Identifying variability in surgical practices and instrumentation for hypospadias repair across the Western Pediatric Urology Consortium (WPUC) network.

BACKGROUND: Although hypospadias outcomes studies typically report a level or type of repair performed, these studies often lack applicability to each surgical practice due to technical variability that is not fully delineated. An example is the tubularized incised plate (TIP) urethroplasty procedure, for which modifications have been associated with significantly decreased complication rates in single center series. However, many studies fail to report specificity in techniques utilized, thereby limiting comparison between series. OBJECTIVE: With the goal of developing a surgical atlas of hypospadias repair techniques, this study examined 1) current techniques used by surgeons in our network for recording operative notes and 2) operative technical details by surgeon for two common procedures, tubularized incised plate (TIP) distal and proximal hypospadias repairs across a multi-institutional surgical network. STUDY DESIGN: A two-part study was completed. First, a survey was distributed to the network to assess surgeon volume and methods of recording hypospadias repair operative notes. Subsequently, an operative template or a representative de-identified operative note describing a TIP and/or proximal repair with urethroplasty was obtained from participating surgeons. Each was analyzed by at least two individuals for natural language that signified specified portions of the procedure. Procedural details from each note were tabulated and confirmed with each surgeon, clarifying that the recorded findings reflected their current practice techniques and instrumentation. RESULTS: Twenty-five surgeons from 12 institutions completed the survey. The number of primary distal hypospadias repairs performed per surgeon in the past year ranged from 1-10 to >50, with 40% performing 1-20. Primary proximal hypospadias repairs performed in the past year ranged from 1-30, with 60% performing 1-10. 96% of surgeons maintain operative notes within an electronic health record. Of these, 66.7% edited a template as their primary method of note entry; 76.5% of these surgeons reported that the template captures their operative techniques very or moderately well. Operative notes or templates from 16 surgeons at 10 institutions were analyzed. In 7 proximal and 14 distal repairs, parameters for chordee correction, urethroplasty suture selection and technique, tissue utilized, and catheter selection varied widely across surgeons. CONCLUSION: Wide variability in technical surgical details of categorically similar hypospadias repairs was demonstrated across a large surgical network. Surgeon-specific modifications of commonly described procedures are common, and further evaluation of short- and long-term outcomes accounting for these technical variations is needed to determine their relative influence.

Telemedicine and patient satisfaction during the COVID-19 pandemic: A case-control study of outpatient pediatric urology patients.

Telemedicine has increased during the coronavirus disease 2019 pandemic. Our objective was to determine if patient satisfaction with telemedicine differed from in-person visits in an academic pediatric urology clinic. Following outpatient telemedicine and in-person pediatric urology visits, the validated NRC Health© Patient Survey was used to assess patient experience. Patient satisfaction was assessed on a 10-point scale with scores of 9-10 considered "satisfied" and 1-8 considered "not satisfied." Satisfaction scores between telemedicine and in-person groups were compared using McNemar's test, Wilcoxon signed rank test, and conditional logistic regression. Fifty-one patients had urology telemedicine visits during April-August 2020 and completed the NRC Health© Patient Survey. Propensity score matching was used to identify 102 in-person controls between January 2019 and March 2020. Ninety-two percent of telemedicine patients were satisfied compared to 87% of in-person patients (OR 1.7 95% CI [0.53-5.6]). Regression analysis adjusting for matching variables demonstrated that patient satisfaction was higher for telemedicine compared to in-person visits but was not statistically significant (OR 1.5 95% CI [0.43-5.6]). Patient satisfaction with telemedicine was similar to in-person visits in the pediatric urology clinic. Reduced waiting time and convenience associated with telemedicine visits provide an opportunity for telemedicine as a useful modality for pediatric urology.

Continuous antibiotic prophylaxis in isolated prenatal hydronephrosis.

