The Role of Suprapubic Superficial Fascial System Reconstruction During Repair of Adult-Acquired Buried Penis.

BACKGROUND: Adult-Acquired Buried Penis is a disorder associated with systemic obesity that confers increased risks of malignancy, sexual dysfunction, urinary abnormalities, and psychological distress. Surgical correction improves patient-reported functional and psychological outcomes and often requires collaboration between plastic and urologic surgeons. To improve postoperative cosmetic outcomes and decrease wound complications following adult-acquired buried penis repair, we performed an anatomic and histologic study of the superficial fascial layers providing support to the external male genitalia and describe our approach for fascial reconstruction. METHODS: We characterized the superficial fascial anatomy in three patients undergoing adult-acquired buried penis repair, including two patients with Wisconsin Type II disease and one patient with Wisconsin Type IV disease. Gross specimens were sent from two patients histologic analysis using H&E and elastin-specific stains to characterize the identity of the superficial fibrofatty tissue. RESULTS: In all three patients, the fundiform ligament overlying the suspensory ligament was identified, isolated, and transected for removal with the suprapubic specimen. We found that reapproximation of this ligament following transection at the time of escutcheonectomy provided significant lift to the penis and genitals via improved support of dartos fascia. Histologic analysis of the superficial fibrofatty tissue located beneath the dermis revealed histologic similarities with the superficial fascial system described previously in abdominal and breast tissue. CONCLUSIONS: Reapproximation of the fundiform ligament and superficial fascial tissue following suprapubic/lower abdominal fat pad removal during adult-acquired buried penis may improve postoperative cosmesis by reducing strain on the dermal closure. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

A Single-resident Experience of Overnight Call Activity at a Multi-site Academic Health System.

OBJECTIVE: To characterize urology resident on-call activities overnight at a multi-site academic medical center and model the expected volume of clinical activity using inpatient beds, emergency room visits, and attendings covered. MATERIALS AND METHODS: On-call activities for 70 13-hour overnight shifts spanning 5 nonconsecutive months between May 2022 and February 2023 were recorded. Clinical coverage included 5 academic hospitals encompassing 1761 staffed inpatient beds and an expected nightly volume of 255 Emergency Department (ED) visits. The time, source, and clinical features of every call were documented. RESULTS: An average of 15 unique calls were received during each shift. Of these, 35% required an in-person evaluation and 12% required a bedside or operative procedure. Approximately a third of calls (36%) were received after midnight. An in-person evaluation occurred within the first hour of 53% of shifts and every shift required at least 1 evaluation. When normalized for inpatient bed volume, an average of 7 unique patient communications occurred per 1000 beds, leading to 2 in-person evaluations. When normalized for an expected number of overnight ED visits, an average of 1 new ED consultation occurred per 100 ED visits. CONCLUSION: After-hours clinical coverage models vary significantly by specialty and institution, and coverage decisions must balance quality clinical care with safe provider workload. Patient needs were appropriately addressed by a single overnight on-call resident, providing a robust clinical experience. The volume of patient care activities in this experience supports the practice of a "night-float" resident with the clear expectation on-site care is required.

Patient Interaction With the Health Care System Following a Nonopioid Discharge Strategy for Anterior Urethroplasty.

INTRODUCTION: Continued efforts have been made to minimize postoperative opioids following urologic interventions. Studies show that patient-reported pain outcomes are similar between those patients discharged with and without opioids following anterior urethroplasty, but we do not know what impact this has on health care utilization. We aim to show that a nonopioid discharge following anterior urethroplasty does not increase postoperative health care utilization. METHODS: Five hundred patients who underwent anterior urethroplasty from January 2016 to October 2022 were identified from retrospective chart review. Patient demographic information, surgical characteristics, and postoperative interactions with the health care system were extracted from the electronic medical record. We then compared these outcomes by discharge opioid prescription status. RESULTS: A total of 253 patients were discharged without an opioid prescription. Patients who received an opioid were more likely to have had a perineal incision (73% vs 64%, P = .02), more likely to have had an overnight hospital stay (30% vs 14%, P < .01), and were more likely to have been prescribed an opioid preoperatively (13% vs 7%, P = .03). There were overall low rates of interaction with the health system in both groups with no significant difference in 30-day unplanned office visits, emergency department visits, or office phone calls. Overall, by the end of our study period 97% were discharged without an opioid and 94% of patients were discharged the same day. CONCLUSIONS: Patients undergoing anterior urethroplasty can safely be discharged home without opioids following surgery without undue postoperative burden on the health care system.

