Quite a Pickle: A 10-Year Increase in Pickleball-Related Hand Injuries.

BACKGROUND: Pickleball popularity has significantly increased in the United States. Wrist, hand, and finger injuries are commonly seen among pickleball players and are on the rise. METHODS: The National Electronic Injury Surveillance System database was queried from 2013 to 2022 tennis- and pickleball-related injuries in the United States. Data were filtered to include wrist, hand, and finger injuries only. RESULTS: There were a total of 12 021 estimated pickleball injuries between 2013 and 2022, most commonly in white women greater than 55 years old. The most common mechanism of injury (90.5%) was a fall. The number of injuries increased by 765.6% between 2013 and 2022. The growth in the number of pickleball-related injuries was found to be statistically significant (P < .05), whereas the growth for tennis-related injuries was found to be not statistically different from 0. There was a statistical difference between the growth of the number of pickleball injuries per year and the number of tennis injuries. The wrist was the most common location of injury (70.0%) compared with the hand (10.5%) and fingers (19.5%). The most common injury diagnosis was fracture (60.3%). Overall, the most common injury was wrist fracture (50.0%). CONCLUSIONS: Hand surgeons should be aware of the increasing prevalence of pickleball-related injuries as an alternative mechanism of injury, particularly among the elderly.

Inferiorly Directed Posterior Cranial Vault Distraction for Treatment of Chiari Malformations.

OBJECTIVE: The authors present a case series of patients with Chiari I malformations treated with distraction osteogenesis of the posterior cranial vault, utilizing a vertical distraction vector for appropriate cranial vault expansion while mitigating the risks of scaphocephaly and cerebellar ptosis. PATIENTS AND METHODS: Patients with syndromic and nonsyndromic Chiari I malformations treated with vertical-vector distraction osteogenesis of the posterior cranial vault were identified from 2008 to 2014. Demographics, preoperative and postoperative clinical symptoms, and perioperative details were assessed. Long-term esthetic outcomes, complications, and symptomatic improvement were evaluated in conjunction with neurosurgery. RESULTS: Nine patients were identified. Five had known syndromes, 2 likely had unidentified syndromes, and 2 were nonsyndromic. Seven had prior Chiari-related surgeries. Most presented with hydrocephalus, motor symptoms, and developmental delay. Operatively, 2 to 3 internal distraction fixators were applied such that the vector of distraction was along a cephalad-caudad axis. Devices were activated on postoperative day 5 and distracted 1 mm per day. Three postoperative complications were found within the first 3 months which included a dislodged distraction arm, a device extrusion, and a local cellulitis. No complications affected the clinical outcome. Radiographic follow-up showed good bone formation, decompression of the posterior fossa, improved cerebrospinal fluid flow, and no cerebellar ptosis. Neurological surveillance showed improvement in intracranial pressure, hydrocephalus, motor symptoms, and behavioral problems. CONCLUSION: The authors have presented 9 patients with Chiari I malformations treated with distraction osteogenesis, along with a novel technique to safely and effectively expand the posterior fossa while minimizing the risk of cerebellar ptosis. LEVEL OF EVIDENCE: Level II (prognostic/risk studies).

Turning Back the Clock: Artificial Intelligence Recognition of Age Reduction after Face-Lift Surgery Correlates with Patient Satisfaction.

BACKGROUND: Patients desire face-lifting procedures primarily to appear younger, more refreshed, and attractive. Because there are few objective studies assessing the success of face-lift surgery, the authors used artificial intelligence, in the form of convolutional neural network algorithms alongside FACE-Q patient-reported outcomes, to evaluate perceived age reduction and patient satisfaction following face-lift surgery. METHODS: Standardized preoperative and postoperative (1 year) images of 50 consecutive patients who underwent face-lift procedures (platysmaplasty, superficial musculoaponeurotic system-ectomy, cheek minimal access cranial suspension malar lift, or fat grafting) were used by four neural networks (trained to identify age based on facial features) to estimate age reduction after surgery. In addition, FACE-Q surveys were used to measure patient-reported facial aesthetic outcome. Patient satisfaction was compared to age reduction. RESULTS: The neural network preoperative age accuracy score demonstrated that all four neural networks were accurate in identifying ages (mean score, 100.8). Patient self-appraisal age reduction reported a greater age reduction than neural network age reduction after a face lift (-6.7 years versus -4.3 years). FACE-Q scores demonstrated a high level of patient satisfaction for facial appearance (75.1 ± 8.1), quality of life (82.4 ± 8.3), and satisfaction with outcome (79.0 ± 6.3). Finally, there was a positive correlation between neural network age reduction and patient satisfaction. CONCLUSION: Artificial intelligence algorithms can reliably estimate the reduction in apparent age after face-lift surgery; this estimated age reduction correlates with patient satisfaction. CLINICAL QUESTION/LEVEL OF EVIDENCE: Diagnostic, IV.