BACKGROUND: Prenatal hydronephrosis (PNH) is one of the most common congenital anomalies and can increase the risk of developing a urinary tract infection (UTI) in the first two years of life. Continuous antibiotic prophylaxis (CAP) has been recommended empirically to prevent UTI in children with PNH, but its use has been controversial. OBJECTIVE: We describe the incidence of UTI in children with isolated PNH of the renal pelvis without ureteral dilation. Our objective was to compare patients receiving and not receiving CAP and determine whether CAP is beneficial at preventing UTI in children with isolated PNH. STUDY DESIGN: Children with confirmed PNH were enrolled between 2008 and 2020 into the Society for Fetal Urology Hydronephrosis Registry. Children with isolated dilation of the renal pelvis without ureteral or bladder abnormality were included. The primary outcome was development of a UTI, comparing patients who were prescribed and not prescribed CAP. RESULTS: In this cohort of 801 children, 76% were male, and 35% had high grade hydronephrosis (SFU grades 3-4). CAP was prescribed in 34% of children. The UTI rate among all children with isolated PNH was 4.2%. Independent predictors of UTI were female sex (HR = 13, 95% CI: 3.8-40, p = 0.0001), intact prepuce (HR = 5.1, 95% CI: 1.4-18, p = 0.01) and high grade hydronephrosis (HR = 2.0, 95% CI: 0.99-4.0, p = 0.05; Table) on multivariable analysis. For patients on CAP, the UTI rate was 4.0% compared to 4.3% without CAP (p = 0.76). The risk of UTI during follow-up was not significantly different between patients who received CAP and patients who were not exposed to CAP; adjusting for sex, circumcision status and hydronephrosis grade (HR = 0.72, 95% CI: 0.34-1.5, p = 0.38). In sub-group analysis of patients at higher risk of UTI (uncircumcised males, females and high grade hydronephrosis), CAP use was not associated with a statistically significant reduction in UTI. CONCLUSIONS: The overall UTI rate in children with isolated PNH is very low at 4.2%. In the overall population of patients with isolated PNH, CAP was not associated with reduction in UTI risk, although the limitations in our study make characterizing CAP effectiveness difficult. Clinicians should consider risk factors prior to placing all patients with isolated PNH on CAP.

Taking "Trans-ano-rectal" out of ASTRA: An anterior sagittal approach without splitting the rectum.

The anterior sagittal trans-ano-rectal approach (ASTRA) provides excellent exposure to the urethra and vagina for partial or total urogenital sinus mobilization and subsequent reconstruction for patients with urogenital sinus anomalies. It is a frequent approach to reconstruction for children with a high confluence. However, the division of the anterior anal external sphincter and the rectal wall in the ASTRA incurs morbidity, which include fecal incontinence if one veers from the midline, and increased risk of wound infection due to fecal soilage. We demonstrate a modified technique to the ASTRA without dividing the anterior anal sphincter and rectal wall, with achievement of comparable exposure and excellent vaginal mobilization and length.

Evaluation of Bi-Layer Silk Fibroin Grafts for Penile Tunica Albuginea Repair in a Rabbit Corporoplasty Model.

The use of autologous tissue grafts for tunica albuginea repair in Peyronie's disease and congenital chordee is often restricted by limited tissue availability and donor site morbidity, therefore new biomaterial options are needed. In this study, bi-layer silk fibroin (BLSF) scaffolds were investigated to support functional tissue regeneration of tunica albuginea in a rabbit corporoplasty model. Eighteen adult male, New Zealand white rabbits were randomized to nonsurgical controls (NSC, N = 3), or subjected to corporoplasty with BLSF grafts (N = 5); decellularized small intestinal submucosa (SIS) matrices (N = 5); or autologous tunica vaginalis (TV) flaps (N = 5). End-point evaluations were cavernosography, cavernosometry, histological, immunohistochemical, and histomorphometric assessments. Maximum intracorporal pressures (ICP) following papaverine-induced erection were similar between all groups. Eighty percent of rabbits repaired with BLSF scaffolds or TV flaps achieved full rigid erections, compared to 40% of SIS reconstructed animals. Five-minute peak erections were maintained in 60% of BLSF rabbits, compared to 20% of SIS and TV flap reconstructed rabbits. Graft perforation occurred in 60% of TV group at maximum ICP compared to 20% of BLSF cohort. Neotissues supported by SIS and BLSF scaffolds were composed of collagen type I and elastin fibers similar to NSC. SIS and TV flaps showed significantly elevated levels of corporal fibrosis relative to NSC with a corresponding decrease in corporal smooth muscle cells expressing contractile proteins. BLSF biomaterials represent emerging platforms for corporoplasty and produce superior functional and histological outcomes in comparison to TV flaps and SIS matrices for tunica albuginea repair.

Risk of urinary tract infection in patients with hydroureter: An analysis from the Society of Fetal Urology Prenatal Hydronephrosis Registry.