Minimally Invasive Management of Posterior Urethral Stricture/Stenosis with DVIU and Mitomycin C Injection.

OBJECTIVE: To demonstrate a technique for minimally invasive endoscopic management of posterior urethral strictures, including those at the bladder neck and vesicourethral anastomosis. METHODS: Herein, we have included endoscopic video footage from 3 patients with posterior urethral strictures, including 1 at the bladder neck, 1 at the vesicourethral anastomosis, and 1 in the bulbomembranous urethra. In each patient, we perform a direct visualization internal urethrotomy (DVIU) with incisions at the 5 and 7 o'clock positions to widen the urethral lumen, followed by injection of 2 mg mitomycin C (MMC) in a total volume of 5 mL sterile water. RESULTS: Herein, we describe our technique for the endoscopic management of posterior urethral strictures, including those in the prostatic urethra and bladder neck. MMC injection, in conjunction with traditional DVIU, adds minimally to the complexity and length of the procedure but may substantially improve long-term surgical outcomes. CONCLUSION: Bladder outlet obstruction due to stenosis or stricture of the posterior urethra is a common urologic diagnosis whose etiology can often be traced to prior urethral manipulation or iatrogenic trauma. While Americal Urological Assicuation (AUA) guidelines state that dilation or direct visualization internal urethrotomy (DVIU) should be offered for bulbar strictures measuring less than 2 cm in length, recent evidence suggests that DVIU with or without MMC injection may have utility in the management of bladder neck or vesicourethral anastomotic contractures. We have found that DVIU with subsequent MMC injection is a viable minimally invasive approach for the treatment of posterior urethral strictures. While more data are needed to better understand the long-term success rates of these procedures, this approach should be considered for patients with a bladder outlet obstruction secondary to a short stricture of the posterior urethra, bladder neck, or vesicourethral anastomosis.

No-Opioid Discharge Following Artificial Urinary Sphincter Placement Does Not Significantly Increase Health Care System Burden.

INTRODUCTION: Postoperative opioid prescriptions are associated with an increased risk of opioid dependance. While studies on no-opioid discharge strategies have been assessed following many urologic procedures, the effect of no-opioid discharges on health care utilization following artificial urinary sphincter placement is unknown. We performed a single-surgeon retrospective comparison of health care system interactions following artificial urinary sphincter implantation between patients who received an opioid prescription on discharge to those who did not. METHODS: We identified 101 male patients who underwent 3-piece artificial urinary sphincter placement or revision by 1 provider between 2015 and 2022. All patients were discharged with acetaminophen and ibuprofen; none received intraoperative local anesthetic. Demographic information, preprocedural opioid use, opioid prescriptions following the procedure, postoperative office communications, unplanned office visits, and emergency department (ED) visits were recorded for each patient for 90 days. RESULTS: Forty-five patients (45%) were discharged without an opioid prescription and 56 patients (55%) were discharged with an opioid prescription. No differences in age, race, BMI, operative time, or presence of a preoperative opioid prescription were observed. Discharge without an opioid did not significantly increase the number of office communications (55% vs 40%, P = .11), unplanned office visits (36% vs 23%, P = .19), or ED visits (20 vs 12, P = .41) within 90 days of implantation/revision. CONCLUSIONS: Opioids can be omitted from the discharge analgesic regimen following artificial urinary sphincter placement without increasing burden to surgical office staff or local EDs. Providers should consider no-opioid discharges for patients undergoing uncomplicated sphincter placement to limit risk of opioid-related morbidity.

Transcorporal Artificial Urinary Sphincter Technique Video.