Comparing Plastic Surgeon Operative Time for DIEP Flap Breast Reconstruction: 2-stage More Efficient than 1-stage?

The deep inferior epigastric perforator flap for breast reconstruction is associated with lengthy operative times that remain an issue for plastic surgeons today. The main objective of this study was to determine if a 2-stage deep inferior epigastric perforator flap reconstruction resulted in a shorter total plastic surgeon operative time compared with an immediate reconstruction. METHODS: A retrospective chart review was conducted on all patients who underwent deep inferior epigastric perforator flap breast reconstruction from February 2013 to July 2020 by the senior author. Patient demographics, medical comorbidities, mastectomy characteristics, expander placement, reconstructive procedures, operative time, and complications were tabulated. RESULTS: The study included a total of 128 patients. For immediate/1-stage flap reconstruction, average operative times for the plastic surgeon were 427.0 minutes for unilateral procedures, and 506.3 minutes for bilateral procedures. For delayed/2-stage reconstruction, average combined plastic surgeon operative times were 351.1 minutes for unilateral expander followed by flap reconstruction (75.9 minutes shorter than immediate unilateral, P = 0.007), and 464.8 minutes for bilateral reconstruction (41.5 minutes shorter than immediate bilateral, P = 0.04). Total patient time under anesthesia was longer for 2-staged bilateral reconstruction (P = 0.0001), but did not differ significantly for unilateral reconstruction. Complications between immediate and delayed groups were not significantly different. CONCLUSIONS: We found that staged reconstruction over 2 procedures resulted in a significant reduction in operative time for the plastic surgeon for both unilateral and bilateral reconstruction. With amenable breast surgeons and patients, the advantages of controlling scheduling and the operating room may encourage plastic surgeons to consider performing free flap reconstruction in a delayed fashion.

Incidence, Impact, and Management of Incidentalomas on Preoperative Computed Tomographic Angiograms for Breast Cancer Patients with and without Genetic Mutations.

BACKGROUND: Preoperative abdominal computed tomographic angiograms for free flap breast reconstruction improve operative safety and efficiency, but incidental findings are common and potentially affect management. In addition, the authors hypothesized that patients with genetic mutations might have a higher rate of significant findings. The authors present the largest series of computed tomographic angiogram "incidentalomas" in these two populations and an evidence-based algorithm for managing common findings. METHODS: All patients undergoing free flap breast reconstruction at Northwell Health between 2009 and 2017 were eligible. Medical history, perioperative details, and radiology reports were examined with abnormal findings recorded. Published literature was reviewed with radiologists to develop standardized guidelines for incidentaloma management. RESULTS: Of 805 patients included, 733 patients had abdominal imaging. One hundred ninety-five (27 percent) had a completely negative examination. In the remaining 538 patients, benign hepatic (22 percent) and renal (17 percent) findings were most common. Sixteen patients (2.2 percent) required additional imaging (n = 15) or procedures (n = 5). One finding was concerning for malignancy-renal cell carcinoma-which interventional radiology ablated postoperatively. Seventy-nine patients (10.8 percent) had a genetic mutation but were not found to have a statistically significant higher rate of incidentalomas. CONCLUSIONS: The authors' rate of computed tomographic angiography incidental findings (73 percent) is consistent with previous studies, but the rate requiring further intervention (2.2 percent) is lower. Incidental findings were no more common or pathologic among genetic mutation carriers. The authors also introduce an evidence-based algorithm for the management of common incidentalomas. Using these guidelines, plastic surgeons can reassure patients, regardless of mutation status, that incidentalomas are most commonly benign and have minimal impact on their surgical plan.

Virtual Planning for Exchange Cranioplasty in Cranial Vault Remodeling.