BACKGROUND: Prenatal hydronephrosis is one of the most common anomalies detected on prenatal ultrasonography. Patients with prenatal hydronephrosis and ureteral dilation are at increased risk of urinary tract infection (UTI) and continuous antibiotic prophylaxis (CAP) is recommended. However, current guidelines do not define the minimum ureteral diameter that would be considered a dilated ureter in these patients. OBJECTIVE: We evaluate the definition of clinically relevant hydroureter, its association with UTI, and the impact of CAP. STUDY DESIGN: Patients with prenatal hydronephrosis from seven centers were enrolled into the Society for Fetal Urology Prenatal Hydronephrosis Registry from 2008 to 2020. Patients with ureteral measurement on ultrasound were included. Patients with ureterocele, ectopic ureter, neurogenic bladder, posterior urethral valves, horseshoe or solitary kidney, known ureteropelvic junction obstruction, or follow-up less than one month were excluded. Primary outcome was UTI. Analyses were performed using Cox regression. RESULTS: Of the 1406 patients enrolled in the registry, 237 were included. Seventy-six percent were male, ureteral diameter ranged from 1 to 34 mm, and median follow-up was 2.2 years. Patients with ureters 7 mm or greater had nearly three times the risk of UTI adjusting for sex, circumcision status, antibiotic prophylaxis and hydronephrosis grade (HR = 2.7, 95% CI: 1.1-6.5, p = 0.03; Figure). In patients who underwent voiding cystourethrogram (VCUG; 200/237), ureteral dilation of 7 mm or more identified patients at increased UTI risk controlling for sex, circumcision status, vesicoureteral reflux and hydronephrosis grade (HR = 2.3, 95% CI: 0.97-5.6, p = 0.06). CAP was significantly protective against UTI (HR = 0.50 (95% CI: 0.28-0.87), p = 0.01). Among patients who underwent VCUG and did not have vesicoureteral reflux, ureteral dilation 7 mm or greater corresponded with higher UTI risk compared to ureteral diameter less than 7 mm on multivariable analysis (HR = 4.6, 95% CI: 1.1-19.5, p = 0.04). CONCLUSIONS: This is the first prospectively collected, multicenter study to demonstrate that hydroureter 7 mm or greater identifies a high-risk group for UTI who benefit from antibiotic prophylaxis. In contrast, patients with prenatal hydronephrosis and non-refluxing hydroureter less than 7 mm may be managed more conservatively.

A multicenter study of acute testicular torsion in the time of COVID-19.

BACKGROUND: Testicular torsion is a surgical emergency, and time to detorsion is imperative for testicular salvage. During the COVID-19 pandemic, patients may delay emergency care due to stay-at-home orders and concern of COVID-19 exposure. OBJECTIVE: To assess whether emergency presentation for testicular torsion was delayed during the COVID-19 pandemic, and whether the rate of orchiectomy increased compared to a retrospective period. STUDY DESIGN: Patients were prospectively enrolled in a multicenter study from seven institutions in the United States and Canada. Inclusion criteria were patients two months to 18 years of age with acute testicular torsion from March through July 2020. The retrospective group included patients from January 2019 through February 2020. Statistical analysis was performed using Kruskal-Wallis tests, Chi-square tests, and logistic regression. RESULTS: A total of 221 patients were included: 84 patients in the COVID-19 cohort and 137 in the retrospective cohort. Median times from symptom onset to emergency department presentation during COVID-19 compared to the retrospective period were 17.9 h (IQR 5.5-48.0) and 7.5 h (IQR 4.0-28.0) respectively (p = 0.04). In the COVID-19 cohort, 42% of patients underwent orchiectomy compared to 29% of pre-pandemic controls (p = 0.06). During COVID-19, 46% of patients endorsed delay in presentation compared to 33% in the retrospective group (p = 0.04). DISCUSSION: We found a significantly longer time from testicular torsion symptom onset to presentation during the pandemic and a higher proportion of patients reported delaying care. Strengths of the study include the number of included patients and the multicenter prospective design during the pandemic. Limitations include a retrospective pre-pandemic comparison group. CONCLUSIONS: In a large multicenter study we found a significantly longer time from testicular torsion symptom onset to presentation during the pandemic and a significantly higher proportion of patients reported delaying care. Based on the findings of this study, more patient education is needed on the management of testicular torsion during a pandemic.

Retractable foreskin reduces urinary tract infections in infant boys with vesicoureteral reflux.

