparison of practice patterns between urologists and family medicine physicians.

Urologists perform the majority of vasectomies in the United States; however, family medicine physicians (FMPs) perform up to 35%. We hypothesized that differences exist in practice patterns and outcomes between urologists and FMPs. Patients who underwent a vasectomy from 2010 to 2016 were identified. Postvasectomy semen analysis (PVSA) practices were compared between urologists and FMPs, before and after release of the 2012 AUA vasectomy guidelines. From 2010 to 2016, FMPs performed 1435 (35.1%) of all vasectomies. PVSA follow-up rates were similar between the two groups (63.4% vs 64.8%, P = 0.18). Of the patients with follow-up, the median number of PVSAs obtained was 1 (range 1-6) in both groups (P = 0.22). Following the release of guidelines, fewer urologists obtained multiple PVSAs (69.8% vs 28.9% pre- and post-2012, P < 0.01). FMPs had a significant but lesser change in the use of multiple PVSAs (47.5% vs 38.4%, P < 0.01). Both groups made appropriate changes in the timing of the first PVSA, but FMPs continued to obtain PVSAs before 8 weeks (15.0% vs 6.5%, P < 0.01). FMPs had a higher rate of positive results in PVSAs obtained after 8 weeks, the earliest recommended by the AUA guidelines (4.1% vs 1.3%, P < 0.01). Significant differences in PVSA utilization between FMPs and urologists were identified and were impacted by the release of AUA guidelines in 2012. In summary, FMPs obtained multiple PVSAs more frequently and continued to obtain PVSAs prior to the 8-week recommendation, suggesting less penetration of AUA guidelines to nonurology specialties. Furthermore, FMPs had more positive results on PVSAs obtained within the recommended window.

Use of Magnetic Induction to Activate a "Touchless" Shape Memory Alloy Implantable Penile Prosthesis.

INTRODUCTION: We describe a novel physiologic penile prosthesis that uses shape memory alloy properties to mimic the transition between a flaccid and erect penis using magnetic induction instead of hydraulic pressure. AIM: To evaluate the parameters of magnetic induction to activate a shape memory alloy implantable penile prosthesis. METHODS: We prototyped an implantable penile prosthesis cylinder using temperature-tuned nickel titanium alloy tubes laser cut to specifications. The device was then tested implanted in an animal tissue model and in cadaveric tissue. Testing consisted of placing the device deactivated in its more malleable and compressed state, then activating it using an external inducer wand while measuring temperature changes that occur on the surface of the device, within the tissue, and at the skin surface. MAIN OUTCOME MEASURES: Our main outcome measures were the efficiency of activation and thermal safety of this approach. RESULTS: Using a handheld magnetic inductor, we were able to successfully activate the SMA penile prosthesis with no direct contact under 45 seconds. This handheld wand produced a magnetic field that penetrated tissue and caused the appropriate phase change within the prosthesis. Tissue temperature (middle and surface probes) in the animal tissue model increased only a few degrees Celsius during the activation process, and never exceeded 28 degrees Celsius from a baseline at room temperature ∼ 25 degrees Celsius. We encountered similar results without a notable change in tissue temperature in the cadaveric tests. The fully activated device resisted buckling forces of 2.66 kgf ± 0.045. CLINICAL IMPLICATIONS: This non-hydraulic shape memory prosthesis obviates the needs for reservoirs and pumps, and the wand-based interaction with the device may be easier to use. STRENGTH & LIMITATIONS: This technology represents a fundamental departure from the hydraulic-based penile prosthesis and has comparable mechanical properties as current-marked devices. It appears to show thermal safety in controlled environments, however real-world use would need further studies. Further optimization of prototypes needs to be done prior to human clinical trials. CONCLUSION: A shape memory penile prosthesis is a promising alternative to hydraulic-based penile prostheses and can be activated safely and efficiently using magnetic induction in our models of the human penis. Le BV, McVary KT, McKenna K, et al. Use of Magnetic Induction to Activate a "Touchless" Shape Memory Alloy Implantable Penile Prosthesis. J Sex Med 2019;16:596-601.

Using Surgeon Hand Motions to Predict Surgical Maneuvers.