ABSTRACT: The use of virtual surgical planning and computer-aided design/computer-aided manufacturing has gained popularity in the surgical correction of craniosynostosis. This study expands the use of virtual surgical planning and computer-aided design/computer-aided manufacturing in cranial vault reconstruction by using these methods to reconstruct the anterior vault using a single endocortically-plated unit constructed from the posterior calvarium. This technique was designed to reduce the risk of undesirable contour deformities that can occur when multiple bone grafts are used to reconstruct the anterior vault and fronto-orbital rim. Six patients were included in this study, all of which had nonsyndromic craniosynostosis. Excellent aesthetic outcomes were obtained in all patients, without complication. Additionally, the placement of a single reconstructive unit constructed from the posterior calvarium was efficient, aesthetically pleasing, and minimized postoperative contour deformities secondary to bone gaps, resorption, and often palpable resorbable plates.

Facelift Surgery Turns Back the Clock: Artificial Intelligence and Patient Satisfaction Quantitate Value of Procedure Type and Specific Techniques.

BACKGROUND: Patients desire facelifting procedures to look younger, refreshed, and attractive. Unfortunately, there are few objective studies assessing the success of types of facelift procedures and ancillary techniques. OBJECTIVES: The authors sought to utilize convolutional neural network algorithms alongside patient-reported FACE-Q outcomes to evaluate perceived age reduction and patient satisfaction following various facelift techniques. METHODS: Standardized preoperative and postoperative (1-year) images of patients who underwent facelift procedures were analyzed by 4 neural networks to estimate age reduction after surgery (n = 105). FACE-Q surveys were employed to measure patient-reported facial aesthetic outcome. We compared (1) facelift procedure type: skin-only vs superficial musculoaponeurotic system (SMAS)-plication, vs SMAS-ectomy; and (2) ancillary techniques: fat grafting (malar) vs no fat grafting. Outcomes were based on complications, estimated age-reduction, and patient satisfaction. RESULTS: The neural network preoperative age accuracy score demonstrated that all neural networks were accurate in identifying our patients' ages (mean score = 100.4). SMAS-ectomy and SMAS-plication had significantly greater age-reduction (5.85 and 5.35 years, respectively) compared with skin-only (2.95 years, P < 0.05). Fat grafting compared to no fat grafting demonstrated 2.1 more years of age reduction. Facelift procedure type did not affect FACE-Q scores; however, patients who underwent fat grafting had a higher satisfaction with outcome (78.1 ± 8 vs 69 ± 6, P < 0.05) and decision to have the procedure (83.0 ± 6 vs 72 ± 9, P < 0.05). CONCLUSIONS: Artificial intelligence algorithms can reliably estimate the reduction in apparent age after facelift surgery. Facelift technique, like SMAS-ectomy or SMAS-plication, and specific technique, like fat grafting, were found to enhance facelifting outcomes and patient satisfaction.

Fore! A 10-year Analysis of Golf-related Facial Fractures.

BACKGROUND: Over 30 million people in the United States play golf, which, while considered a low-impact sport, involves balls and clubs moving >100 miles/h (>160.93 km/h), creating potential for a significant facial trauma. The objective of this study was to characterize the epidemiology of golf-related facial fractures in the United States. METHODS: The National Electronic Injury Surveillance System, which records injuries from approximately 100 US emergency departments, was queried for golf-related facial fractures from 2009 to 2018. Entries were tabulated for mechanism of injury and fracture location. Age, gender, seasonality of injury, type of fracture, and injury mechanism were analyzed using SPSS. Because of the different nature and management of pediatric fractures, we looked at pediatric patients as a distinct subgroup. RESULTS: There were 114 reported cases of golf-related craniofacial fractures in patients 2-91 years of age with male predominance (73.7%). This rate extrapolates to an estimated 3,850 ED presentations. Although accounting for about 10% of total players, patients under 18, commonly referred to as "junior golfers," comprised 55.3% of the cohort. Nasal fractures (25.4%), skull fractures (23.7%), unspecified facial fractures (17.5%), and mandible fractures (10.5%) predominated, which most commonly result from clubs (63.2%), balls (23.7%), and falls (11.4%). CONCLUSIONS: Facial fractures resulting from golf clubs and balls may not be as common as in other traditionally identified "high-impact sports," but they affect pediatric patients disproportionately and with a greater morbidity. Parents and children engaging the sport should be aware of the risk of head trauma, especially with any form of off-course participation.