BACKGROUND: Uncircumcised males are at higher risk of urinary tract infection (UTI) in the first year of life and circumcision is recommended as an option for males with vesicoureteral reflux (VUR). Uncircumcised males treated successfully with topical corticosteroid cream have decreased risk of UTI but the role of preputial management has not been explored previously in males with VUR. OBJECTIVE: We hypothesized that among uncircumcised boys with VUR, those with retractable foreskin would be at reduced risk of UTI compared to those with non-retractable foreskin. STUDY DESIGN: Males less than one year of age with primary VUR were prospectively enrolled. Patients with concomitant urologic diagnoses or less than one month of follow-up were excluded. Phimosis severity was graded on a 0-5 scale. Primary outcome was UTI during follow-up. Patients were divided into three groups for analysis: circumcised, low grade phimosis (grades 0-3) and high grade phimosis (grades 4-5). Multivariable Cox proportional hazards regression was used to estimate UTI risk adjusting for risk factors. RESULTS: One-hundred and five boys (24 circumcised and 81 uncircumcised) with VUR were included. Median age at enrollment was 4.4 months (IQR 2.2-6.6) and median follow-up was 1.1 years (IQR 0.53-2.9). Males with phimosis grades 4-5 had a higher UTI rate (29%) compared to phimosis grade 0-3 (4%). Based on Kaplan-Meier curves, boys with initial phimosis grades 4-5 were significantly more likely to develop a UTI than boys who were circumcised or had phimosis grades 0-3 (p = 0.005). On multivariable analysis, boys with phimosis grades 4-5 were significantly more likely to develop UTI when compared to boys with grades 0-3 phimosis (HR = 8.4, 95% CI: 1.1-64, p = 0.04). DISCUSSION: Males with a retractable prepuce had a lower UTI risk compared to males with non-retractable prepuce (high grade phimosis) and this remained significant on multivariable analysis. This is concordant with prior studies demonstrating that a retractable prepuce is associated with decreased UTI risk. Limitations of our study include using phimosis grade at time of study enrollment and heterogenous prophylactic antibiotic use in our population. CONCLUSIONS: Retractable foreskin reduces UTI risk in uncircumcised boys less than one year of age with VUR. Medical phimosis treatment to achieve a retractable prepuce offers an alternative and less invasive modality to reduce UTI risk in males with VUR.

Crizotinib-associated Renal Cyst Formation in a Pediatric Patient With ALK+ Epithelioid Inflammatory Myofibroblastic Sarcoma.

BACKGROUND: Crizotinib is a first-generation tyrosine kinase inhibitor used for anaplastic lymphoma kinase (ALK) positive cancers. Simple and complex renal cyst formation is a rare complication of crizotinib use that has been reported previously in the adult population. CASE: We report a case of a right renal mass in a 17-year-old with ALK-positive epithelioid inflammatory myofibroblastic sarcoma treated with Crizotinib. After cessation of Crizotinib and initiating Alectenib, a second generation ALK inhibitor, the mass decreased in size and the patient remained asymptomatic without evidence of recurrence at three months of follow-up.

Papers presented at the fall 2020 Pediatric Urologic Oncology Work Group of the Societies of Pediatric Urology meetingPrenatally Diagnosed Solid Renal Mass.

Congenital mesoblastic nephroma is the most common renal neoplasm diagnosed in the first month of life of which 15% occur prenatally. We present a prenatal diagnosis of a 5.8 cm solid renal mass identified on the 36-week ultrasound. Labor was induced at 38 weeks and a female infant was delivered vaginally without complications. The postnatal ultrasound demonstrated a 6.3 cm heterogeneous mass nearly replacing the kidney. The infant underwent a radical nephrectomy on the first day of life and pathology confirmed stage II classic CMN with negative margins and nodes. She is otherwise healthy at follow-up of 1 year with no evidence of recurrence.

High incidence of abnormal imaging findings in circumcised boys diagnosed with urinary tract infections.