OBJECTIVE: This study explores how common machine learning techniques can predict surgical maneuvers from a continuous video record of surgical benchtop simulations. BACKGROUND: Automatic computer vision recognition of surgical maneuvers (suturing, tying, and transition) could expedite video review and objective assessment of surgeries. METHOD: We recorded hand movements of 37 clinicians performing simple and running subcuticular suturing benchtop simulations, and applied three machine learning techniques (decision trees, random forests, and hidden Markov models) to classify surgical maneuvers every 2 s (60 frames) of video. RESULTS: Random forest predictions of surgical video correctly classified 74% of all video segments into suturing, tying, and transition states for a randomly selected test set. Hidden Markov model adjustments improved the random forest predictions to 79% for simple interrupted suturing on a subset of randomly selected participants. CONCLUSION: Random forest predictions aided by hidden Markov modeling provided the best prediction of surgical maneuvers. Training of models across all users improved prediction accuracy by 10% compared with a random selection of participants. APPLICATION: Marker-less video hand tracking can predict surgical maneuvers from a continuous video record with similar accuracy as robot-assisted surgical platforms, and may enable more efficient video review of surgical procedures for training and coaching.

Vasectomy Practice Patterns Among Family Medicine Physicians and Compliance With the American Urological Association 2012 Vasectomy Guidelines.

OBJECTIVE: To survey urologists and family medicine physicians (FMPs) within a single institution to determine current vasectomy practice patterns and determine compliance with 2012 American Urological Association (AUA) vasectomy guidelines. METHODS: In 2016, a single-institution survey was conducted to understand the vasectomy practice patterns among urologists and nonurologists. The survey questions and 3 clinical scenarios were designed based on the 2012 AUA vasectomy guidelines. Results of the survey were compiled between urologists and nonurologists and then compared with the guideline recommendations. RESULTS: A total of 23 FMPs and 6 urologists responded. Fewer prevasectomy counseling topics were discussed by FMPs compared with urologists. A variety of vasectomy techniques were used among FMPs. Vas deferens segments were more likely to be sent for histology by FMPs than urologists (65% vs 17%, P = .02). FMPs were more likely to send postvasectomy semen analyses earlier than urologists (P = .02) and more likely to send multiple postvasectomy semen analyses (P = .006) before forgoing alternative contraceptive methods. Regarding the clinical scenario questions, FMPs were more likely to answer discordantly from guideline recommendations compared with urologists. CONCLUSION: Significant vasectomy practice pattern heterogeneity still exists among nonurologists surveyed within our institution. The 2012 AUA vasectomy guidelines have yet to be broadly implemented within nonurology practices. Further studies are warranted to investigate national trends in nonurologist vasectomy practice patterns and determine how the guidelines can be better implemented in nonurologic practices.

How I treat priapism.

Priapism is a disorder of persistent penile erection unrelated to sexual interest or desire. This pathologic condition, specifically the ischemic variant, is often associated with devastating complications, notably erectile dysfunction. Because priapism demonstrates high prevalence in patients with hematologic disorders, most commonly sickle cell disease (SCD), there is significant concern for its sequelae in this affected population. Thus, timely diagnosis and management are critical for the prevention or at least reduction of cavernosal tissue ischemia and potential damage consequent to each episode. Current guidelines and management strategies focus primarily on reactive treatments. However, an increasing understanding of the molecular pathophysiology of SCD-associated priapism has led to the identification of new potential therapeutic targets. Future agents are being developed and explored for use in the prevention of priapism.

A contemporary analysis of Fournier gangrene using the National Surgical Quality Improvement Program.

OBJECTIVE: To determine a nationwide contemporary description of surgical Fournier gangrene (FG) and necrotizing fasciitis of the genitalia (NFG) outcomes because historically reported mortality rates for FG and NFG are based on small single-institution studies from the 1980s and the 1990s. METHODS: The National Surgical Quality Improvement Program is a risk-adjusted surgical database used by nearly 400 hospitals nationwide, which tracks preoperative, intraoperative, and 30-day postoperative clinical variables. Data are extracted from patient charts by an independent surgical clinical reviewer at each hospital. Using the National Surgical Quality Improvement Program data from 2005 to 2009, we calculated 30-day mortality rates and identified preoperative factors associated with increased mortality. RESULTS: A total of 650 patients were identified with surgery for FG or NFG. Fourteen patients with do not resuscitate orders placed preoperatively were excluded from analyses. For the remaining 636 patients, the overall 30-day mortality was 10.1% (64 of 636). Fifty-seven percent of patients (360 of 636) were men, 70% (446 of 636) were white, and 13% (81 of 636) were African American. Multivariate logistic regression indicated that increased age (odds ratio [OR], 1.041; P = .004), body mass index (OR, 1.045; P <.001), and preoperative white blood cell count (OR, 1.061; P = .001), and decreased platelet count (OR, 0.993; P <.001) were all associated with increased risk of death. CONCLUSION: We determined a surgical mortality rate for FG-NFG of 10.1%. This rate is about half of historically published estimates and similar to recent studies. The lower rate may indicate improvements in therapy. Increased age, body mass index, and white blood cell count, and decreased platelet count were all associated with an increased risk of 30-day mortality.