Use of Pericranial Flaps with Dermal Substitute for Scalp Reconstruction: A Case Series.

Skin cancer incidence has been rapidly increasing over the past 2 decades, and the resulting defects from excision have significant aesthetic and functional implications. In particular, wound coverage for large scalp and forehead defects with calvarial exposure can lead to hairline distortion, contour irregularities, and alopecia. We describe a 2-stage technique for scalp reconstruction, which preserves the normal hairline, covers exposed bone with vascularized tissue, and restores an aesthetic soft-tissue contour. METHODS: This is a retrospective case series of 13 adults with ages ranging from 50 to 89 years. All patients underwent Mohs surgery on the forehead or scalp between July 2014 and April 2017. Patients underwent a 2-staged reconstruction with an initial pericranial flap and dermal substitute placement followed by the placement of a split-thickness skin graft within 4-6 weeks. RESULTS: Over a 3-year period, 13 patients had successful reconstruction of the scalp defect without alteration of the hairline or contour irregularity. Two patients had minor complications after the first-stage procedure with successful aesthetic reconstruction. CONCLUSIONS: Full-thickness defects of the scalp and forehead with bone exposure provide a reconstructive challenge for plastic surgeons. Reconstructive algorithms continue to evolve and should be tailored to best suit patients' needs and medial comorbidities. Two-staged reconstruction with local pericranial flap provides a safe and efficacious reconstruction that minimizes hairline distortion, contour irregularity, and donor site morbidity.

Facial Recognition Neural Networks Confirm Success of Facial Feminization Surgery.

BACKGROUND: Male-to-female transgender patients desire to be identified, and treated, as female, in public and social settings. Facial feminization surgery entails a combination of highly visible changes in facial features. To study the effectiveness of facial feminization surgery, we investigated preoperative/postoperative gender-typing using facial recognition neural networks. METHODS: In this study, standardized frontal and lateral view preoperative and postoperative images of 20 male-to-female patients who completed hard- and soft-tissue facial feminization surgery procedures were used, along with control images of unoperated cisgender men and women (n = 120 images). Four public neural networks trained to identify gender based on facial features analyzed the images. Correct gender-typing, improvement in gender-typing (preoperatively to postoperatively), and confidence in femininity were analyzed. RESULTS: Cisgender male and female control frontal images were correctly identified 100 percent and 98 percent of the time, respectively. Preoperative facial feminization surgery images were misgendered 47 percent of the time (recognized as male) and only correctly identified as female 53 percent of the time. Postoperative facial feminization surgery images were gendered correctly 98 percent of the time; this was an improvement of 45 percent. Confidence in femininity also improved from a mean score of 0.27 before facial feminization surgery to 0.87 after facial feminization surgery. CONCLUSIONS: In the first study of its kind, facial recognition neural networks showed improved gender-typing of transgender women from preoperative facial feminization surgery to postoperative facial feminization surgery. This demonstrated the effectiveness of facial feminization surgery by artificial intelligence methods. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Facial Feminization Surgery Changes Perception of Patient Gender.

BACKGROUND: The goal of facial feminization surgery (FFS) is to feminize the sexually dimorphic characteristics of the face and enable transwomen to be correctly gendered as female. Studies have demonstrated high patient satisfaction with FFS. However, the correct gendering of patients after FFS has never been objectively studied. OBJECTIVES: The aim of this study was to determine if FFS changed the perceived gender of patients in the public eye. METHODS: An online survey platform with control photographs of cis-gender males and cis-gender females as well as preoperative and postoperative FFS patients was created. Respondents were asked to identify patients as "male" or "female" and to assign a confidence score ranging from -10 (masculine) to +10 (feminine) (n = 802). RESULTS: Cis-gender male and female controls were gendered correctly 99% and 99.38% of the time and with a confidence metric (CM) of -8.96 and 8.93, respectively. Preoperative FFS patients were gendered as female 57.31% of the time with a CM of 1.41 despite hormone therapy, makeup, and hairstyle. Postoperative FFS patients were gendered as female 94.27% of the time with a CM of 7.78. Ninety-five percent of patients showed a significant improvement in CM after FFS. CONCLUSIONS: This study illustrates that FFS changes the social perception of a patient's gender. Patients after FFS are more likely to be identified as female and with greater confidence than before surgery. This is despite preoperative female hormone therapy, and nonsurgical methods that patients use to feminize their appearance.