OBJECTIVE: The artificial urethral sphincter (AUS) is the gold standard treatment for male stress urinary incontinence which commonly results from prostatectomy or pelvic radiation for prostate cancer. Patients with prior pelvic radiation history experience increased risk of developing urethral erosion. Transcorporal AUS (TAUS) placement can be used as an alternative for compromised urethras to incorporate a small portion of the corporal bodies for additional support. The inclusion of an additional tissue barrier has been shown to improve outcomes. Patients who undergo this technique require device explanation and AUS revision less often than those with AUS devices placed in the standard fashion. Additionally, TAUS placement has been shown to improve functional urinary outcomes such as postoperative Internal prostate symptom score (IPSS), and postoperative IPSS Quality of Life (QoL) scores. MATERIALS AND METHODS: A 67-year-old male with a past medical history of prostate cancer treated with surgery and radiation underwent a TAUS placement which was filmed to demonstrate placement technique and tips. Informed consent was obtained prior to filming this video. RESULTS: This technique can serve as a successful primary or salvage AUS placement technique as seen in this video. CONCLUSION: This video is used to demonstrate the technique of TAUS placement.

Surgical Outcomes and Prediction of Complications Following High-complexity Buried Penis Reconstruction.

PURPOSE: With uniform modern approaches to adult acquired buried penis reconstruction, this study provides updated results on surgical outcomes for complex cases while evaluating the relative influence of medical, surgical, and socioeconomic factors on these results. MATERIALS AND METHODS: Retrospective review was conducted of all patients undergoing initial buried penis reconstruction including escutcheonectomy and penile skin grafting at 1 tertiary center from 2015 through 2022. Summary scores for frailty and socioeconomic status were calculated with the Modified Frailty Index and Area Deprivation Index, respectively. RESULTS: The cohort included 103 patients. Median age was 51 years (IQR 44-65), and median BMI was 43 (IQR 38-49). Frail patients (≥2 Modified Frailty Index risk factors) accounted for 27% of the population while socioeconomic disadvantage (≥85th percentile on Area Deprivation Index) affected 33% of patients. Twenty-eight percent of repairs included a panniculectomy. Rate of revision for a poor outcome was 3.9% with median follow-up of 11 months. Complications were frequent (50%) with most being Clavien I or II (41%) and related to wound dehiscence (31%) or infection (30%). Frail patients had a higher rate of complication (71% vs 41%, P = .01) and were 6 times more likely to experience a complication on multivariable logistic regression (OR 6.41, 95% CI: 1.77-23.22, P = .005). CONCLUSIONS: The modern approach to complex buried penis reconstruction results in a low revision rate; however, low-grade complications are frequent. Patient frailty identifies those at highest risk for complication, offering an opportunity for counseling and preoperative preparation.

RNA Sequencing Identifies Novel NRG1 Fusions in Solid Tumors that Lack Co-Occurring Oncogenic Drivers.

NRG1 gene fusions are rare, therapeutically relevant, oncogenic drivers that occur across solid tumor types. To understand the landscape of NRG1 gene fusions, 4397 solid tumor formalin-fixed, paraffin-embedded samples consecutively tested by comprehensive genomic and immune profiling during standard care were analyzed. Nineteen NRG1 fusions were found in 17 unique patients, across multiple tumor types, including non-small-cell lung (n = 7), breast (n = 2), colorectal (n = 3), esophageal (n = 2), ovarian (n = 1), pancreatic (n = 1), and unknown primary (n = 1) carcinomas, with a cumulative incidence of 0.38%. Fusions were identified with breakpoints across four NRG1 introns spanning 1.4 megabases, with a mixture of known (n = 8) and previously unreported (n = 11) fusion partners. Co-occurring driver alterations in tumors with NRG1 fusions were uncommon, except colorectal carcinoma, where concurrent alterations in APC, BRAF, and ERBB2 were present in a subset of cases. The overall lack of co-occurring drivers highlights the importance of identifying NRG1 gene fusions, as these patients are unlikely to harbor other targetable alterations. In addition, RNA sequencing is important to identify NRG1 gene fusions given the variety of fusion partners and large genomic areas where breakpoints can occur.

An institutional review of genomic sequencing in pediatric solid tumors.