Suprapubic cystostomy for the management of urethral injuries during penile prosthesis implantation.

INTRODUCTION: Urethral injury is an uncommon surgical complication of penile prosthesis (PP) surgery. Conventional dogma requires abortion of the procedure if the adjacent corporal body is involved or delayed implantation to avert device infection associated with urinary extravasation. Besides the setback of the aborted surgery, this management approach also presents the possible difficulty of encountering corporal fibrosis at the time of reoperation. AIM: We report an approach using primary urethral repair and temporary suprapubic cystostomy for the management of incidental urethral injuries in a cohort of patients allowing for successful completion of unaborted PP implantation. MATERIALS AND METHODS: We performed a retrospective analysis of all patients receiving PPs from 1990 to 2014 in which incidental urethral injuries were repaired and PP implantation was completed with suprapubic cystostomy (suprapubic tube [SPT] insertion). After allowing for urethral healing and urinary diversion via SPT for 4-8 weeks, the PP was activated. MAIN OUTCOME MEASURES: Successful management was determined by the absence of perioperative complications within 6 months of implantation. RESULTS: We identified four cases, all receiving inflatable PPs, managed with temporary suprapubic cystostomy. These patients sustained urethral injuries during corporal dissection (one patient), corporal dilation (one patient), and penile straightening (two patients). All patients were managed safely and successfully. CONCLUSION: Primary urethral repair followed by temporary suprapubic cystostomy offers a surgical approach to complete PP implantation successfully in patients who sustain urethral injury complications, particularly for complex PP surgeries. Anele UA, Le BV, and Burnett AL. Suprapubic cystostomy for the management of urethral injuries during penile prosthesis implantation.

External validation of a prediction model for penile prosthesis implantation for erectile dysfunction management.

INTRODUCTION: Penile prosthesis implantation (PPI) is the definitive surgical treatment for erectile dysfunction (ED), yet it is often delayed for a variety of reasons. From commercial and Medicare claims data, we previously developed a tool for determining a patient's likelihood of eventually receiving PPI. We validated this instrument's utility by comparing cohorts receiving surgical (PPI) versus non-surgical ED management at a single institution. MATERIAL AND METHODS: The prediction model was based on a logistic regression incorporating claims data on demographics, comorbidities and ED therapy. A risk score is calculated from the model as the product of relative risks for the individual variables. The current validation was a retrospective analysis of ED patients seen at this institution from January to December 2012. Inclusion criteria included ED diagnosis and either first-time PPI or non-surgical treatment (controls). Risk scores for patients receiving PPI were compared to those of non-surgical controls. RESULTS: We established a cohort of 60 PPI patients (mean age 54.4 ± 9.5) and compared them with 120 non-PPI patients (mean age 53.4 ± 11.2 years). The median score of the PPI cohort was 5.7 (IQR 2.8-9.9) versus the non-PPI cohort's 1.8 (IQR 0.9-5.5) (p < 0.0001). The area under the receiver operator characteristic curve for predicting eventual PPI was 0.72 (95% CI, 0.64-0.79) (p < 0.0001). CONCLUSION: The prediction model risk-stratified men who ultimately underwent PPI compared to non-surgically managed controls. This external validation study suggests that the prediction model may be used on an individual patient basis to support a recommendation of PPI for managing ED.

National trends in the treatment of penile prosthesis infections by explantation alone vs. immediate salvage and reimplantation.