The Public Face of Rhinoplasty: Impact on Perceived Attractiveness and Personality.

BACKGROUND: The authors assess the impact of rhinoplasty on public perception of a patient's appearance and personality. METHODS: A survey was created using standardized before-and-after photographs of 10 Caucasian women who had undergone primary rhinoplasty. Photographs of two additional women who had not undergone facial surgery were randomly included as controls, for a total of 12 survey items. Preoperative and postoperative photographs were placed side by side. The survey was administered by means of crowd-sourcing. Respondents were asked to evaluate which photograph better represented 11 traits of appearance or personality, according to a seven-point Likert scale. A score of 1 meant the preoperative photograph was much better, 7 meant the postoperative photograph was much better, and 4 meant no difference. T tests and analyses of variance were used to evaluate rating changes for each trait and differences between demographic groups. RESULTS: There were 264 responses received. Averaged scores across the 10 survey patients produced a value for each appearance or personality trait. In 10 of 11 categories (i.e., symmetry, youthfulness, facial harmony, likeability, trustworthiness, confidence, femininity, attractiveness, approachability, and intelligence), the postoperative photograph was significantly favorable compared with the preoperative photograph (p < 0.00001). The preoperative photograph was rated higher only in aggressiveness (p < 0.001). The same scores were calculated for the controls; no significant difference in any category was seen except confidence, where the right image was viewed as more confident (mean, 4.19; p < 0.005). CONCLUSION: Aesthetic rhinoplasty improves the public perception of a person's appearance and personality in multiple aspects.

On facial asymmetry and self-perception.

Self-perception has been an enduring human concern since ancient times and remains a significant component of the preoperative and postoperative consultation. Despite modern technological attempts to reproduce the first-hand experience, there is no perfect substitute for human, stereoscopic, three-dimensional vision in evaluating appearance. Nowadays, however, the primary tools available to a patient for examining his or her own appearance, particularly the face, are photographs and mirrors. Patients are often unaware of how cameras and photographs can distort and degrade image quality, leading to an inaccurate representation of true appearance. Everyone knows that mirrors reverse an image, left and right, and most people recognize their own natural facial asymmetry at some level. However, few realize that emotions are not only expressed unequally by the left and right sides of the face but also perceived unequally by others. The impact and effect of this "facedness" is completely reversed by mirrors, potentially creating a significant discrepancy between what a patient perceives of himself or herself and what the surgeon or other third party sees. This article ties together the diverse threads leading to this problem and suggests several ways of mitigating the issue through technology and patient counseling.

The impact of complications on function, health, and satisfaction following abdominally based autologous breast reconstruction: a prospective evaluation.

BACKGROUND: The impact of surgical complications following autologous reconstruction on abdominal strength, health, and satisfaction is not completely understood. We prospectively examined the effect of complications on these aspects in patients undergoing abdominally-based autologous reconstruction. METHODS: A prospective study of patients who underwent autologous breast reconstruction between 2005 and 2010 was performed at a single teaching hospital. Patients enrolled in the study completed an abdominal strength functional assessment, the Short Form 36 (SF-36), and a satisfaction survey. Data were obtained at preoperative, early (<90 d), intermediate (90-365 d), and late (>365 d) follow-up visits. Patients who experienced surgical complications were compared with patients who did not. A subgroup analysis examined the specific impact of abdominal complications. RESULTS: Overall, 97 enrolled patients had preoperative, early and intermediate follow up. Forty of these patients had late follow-up. Fifty-six (58%) experienced surgical complications. After reconstruction, the complications group had decreased upper abdominal strength and function scores through early (p = 0.009, p = 0.01) and intermediate (p = 0.01, p = 0.06) follow-up. SF-36 physical health (p = 0.053) trended towards being lower in the early follow-up period. The complications group was less satisfied with the overall cosmetic result (p = 0.01) and shape of breasts (p = 0.02) through intermediate follow-up. At late follow-up, both cohorts recovered to baseline values in all study aspects. Patients with abdominal complications alone followed similar trends, with decreased upper abdominal strength and FIM scores through intermediate follow up. CONCLUSIONS: Having a major postoperative complication can significantly impact early physical health, mental health, abdominal strength, and patient satisfaction. Beyond one year, recovery towards baseline may occur in the majority of patients. LEVEL OF EVIDENCE: Prognostic/Risk Study, Level II.