BACKGROUND: Although tumor genomic profiling has aided the advancement of targeted genetic therapy, its clinical integration remains a challenge in pediatric cancers due to lower mutation frequency and less available targeted drugs. There have been multiple novel studies examining molecular sequencing in pediatrics; however, many of these studies primarily utilized large-scale, genome-wide screening applications that limit applicable use due to the availability of testing. This study examined the institutional use of a targeted, clinically available approach to tumor genomic sequencing. METHODS: A retrospective chart review was performed on pediatric patients with solid tumors who were managed at Roswell Park Comprehensive Cancer Center and underwent molecular testing of their tumor biopsy via OmniSeq from August 2016 to July 2021. Results were reviewed for mutations considered to be "actionable" by targeted therapy. Patients with actionable mutations were further examined to evaluate treatment course, receival of targeted therapy, and clinical outcomes. RESULTS: We identified 64 pediatric patients consisting of 20 (31%) with CNS tumors and 44 (69%) with non-CNS tumors, ranging in age from 9 months to 21 years. Thirty-five total actionable mutations were identified amongst 27 patients (42%). Of these 27, 12 patients (44%) received at least 1 targeted drug against a respective actionable mutation, of which 6 patients (50%) achieved clinical benefit to therapy, including 1 complete response. CONCLUSIONS: The use of a clinically focused and readily available targeted molecular sequencing panel identified actionable mutations at a comparable rate to the large-scale, less readily available sequencing panels utilized in other studies. Half of our patients who received targeted therapy achieved a complete response or clinical benefit from therapy. Although targeted therapy has a role in pediatric cancer treatment, many newer drugs require further research on their safety and efficacy.

Effect of Plasma and Blood Donations on Levels of Perfluoroalkyl and Polyfluoroalkyl Substances in Firefighters in Australia: A Randomized Clinical Trial.

Importance: Elevated levels of blood perfluoroalkyl and polyfluoroalkyl substances (PFASs) have been associated with a range of adverse health outcomes. Firefighters have been exposed to PFASs in firefighting foams and have previously been shown to have higher PFAS levels in blood samples than the general population. No interventions have been shown to reduce PFAS levels. Objective: To examine the effect of blood or plasma donations on PFAS levels in firefighters in Australia. Design, Setting, and Participants: This 52-week, open-label, randomized clinical trial enrolled participants from May 23 to August 23, 2019. Participants were 285 Fire Rescue Victoria staff or contractors with serum levels of perfluorooctane sulfonate (PFOS) of 5 ng/mL or more who were eligible to donate blood, had not donated blood in the 3 months prior to randomization, and were able to provide written informed consent. Analysis was performed on an intention-to-treat basis from May to July 2021. Interventions: Firefighters with baseline PFOS levels of 5 ng/mL or more were randomly assigned to donate plasma every 6 weeks for 12 months, donate blood every 12 weeks for 12 months, or be observed only. Main Outcomes and Measures: The primary end points were changes in the serum PFOS and perfluorohexane sulfonic acid (PFHxS) levels after 12 months of plasma or blood donations or after observation only. Secondary end points included changes in serum PFAS levels from week 52 to week 64, changes in other PFASs, and changes in complete blood count, biochemistry, thyroid function, and lipid profile from screening to week 52. Results: A total of 285 firefighters (279 men [97.9%]; mean [SD] age, 53.0 [8.4] years) were enrolled; 95 were randomly assigned to donate plasma, 95 were randomly assigned to donate blood, and 95 were randomly assigned to be observed. The mean level of PFOS at 12 months was significantly reduced by plasma donation (-2.9 ng/mL; 95% CI, -3.6 to -2.3 ng/mL; P < .001) and blood donation (-1.1 ng/mL; 95% CI, -1.5 to -0.7 ng/mL; P < .001) but was unchanged in the observation group. The mean level of PFHxS was significantly reduced by plasma donation (-1.1 ng/mL; 95% CI, -1.6 to -0.7 ng/mL; P < .001), but no significant change was observed in the blood donation or observation groups. Analysis between groups indicated that plasma donation had a larger treatment effect than blood donation, but both were significantly more efficacious than observation in reducing PFAS levels. Conclusions and Relevance: Plasma and blood donations caused greater reductions in serum PFAS levels than observation alone over a 12-month period. Further research is needed to evaluate the clinical implications of these findings. Trial Registration: anzctr.org.au Identifier: ACTRN12619000204145.

A scalable high-throughput targeted next-generation sequencing assay for comprehensive genomic profiling of solid tumors.