INTRODUCTION: A penile prosthesis infection (PPI) is either treated with explantation of the prosthesis with a possible delayed reimplantation or a salvage procedure with an immediate reimplantation of the prosthesis. AIM: We used a large, all-payer national database to investigate the use of the salvage procedure in the setting of PPI. METHODS: The study used years 2000-2009 of the Nationwide Inpatient Sample to identify PPIs treated with immediate salvage or explantation alone. Admissions were then stratified by various parameters to compare differences in the salvage rates. MAIN OUTCOME MEASURES: Salvage Rate of Penile Prosthesis infection. RESULTS: A total of 1,557 patients were treated with an explantation only (82.7%) or salvage (17.3%) for PPI, a proportion that remained stable over the study period. The patients treated with salvage were younger (60.4 vs. 65.1 years), more likely to be discharged home (87.3% vs. 61.9%), and were less likely to have a severe presentation (7.2% vs. 31.6%) than those who were explanted only (P < 0.001). These factors were confirmed on multivariate regression analysis. The regression also revealed that treatment at rural hospitals had lower odds of salvage than treatment at urban teaching hospitals. Race, comorbid diabetes, and insurance status did not independently affect the salvage rate. There was no significant difference in total hospital charges between groups. CONCLUSIONS: Salvage rates have remained low over the past decade. Our study elucidated several factors decreasing the chances of salvage after PPI including age, severity of presentation, and hospital setting.

Relating economic conditions to vasectomy and vasectomy reversal frequencies: a multi-institutional study.

PURPOSE: It was theorized that the use of permanent contraceptive methods may vary with economic conditions. We evaluated the relationship between vasectomy/vasectomy reversal frequencies at several large referral centers and national economic indicators during 2 recessions spanning 2001 to 2011. MATERIALS AND METHODS: We performed an institutional review board approved, retrospective chart review to identify the number of vasectomies/vasectomy reversals per month at several large referral centers from January 2001 to July 2011. The rates of these procedures were pooled, correlated with national economic data and analyzed in a multivariate linear regression model. RESULTS: A total of 4,599 vasectomies and 1,549 vasectomy reversals were performed at our institutions during the study period. The number of vasectomies per month positively correlated with the unemployment rate (r=0.556, p<0.001) and personal income per capita (r=0.276, p=0.002). The number of reversals per month negatively correlated with the unemployment rate (r=-0.399, p<0.001) and personal income per capita (r=-0.305, p<0.001). Neither vasectomy nor vasectomy reversal frequency significantly correlated with the inflation rate or the S&P 500®. Regression models confirmed that the unemployment rate explained more of the variance in vasectomy/vasectomy reversal frequencies than other indicators. CONCLUSIONS: We noted a correlation between the number of vasectomies/vasectomy reversals performed at our institutions and national economic indicators. The strongest association was with the unemployment rate. This points to the importance of financial pressure on family planning decisions.

Vasectomy demographics and postvasectomy desire for future children: results from a contemporary national survey.

OBJECTIVE: To describe the longitudinal demographics and family planning attitudes of vasectomized men with the use of the National Survey for Family Growth (NSFG). DESIGN: Retrospective cohort analysis of the NSFG with the use of national projections and multivariable regressions. SETTING: In-home survey. PATIENT(S): The NSFG sampled 10,403 men aged 15-45 years from 2006 to 2010 regarding family planning attitudes. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Vasectomy and desire for children. RESULT(S): There were 3,646,339 (6.6%) vasectomized men aged 18-45 years in the U.S. On multivariable regression the following factors increased the odds of having a vasectomy: currently married (odds ratio [OR] 7.814), previously married (OR 5.865), and increased age (OR 1.122) and income (OR 1.003). The odds of having a vasectomy increased with number of children. The following factors decreased the odds of having a vasectomy: immigrant status (OR 0.186), African American (OR 0.226), Hispanic (OR 0.543), Catholic (OR 0.549), and other non-Protestant religion (OR 0.109). Surprisingly, an estimated 714,682 (19.6%) vasectomized men in the U.S. desire future children. Men practicing a religion (OR 8.575-15.843) were more likely than atheists to desire children after vasectomy. 71,886 (2.0%) vasectomized men reported having a vasectomy reversal. CONCLUSION(S): This study highlights the importance of preoperative counseling for permanency of vasectomy and reveals an opportunity to counsel couples about vasectomy versus tubal ligation.

[-2]Proenzyme prostate specific antigen is more accurate than total and free prostate specific antigen in differentiating prostate cancer from benign disease in a prospective prostate cancer screening study.