Myotonic dystrophy protein kinase monoclonal antibody generation from a coiled-coil template.

Myotonic dystrophy protein kinase (DMPK) was the initial representative of a ubiquitous protein kinase family that regulates cell size and shape. DMPK is highly expressed in heart and skeletal muscle and transgenic over-expression induces cardiac hypertrophy. The characterization of DMPK has been limited by the paucity of immunological reagents with high affinity and well-defined specificity. Amino acid sequence data was used to predict the surface exposure of the coil-coiled domain of DMPK. These exposed amino acids were substituted into an extremely stable coiled-coil template to produce a peptide antigen. Sera from mice immunized with the peptide conjugated to keyhole limpet hemocyanin were screened against recombinant DMPK using Western blots. Murine spleens expressing DMPK antibodies were used to produce hybridoma cell lines. Hybridoma supernatants were further screened against recombinant DMPK and four clonal hybridoma cell lines expressing DMPK antibodies were generated. These four monoclonal antibodies recognized recombinant DMPK in Western blots of COS-1 cell lysates expressing high levels of recombinant DMPK and immunoprecipitated recombinant DMPK from COS-1 cell lysates. The identity of the immunoprecipitated DMPK was confirmed by MALDI-TOF mass spectrometry and peptide mass fingerprinting. DMPK was the only protein detected in the immunoprecipitates, indicating the high specificity of the antibodies. Western blots immunostained with two of the monoclonal antibodies specifically recognized the two isoforms of endogenous DMPK, DMPK-1 and DMPK-2, that are expressed at low levels in the human heart. The recognition of low amounts of DMPK-1 and DMPK-2 indicates the high affinity of these antibodies. A human heart lysate was subjected to ammonium sulfate precipitation and column chromatography to produce a fraction that was enriched in DMPK. One of the monoclonal antibodies immunoprecipitated endogenous DMPK from this fraction. This antibody was used for immuno-localization studies of an adenoviral DMPK construct, expressed in adult mouse cardiac myocytes. This construct was localized to the intercalated disc, the site of endogenous DMPK, indicating that this antibody is applicable to immuno-localization studies. This study demonstrates the utility of the described procedure for generation of specific monoclonal antibodies with high affinity for epitopes in coiled-coiled domains of mammalian proteins expressed at low levels.

Functional evolution within a protein superfamily.

The ability to predict and characterize distributions of reactivities over families and even superfamilies of proteins opens the door to an array of analyses regarding functional evolution. In this article, insights into functional evolution in the Kazal inhibitor superfamily are gained by analyzing and comparing predicted association free energy distributions against six serine proteinases, over a number of groups of inhibitors: all possible Kazal inhibitors, natural avian ovomucoid first and third domains, and sets of Kazal inhibitors with statistically weighted combinations of residues. The results indicate that, despite the great hypervariability of residues in the 10 proteinase-binding positions, avian ovomucoid third domains evolved to inhibit enzymes similar to the six enzymes selected, whereas the orthologous first domains are not inhibitors of these enzymes on purpose. Hypervariability arises because of similarity in energetic contribution from multiple residue types; conservation is in terms of functionality, with "good" residues, which make positive or less deleterious contributions to the binding, selected more frequently, and yielding overall the same distributional characteristics. Further analysis of the distributions indicates that while nature did optimize inhibitor strength, the objective may not have been the strongest possible inhibitor against one enzyme but rather an inhibitor that is relatively strong against a number of enzymes.

Defining the minimum size of a hydrophobic cluster in two-stranded alpha-helical coiled-coils: effects on protein stability.