Timely and accurate identification of molecular alterations in solid tumors is essential for proper management of patients with advanced cancers. This has created a need for rapid, scalable comprehensive genomic profiling (CGP) systems that detect an increasing number of therapeutically-relevant variant types and molecular signatures. In this study, we assessed the analytical performance of the TruSight Oncology 500 High-Throughput assay for detection of somatic alterations from formalin-fixed paraffin-embedded tissue specimens. In parallel, we developed supporting software and automated sample preparation systems designed to process up to 70 clinical samples in a single NovaSeq 6000TM sequencing run with a turnaround time of <7 days from specimen receipt to report. The results demonstrate that the scalable assay accurately and reproducibly detects small variants, copy number alterations, microsatellite instability (MSI) and tumor mutational burden (TMB) from 40ng DNA, and multiple gene fusions, including known and unknown partners and splice variants from 20ng RNA. 717 tumor samples and reference materials with previously known alterations in 96 cancer-related genes were sequenced to evaluate assay performance. All variant classes were reliably detected at consistent and reportable variant allele percentages with >99% overall accuracy and precision. Our results demonstrate that the high-throughput CGP assay is a reliable method for accurate detection of molecular alterations in support of precision therapeutics in oncology. The supporting systems and scalable workflow allow for efficient interpretation and prompt reporting of hundreds of patient cancer genomes per week with excellent analytical performance.

Association Between Dense Breast Legislation and Cancer Stage at Diagnosis.

INTRODUCTION: Many states have mandated breast density notification and insurance coverage for additional screening; yet, the association between such legislation and stage of diagnosis for breast cancer is unclear. This study investigates this association and examines the differential impacts among different age and race/ethnicity subgroups. METHODS: The Surveillance, Epidemiology, and End Results database was queried to identify patients with breast cancer aged 40-74 years diagnosed between 2005 and 2016. Using a difference-in-differences multinomial logistic model, the odds of being diagnosed at different stages of cancer relative to the localized stage depending on legislation and individual characteristics were examined. Analyses were conducted in 2020-2021. RESULTS: The study included 689,641 cases. Overall, the impact of notification legislation was not significant, whereas insurance coverage legislation was associated with 6% lower odds (OR=0.94, 95% CI=0.91, 0.96) of being diagnosed at the regional stage. The association between insurance coverage legislation and stage of diagnosis was even stronger among women aged 40-49 years, with 11% lower odds (OR=0.89, 95% CI=0.82, 0.96) of being diagnosed at the regional stage and 12% lower odds (OR=0.88, 95% CI=0.81, 0.96) of being diagnosed at the distant stage. Hispanic women benefited from notification laws, with 11% lower odds (OR=0.89, 95% CI=0.82, 0.97) of being diagnosed at distant stage. Neither notification nor supplemental screening insurance coverage legislation showed a substantial impact on Black women. CONCLUSIONS: The findings imply that improving insurance coverage is more important than being notified overall. Raising awareness is important among Hispanic women; improving communication about dense breasts and access to screening might be more important than legislation among Black women.

Integration of tumor inflammation, cell proliferation, and traditional biomarkers improves prediction of immunotherapy resistance and response.

BACKGROUND: Contemporary to the rapidly evolving landscape of cancer immunotherapy is the equally changing understanding of immune tumor microenvironments (TMEs) which is crucial to the success of these therapies. Their reliance on a robust host immune response necessitates clinical grade measurements of immune TMEs at diagnosis. In this study, we describe a stable tumor immunogenic profile describing immune TMEs in multiple tumor types with ability to predict clinical benefit from immune checkpoint inhibitors (ICIs). METHODS: A tumor immunogenic signature (TIGS) was derived from targeted RNA-sequencing (RNA-seq) and gene expression analysis of 1323 clinical solid tumor cases spanning 35 histologies using unsupervised analysis. TIGS correlation with ICI response and survival was assessed in a retrospective cohort of NSCLC, melanoma and RCC tumor blocks, alone and combined with TMB, PD-L1 IHC and cell proliferation biomarkers. RESULTS: Unsupervised clustering of RNA-seq profiles uncovered a 161 gene signature where T cell and B cell activation, IFNg, chemokine, cytokine and interleukin pathways are over-represented. Mean expression of these genes produced three distinct TIGS score categories: strong (n = 384/1323; 29.02%), moderate (n = 354/1323; 26.76%), and weak (n = 585/1323; 44.22%). Strong TIGS tumors presented an improved ICI response rate of 37% (30/81); with highest response rate advantage occurring in NSCLC (ORR = 36.6%; 16/44; p = 0.051). Similarly, overall survival for strong TIGS tumors trended upward (median = 25 months; p = 0.19). Integrating the TIGS score categories with neoplastic influence quantified via cell proliferation showed highly proliferative and strong TIGS tumors correlate with significantly higher ICI ORR than poorly proliferative and weak TIGS tumors [14.28%; p = 0.0006]. Importantly, we noted that strong TIGS and highly [median = not achieved; p = 0.025] or moderately [median = 16.2 months; p = 0.025] proliferative tumors had significantly better survival compared to weak TIGS, highly proliferative tumors [median = 7.03 months]. Importantly, TIGS discriminates subpopulations of potential ICI responders that were considered negative for response by TMB and PD-L1. CONCLUSIONS: TIGS is a comprehensive and informative measurement of immune TME that effectively characterizes host immune response to ICIs in multiple tumors. The results indicate that when combined with PD-L1, TMB and cell proliferation, TIGS provides greater context of both immune and neoplastic influences on the TME for implementation into clinical practice.