BACKGROUND: The American Academy of Pediatrics guidelines recommend delaying voiding cystourethrogram (VCUG) until the second febrile urinary tract infection (UTI). Similarly, the NICE Guidelines do not recommend VCUG after the first febrile UTI in infants and children under three years of age. Currently, there is no good clinical indicator to determine which patients would benefit from an earlier cystogram. Here, we sought to identify clinical and bacteriologic findings that are associated with an increased risk of urinary tract anatomic anomalies in pediatric males presenting to the Emergency Department with a UTI. METHODS: A retrospective review was performed of all urine cultures from the Emergency Department at our institution between 2006 and 2015. Males under 18 years of age with ≥50,000 CFU/mL of Proteus, Klebsiella, Escherichia coli, Staphylococcus, Streptococcus and Enterococcus were included. Clean catch, catheterized and suprapubic aspiration specimens were included. Bagged specimens and specimens from patients on intermittent catheterization were excluded. Ultrasound and cystogram images were reviewed when available. Univariate and multivariable Poisson regression with robust variance was used to calculate and compare prevalence ratios. Statistical analysis was performed with Stata software, version 15.1 MP (Stata Corporation, College Station, Texas, USA). RESULTS: We reviewed 1585 urine cultures, of which 812 met eligibility criteria including 619 (76.2%) E. coli, 84 (10.3%) Proteus, 55 (6.8%) Klebsiella, 29 (3.6%) Staphylococcus, 13 (1.6%) Enterococcus and 12 (1.5%) Streptococcus. Median age was six months (2.4-36 months IQR). After adjusting for age, ethnicity and bacterial species, the prevalence of dilating vesicoureteral reflux (VUR, defined as grades III-V) was 5.3 times higher in circumcised males compared to uncircumcised males (95% CI: 2.4-11.4, p = 0.001). Circumcised males had a 3.1 times increased prevalence of high-grade hydronephrosis (defined as SFU grades 3 and 4) compared to uncircumcised males (prevalence ratio: 3.1, 95% CI: 1.0-10.0, p = 0.05). Among bacterial pathogens, no patients with Proteus UTI had high-grade hydronephrosis or dilating VUR. In contrast, Staphylococcal infections were associated significantly with dilating VUR (prevalence ratio 10.1, 95% CI: 2.3, 44.8; p = 0.002) and high-grade hydronephrosis (prevalence ratio 8.1, 95% CI: 1.7, 39.7; p = 0.010). CONCLUSIONS: Circumcision status is an independent predictor of structural urinary anomalies of the bladder and upper tracts in pediatric males with UTI, therefore circumcised males should be considered for early imaging, including VCUG, after their first UTI. Furthermore, Staphylococcal infections were associated with an even higher prevalence of high-grade hydronephrosis and dilating VUR and therefore warrant early investigation with VCUG. In contrast, none of the Proteus infections, which were seen almost exclusively in uncircumcised males, were associated with dilating VUR or hydronephrosis, making initial observation more appropriate in these patients.

Ruling out clinically significant prostate cancer with negative multi-parametric MRI.

PURPOSE: To evaluate the negative predictive value (NPV) of a negative prostate multi-parametric magnetic resonance imaging (mpMRI) in ruling out clinically significant prostate upon 12-core systematic biopsy. METHODS: We retrospectively reviewed 114 men evaluated at our institution who underwent systematic 12-core biopsy within 1 year of a negative prostate mpMRI. Clinicopathologic features were evaluated and NPV was calculated for detection of clinically significant (Gleason ≥ 7) cancer. Regression analysis was performed to identify clinical predictors of biopsy outcome. RESULTS: Overall, 88 (77.2%) patients in our cohort had no cancer detected upon biopsy. The highest pathologic grade was Gleason 6 (3 + 3) in 22 (19.3%) patients, and Gleason ≥ 7 in 4 (3.6%) patients. NPV for detecting Gleason ≥ 7 cancer was 96.5% (95% CI 93.1-99.9%) in the entire negative MRI cohort, 100% in those who were prostate biopsy naïve (n = 20), 100% in those with a prior negative biopsy (n = 53), and 90% in those who have had a previous positive biopsy and on active surveillance (n = 41). Regression analysis identified no predictors of significant cancer in our cohort. CONCLUSION: In our cohort of men with no lesions detected on prostate mpMRI, we found very low rates of clinically significant cancer on systematic 12-core biopsy. In the few patients who diagnosed with prostate cancer, the majority had low-risk disease and could remain on active surveillance. Although validation studies and greater sample size is needed before clinical recommendations can be made, our data suggest patients with negative mpMRI evaluated by experienced radiologists may avoid unnecessary prostate biopsy and potential overtreatment.

Major histocompatibility complex I upregulation in clear cell renal cell carcinoma is associated with increased survival.