The alpha-helical coiled-coil motif is characterized by a heptad repeat pattern (abcdefg)(n) in which residues a and d form the hydrophobic core. Long coiled-coils (e.g., tropomyosin, 284 residues per polypeptide chain) typically do not have a continuous hydrophobic core of stabilizing residues, but rather one that consists of alternating clusters of stabilizing and destabilizing residues. We have arbitrarily defined a cluster as a minimum of three consecutive stabilizing or destabilizing residues in the hydrophobic core. We report here on a series of two-stranded, disulfide-bridged parallel alpha-helical coiled-coils that contain a central cassette of three consecutive hydrophobic core positions (d, a, and d) with a destabilizing cluster of three consecutive Ala residues in the hydrophobic core on each side of the cassette. The effect of adding one to three stabilizing hydrophobes in these positions (Leu or Ile; denoted as [see text]) was investigated. Alanine residues (denoted as [see text]) are used to represent destabilizing residues. The peptide with three Ala residues in the d a d cassette positions ([see text]) was among the least stable coiled-coil (T(m) = 39.3 degrees C and Urea(1/2) = 1.9 M). Surprisingly, the addition of one stabilizing hydrophobe (Leu) to the cassette or two stabilizing hydrophobes (Leu), still interspersed by an Ala in the cassette ([see text]), also did not lead to any gain in stability. However, peptides with two adjacent hydrophobes in the cassette ([see text])([see text]) did show a gain in stability of 0.9 kcal/mole over the peptide with two interspersed hydrophobes ([see text]). Because the latter three peptides have the same inherent hydrophobicity, the juxtaposition of stabilizing hydrophobes leads to a synergistic effect, and thus a clustering effect. The addition of a third stabilizing hydrophobe to the cassette ([see text]) resulted in a further synergistic gain in stability of 1.7 kcal/mole (T(m) = 54.1 degrees C and Urea(1/2) = 3.3M). Therefore, the role of hydrophobicity in the hydrophobic core of coiled-coils is extremely context dependent and clustering is an important aspect of protein folding and stability.

Testing of the additivity-based protein sequence to reactivity algorithm.

The standard free energies of association (or equilibrium constants) are predicted for 11 multiple variants of the turkey ovomucoid third domain, a member of the Kazal family of protein inhibitors, each interacting with six selected enzymes. The equilibrium constants for 38 of 66 possible interactions are strong enough to measure, and for these, the predicted and measured free energies are compared, thus providing an additional test of the additivity-based sequence to reactivity algorithm. The test appears to be unbiased as the 11 variants were designed a decade ago to study furin inhibition and the specificity of furin differs greatly from the specificities of our six target enzymes. As the contact regions of these inhibitors are highly positive, nonadditivity was expected. Of the 11 variants, one does not satisfy the restriction that either P(2) Thr or P(1)' Glu should be present and all three measurable results on it, as expected, are nonadditive. For the remaining 35 measurements, 22 are additive, 12 are partially additive, and only one is (slightly) nonadditive. These results are comparable to those obtained for a set of 398 equilibrium constants for natural variants of ovomucoid third domains. The expectation that clustering of charges would be nonadditive is modified to the expectation that major nonadditivity will be observed only if the combining sites in both associating proteins involve large charge clusters of the opposite sign. It is also shown here that an analysis of a small variant set can be accomplished with a smaller subset, in this case 13 variants, rather than the whole set of 191 members used for the complete algorithm.

A de novo designed template for generating conformation-specific antibodies that recognize alpha-helices in proteins.

The generation of antibodies directed toward the surface-exposed regions of a protein using synthetic peptides as immunogens representing surface loops and turns has been widely successful. However, peptides representing alpha-helical regions are typically unstructured in solution and unable to produce antibodies that recognize alpha-helices in native proteins. We describe a de novo designed parallel two-stranded alpha-helical coiled-coil template for immunization to prepare antibodies that recognize alpha-helical protein sequences in the native protein. This template was designed for maximum stability through an Ile/Leu hydrophobic core and an interchain disulfide bridge. Surface-exposed helical residues are inserted into the template and used for immunization to generate polyclonal antibodies. To demonstrate the feasibility of this approach, 15 residues of the yeast transcription factor GCN4 were inserted into this template, and the resultant antibodies were screened for conformational specificity. Peptide antigens that contain the same surface-exposed residues but differ in structure were used as competitors in a competition assay. Direct competition between the capture peptide immobilized on a biosensor chip, the peptide antigens, and the antibodies generated by the template demonstrated that the antibodies were specific for helical structure in the native coiled-coil (synthetic GCN4 residues 250-280). These antibodies were unable to recognize the same inserted sequence in an unstructured analog. The helix-specific antibodies were also able to identify native GCN4 (31.3 kDa) from yeast whole cell extracts.