Urinary tract infections: microbial pathogenesis, host-pathogen interactions and new treatment strategies.

Urinary tract infections (UTIs) are common, recurrent infections that can be mild to life-threatening. The continued emergence of antibiotic resistance, together with our increasing understanding of the detrimental effects conferred by broad-spectrum antibiotic use on the health of the beneficial microbiota of the host, has underscored the weaknesses in our current treatment paradigm for UTIs. In this Review, we discuss how recent microbiological, structural, genetic and immunological studies have expanded our understanding of host-pathogen interactions during UTI pathogenesis. These basic scientific findings have the potential to shift the strategy for UTI treatment away from broad-spectrum antibiotics targeting conserved aspects of bacterial replication towards pathogen-specific antibiotic-sparing therapeutics that target core determinants of bacterial virulence at the host-pathogen interface.

Factors Associated With Hospital Decisions to Purchase Robotic Surgical Systems.

Background. Robotic surgical systems are expensive to own and operate, and the purchase of such technology is an important decision for hospital administrators. Most prior literature focuses on the comparison of clinical outcomes between robotic surgery and other laparoscopic or open surgery. There is a knowledge gap about what drives hospitals' decisions to purchase robotic systems. Objective. To identify factors associated with a hospital's acquisition of advanced surgical systems. Method. We used 2002 to 2011 data from the State of California Office of Statewide Health Planning and Development to examine robotic surgical system purchase decisions of 476 hospitals. We used a probit estimation allowing heteroscedasticity in the error term including a set of two equations: one binary response equation and one heteroscedasticity equation. Results. During the study timeframe, there were 78 robotic surgical systems purchased by hospitals in the sample. Controlling for hospital characteristics such as number of available beds, teaching status, nonprofit status, and patient mix, the probit estimation showed that market-level directly relevant surgery volume in the previous year (excluding the hospital's own volume) had the largest impact. More specifically, hospitals in high volume (>50,000 surgeries v. 0) markets were 12 percentage points more likely to purchase robotic systems. We also found that hospitals in less competitive markets (i.e., Herfindahl index above 2500) were 2 percentage points more likely to purchase robotic systems. Limitations. This study has limitations common to observational database studies. Certain characteristics such as cultural factors cannot be accurately quantified. Conclusions. Our findings imply that potential market demand is a strong driver for hospital purchase of robotic surgical systems. Market competition does not significantly increase the adoption of new expensive surgical technologies.

Current Policy Challenges in Genomic Medicine.