OBJECTIVE: To examine the prognostic value of tumor major histocompatibility complex I (MHCI) expression on survival and recurrence in patients with clear cell renal cell carcinoma (RCC). METHODS: Fifty-three patients that underwent nephrectomy at our institution for clear cell RCC (T1-T3) with ≥4 years of follow-up were queried from our nephrectomy database. Immunohistochemical staining for MHCI was performed on tumor specimens and MHCI expression was quantified with an automated image analysis technique. Patients were divided into high and low MHCI expression groups in order to study the relationship between MHCI expression and prognosis using the Kaplan-Meier method and log-rank test. RESULTS: Overall survival and recurrence free survival were increased in the high MHCI expression group compared to the low MHCI expression group (log-rank, p = 0.036 and p = 0.028, respectively). Patients alive at the end of the study had higher MHCI expression (mean positivity score 0.82) than those that died of disease (mean positivity score 0.76, t test, p = 0.030). Patients that did not develop recurrence during the study period had higher MHCI expression (mean positivity score 0.83) than those that did develop recurrence (mean positivity score 0.78), but this difference was not significant (t test, p = 0.079). CONCLUSION: Our data demonstrate that high MHCI expression confers improved overall and recurrence free survival in patients with clear cell RCC and could serve as an important prognostic tool in identifying high-risk patients.

High Expression of Major Histocompatibility Complex Class I in Clear Cell Renal Cell Carcinoma Is Associated with Improved Prognosis.

INTRODUCTION: In this study we analyzed major histocompatibility complex class I (MHCI) expression as a potential prognostic immune marker for patients with clear cell renal cell carcinoma (ccRCC). PATIENTS AND METHODS: 34 patients with localized ccRCC (pT1-pT3) who had undergone nephrectomy and had at least 4 years of clinical follow-up data were included in the study. Immunohistochemical staining for MHCI was performed on tumor sections. An automated image analysis algorithm was applied to representative tumor areas to quantitate the proportion of stained pixels (positivity score = positive pixels/total pixels) on scanned digital slides. RESULTS: At the end of the study, the patients who were alive had increased MHCI expression (mean positivity score 0.80) compared to those who died of the disease (mean positivity score 0.53; p < 0.0001, t test). Patients who were alive with recurrence had increased MHCI expression (positivity score 0.81) compared to those who succumbed to their disease recurrence (positivity score 0.53; p < 0.0001, t test). Survival was higher among patients with high MHCI expression compared to patients with low MHCI expression (p < 0.0001, Mantel-Cox). CONCLUSIONS: With an automated high-throughput image analysis technique, this study shows that higher tumor cell MHCI expression promotes increased survival and reduced incidence of recurrence compared to patients with lower tumor cell MHCI expression.

Functional supernumerary testis in a child with testicular torsion and review of polyorchidism.

Histologically confirmed polyorchidism is an uncommon condition. The majority of cases are asymptomatic and associated with inguinal hernia or cryptorchidism. There are few reported cases of pediatric supernumerary testis presenting with testicular torsion. We present a case of missed torsion with incidentally detected triorchidism as well as an overview of pediatric polyorchidism.

Specific ligand binding domain residues confer low dioxin responsiveness to AHR1ß of Xenopus laevis.

The aryl hydrocarbon receptor (AHR) is a Per-ARNT-Sim (PAS) family protein that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vertebrates. Frogs are remarkably insensitive to TCDD, and AHRs from Xenopus laevis bind TCDD with low affinity. We sought to identify structural features of X. laevis AHR1ß associated with low TCDD sensitivity. Substitution of the entire ligand binding domain (LBD) with the corresponding sequence from mouse AHR(b-1) dramatically increased TCDD responsiveness in transactivation assays. To identify the amino acid residues responsible, we constructed a comparative model of the AHR1ß LBD using homologous domains of PAS proteins HIF2α and ARNT. The model revealed an internal cavity with dimensions similar to those of the putative binding cavity of mouse AHR(b-1), suggesting the importance of side chain interactions over cavity size. Of residues with side chains clearly pointing into the cavity, only two differed from the mouse sequence. When A354, located within a conserved ß-strand, was changed to serine, the corresponding mouse residue, the EC50 for TCDD decreased more than 15-fold. When N325 was changed to serine, the EC50 decreased 3-fold. When the mutations were combined, the EC50 decreased from 18.6 to 0.8 nM, the value nearly matching the TCDD sensitivity of mouse AHR. Velocity sedimentation analysis confirmed that mutant frog AHRs exhibited correspondingly increased levels of TCDD binding. We also assayed mutant AHRs for responsiveness to a candidate endogenous ligand, 6-formylindolo[3,2-b]carbazole (FICZ). Mutations that increased sensitivity to TCDD also increased sensitivity to FICZ. This comparative study represents a novel approach to discerning fundamental information about the structure of AHR and its interactions with biologically important agonists.