PURPOSE: Due to the limited specificity of prostate specific antigen for prostate cancer screening, there is an ongoing search for adjunctive biomarkers. Retrospective studies have suggested that an isoform of proenzyme prostate specific antigen called [-2]proenzyme prostate specific antigen may enhance the specificity of prostate specific antigen based screening. We examined the usefulness of this isoform in a prospective prostate cancer screening study. MATERIALS AND METHODS: From a population of 2,034 men undergoing prostate cancer screening we examined the relationship between the measurement of the [-2]isoform of proenzyme prostate specific antigen (p2PSA) and prostate cancer detection. Specifically we compared the usefulness of total prostate specific antigen, the ratio of free-to-total prostate specific antigen, the ratio of p2PSA-to-free prostate specific antigen, and a formula combining prostate specific antigen, free prostate specific antigen and p2PSA (the Beckman Coulter prostate health index or phi) to predict prostate cancer in men from the study undergoing prostate biopsy with a prostate specific antigen of 2.5 to 10 ng/ml and nonsuspicious digital rectal examination. RESULTS: Despite similar total prostate specific antigen (p = 0.88), percent free prostate specific antigen (p = 0.02) and %p2PSA (p = 0.0006) distinguished between positive and negative biopsy results. On ROC analysis %p2PSA (AUC 0.76) outperformed prostate specific antigen (AUC 0.50) and percent free prostate specific antigen (AUC 0.68) for differentiating between prostate cancer and benign disease. Setting the sensitivity at 88.5%, p2PSA led to a substantial improvement in specificity as well as positive and negative predictive values. The Beckman Coulter prostate health index (AUC 0.77) had the best overall performance characteristics. CONCLUSIONS: This is the first prospective study to our knowledge to demonstrate that p2PSA provides improved discrimination between prostate cancer and benign disease in screened men with a prostate specific antigen of 2.5 to 10 ng/ml and a negative digital rectal examination.

Genitourinary pain syndromes, prostatitis, and lower urinary tract symptoms.

The overlap of pain and urinary voiding symptoms is common for urologic patients. The etiology of these syndromes is frequently multifactorial and due to disorders of the bladder and/or prostate. The evaluation and treatment of these syndromes continues to evolve. Here we summarize the general approach to evaluation and treatment of these pain syndromes.

Directed evolution of protein enzymes using nonhomologous random recombination.

We recently reported the development of nonhomologous random recombination (NRR) as a method for nucleic acid diversification and applied NRR to the evolution of DNA aptamers. Here, we describe a modified method, protein NRR, that enables proteins to access diversity previously difficult or impossible to generate. We investigated the structural plasticity of protein folds and the ability of helical motifs to function in different contexts by applying protein NRR and in vivo selection to the evolution of chorismate mutase (CM) enzymes. Functional CM mutants evolved using protein NRR contained many insertions, deletions, and rearrangements. The distribution of these changes was not random but clustered in certain regions of the protein. Topologically rearranged but functional enzymes also emerged from these studies, indicating that multiple connectivities can accommodate a functional CM active site and demonstrating the ability to generate new domain connectivities through protein NRR. Protein NRR was also used to randomly recombine CM and fumarase, an unrelated but also alpha-helical protein. Whereas the resulting library contained fumarase fragments in many contexts before functional selection, library members surviving selection for CM activity invariably contained a CM core with fumarase sequences found only at the termini or in one loop. These results imply that internal helical fragments cannot be swapped between these proteins without the loss of nearly all CM activity. Our findings suggest that protein NRR will be useful in probing the functional requirements of enzymes and in the creation of new protein topologies.

Nucleic acid evolution and minimization by nonhomologous random recombination.

We have developed a simple method for exploring nucleic acid sequence space by nonhomologous random recombination (NRR) that enables DNA fragments to randomly recombine in a length-controlled manner without the need for sequence homology. We compared the results of using NRR and error-prone PCR to evolve DNA aptamers that bind streptavidin. Starting with two parental sequences of modest avidin affinity, evolution using NRR resulted in aptamers with 15- to 20-fold higher affinity than the highest-affinity aptamers evolved using error-prone PCR, and 27- or 46-fold higher affinities than parental sequences derived using systematic evolution of ligands by exponential enrichment (SELEX). NRR also facilitates the identification of functional regions within evolved sequences. Inspection of a small number of NRR-evolved clones identified a 40-base DNA sequence, present in multiple copies in each clone, that binds streptavidin. Our findings suggest that NRR may enhance the effectiveness of nucleic acid evolution and the ease of identifying structure-activity relationships among evolved sequences.