BACKGROUND: Molecular genetic testing has raised a variety of policy issues, ranging from privacy to reimbursement. Recently, payment policies have become of paramount importance as Medicare implemented the first significant change to test pricing since 1984 and announced a broad national coverage policy for the use of next-generation sequencing (NGS) in cancer patients that contains significant restrictions. Regulatory and oversight concerns have been important topics for discussion as the US Food and Drug Administration (FDA), Congress, and stakeholders have focused on new approaches to regulation of laboratory-developed tests (LDTs). Patents on gene sequences and relationships between genetic variants and clinical phenotypes have been points of contention since the field's inception. Two Supreme Court cases invalidated patents on gene sequences and biological relationships, ushering in the era of NGS and precision medicine. However, a recent legislative proposal threatens to reverse these gains and restore gene patents as barriers to progress in genetic and genomic testing and the implementation of genomic medicine. CONTENT: This review discusses current issues in payment policy, laboratory oversight, and gene patenting and their potential impacts on genetic and genomic testing. SUMMARY: Coverage and reimbursement policies present serious challenges to genetic and genomic testing. The potential for FDA regulation of LDTs looms as a significant threat to diagnostic innovation, patient access, and the viability of molecular genetic testing laboratories. Changes in patent law could cause gene patents to reemerge as barriers to the advancement of genomic medicine.

Biphenyl Gal and GalNAc FmlH Lectin Antagonists of Uropathogenic E. coli (UPEC): Optimization through Iterative Rational Drug Design.

The F9/Yde/Fml pilus, tipped with the FmlH adhesin, has been shown to provide uropathogenic Escherichia coli (UPEC) a fitness advantage in urinary tract infections (UTIs). Here, we used X-ray structure guided design to optimize our previously described ortho-biphenyl Gal and GalNAc FmlH antagonists such as compound 1 by replacing the carboxylate with a sulfonamide as in 50. Other groups which can accept H-bonds were also tolerated. We pursued further modifications to the biphenyl aglycone resulting in significantly improved activity. Two of the most potent compounds, 86 (IC50 = 0.051 µM) and 90 (IC50 = 0.034 µM), exhibited excellent metabolic stability in mouse plasma and liver microsomes but showed only limited oral bioavailability (<1%) in rats. Compound 84 also showed a good pharmacokinetic (PK) profile in mice after IP dosing with compound exposure above the IC50 for 6 h. These new FmlH antagonists represent new antivirulence drugs for UTIs.

Structure-Function Analysis of the Curli Accessory Protein CsgE Defines Surfaces Essential for Coordinating Amyloid Fiber Formation.

Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed primarily of the major structural subunit CsgA. The CsgE chaperone facilitates the secretion of CsgA through CsgG by forming a cap at the base of the nonameric CsgG outer membrane pore. We elucidated a series of finely tuned nonpolar and charge-charge interactions that facilitate the oligomerization of CsgE and its ability to transport unfolded CsgA to CsgG for translocation. CsgE oligomerization in vitro is temperature dependent and is disrupted by mutations in the W48 and F79 residues. Using nuclear magnetic resonance (NMR), we identified two regions of CsgE involved in the CsgE-CsgA interaction: a head comprising a positively charged patch centered around R47 and a stem comprising a negatively charged patch containing E31 and E85. Negatively charged residues in the intrinsically disordered N- and C-terminal "tails" were not implicated in this interaction. Head and stem residues were mutated and interrogated using in vivo measurements of curli production and in vitro amyloid polymerization assays. The R47 head residue of CsgE is required for stabilization of CsgA- and CsgE-mediated curli fiber formation. Mutation of the E31 and E85 stem residues to positively charged side chains decreased CsgE-mediated curli fiber formation but increased CsgE-mediated stabilization of CsgA. No single-amino-acid substitutions in the head, stem, or tail regions affected the ability of CsgE to cap the CsgG pore as determined by a bile salt sensitivity assay. These mechanistic insights into the directed assembly of functional amyloids in extracellular biofilms elucidate possible targets for biofilm-associated bacterial infections.IMPORTANCE Curli represent a class of functional amyloid fibers produced by Escherichia coli and other Gram-negative bacteria that serve as protein scaffolds in the extracellular biofilm matrix. Despite the lack of sequence conservation among different amyloidogenic proteins, the structural and biophysical properties of functional amyloids such as curli closely resemble those of amyloids associated with several common neurodegenerative diseases. These parallels are underscored by the observation that certain proteins and chemicals can prevent amyloid formation by the major curli subunit CsgA and by alpha-synuclein, the amyloid-forming protein found in Lewy bodies during Parkinson's disease. CsgA subunits are targeted to the CsgG outer membrane pore by CsgE prior to secretion and assembly into fibers. Here, we use biophysical, biochemical, and genetic approaches to elucidate a mechanistic understanding of CsgE function in curli biogenesis.