Structural Basis for the Interaction of Redß Single-Strand Annealing Protein with Escherichia coli Single-Stranded DNA-Binding Protein.

Redß is a protein from bacteriophage λ that binds to single-stranded DNA (ssDNA) to promote the annealing of complementary strands. Together with λ-exonuclease (λ-exo), Redß is part of a two-component DNA recombination system involved in multiple aspects of genome maintenance. The proteins have been exploited in powerful methods for bacterial genome engineering in which Redß can anneal an electroporated oligonucleotide to a complementary target site at the lagging strand of a replication fork. Successful annealing in vivo requires the interaction of Redß with E. coli single-stranded DNA-binding protein (SSB), which coats the ssDNA at the lagging strand to coordinate access of numerous replication proteins. Previous mutational analysis revealed that the interaction between Redß and SSB involves the C-terminal domain (CTD) of Redß and the C-terminal tail of SSB (SSB-Ct), the site for binding of numerous host proteins. Here, we have determined the x-ray crystal structure of Redß CTD in complex with a peptide corresponding to the last nine residues of SSB (MDFDDDIPF). Formation of the complex is predominantly mediated by hydrophobic interactions between two phenylalanine side chains of SSB (Phe-171 and Phe-177) and an apolar groove on the CTD, combined with electrostatic interactions between the C-terminal carboxylate of SSB and Lys-214 of the CTD. Mutation of any of these residues to alanine significantly disrupts the interaction of full-length Redß and SSB proteins. Structural knowledge of this interaction will help to expand the utility of Redß-mediated recombination to a wider range of bacterial hosts for applications in synthetic biology.

Structure of a RecT/Redß family recombinase in complex with a duplex intermediate of DNA annealing.

Some bacteriophage encode a recombinase that catalyzes single-stranded DNA annealing (SSA). These proteins are apparently related to RAD52, the primary human SSA protein. The best studied protein, Redß from bacteriophage λ, binds weakly to ssDNA, not at all to dsDNA, but tightly to a duplex intermediate of annealing formed when two complementary DNA strands are added to the protein sequentially. We used single particle cryo-electron microscopy (cryo-EM) to determine a 3.4 Å structure of a Redß homolog from a prophage of Listeria innocua in complex with two complementary 83mer oligonucleotides. The structure reveals a helical protein filament bound to a DNA duplex that is highly extended and unwound. Native mass spectrometry confirms that the complex seen by cryo-EM is the predominant species in solution. The protein shares a common core fold with RAD52 and a similar mode of ssDNA-binding. These data provide insights into the mechanism of protein-catalyzed SSA.

Mutational Analysis of Redß Single Strand Annealing Protein: Roles of the 14 Lysine Residues in DNA Binding and Recombination In Vivo.

Redß is a 261 amino acid protein from bacteriophage λ that promotes a single-strand annealing (SSA) reaction for repair of double-stranded DNA (dsDNA) breaks. While there is currently no high-resolution structure available for Redß, models of its DNA binding domain (residues 1-188) have been proposed based on homology with human Rad52, and a crystal structure of its C-terminal domain (CTD, residues 193-261), which binds to λ exonuclease and E. coli single-stranded DNA binding protein (SSB), has been determined. To evaluate these models, the 14 lysine residues of Redß were mutated to alanine, and the variants tested for recombination in vivo and DNA binding and annealing in vitro. Most of the lysines within the DNA binding domain, including K36, K61, K111, K132, K148, K154, and K172, were found to be critical for DNA binding in vitro and recombination in vivo. By contrast, none of the lysines within the CTD, including K214, K245, K251, K253, and K258 were required for DNA binding in vitro, but two, K214 and K253, were critical for recombination in vivo, likely due to their involvement in binding to SSB. K61 was identified as a residue that is critical for DNA annealing, but not for initial ssDNA binding, suggesting a role in binding to the second strand of DNA incorporated into the complex. The K148A variant, which has previously been shown to be defective in oligomer formation, had the lowest affinity for ssDNA, and was the only variant that was completely non-cooperative, suggesting that ssDNA binding is coupled to oligomerization.

Oligomeric complexes formed by Redß single strand annealing protein in its different DNA bound states.

Redß is a single strand annealing protein from bacteriophage λ that binds loosely to ssDNA, not at all to pre-formed dsDNA, but tightly to a duplex intermediate of annealing. As viewed by electron microscopy, Redß forms oligomeric rings on ssDNA substrate, and helical filaments on the annealed duplex intermediate. However, it is not clear if these are the functional forms of the protein in vivo. We have used size-exclusion chromatography coupled with multi-angle light scattering, analytical ultracentrifugation and native mass spectrometry (nMS) to characterize the size of the oligomers formed by Redß in its different DNA-bound states. The nMS data, which resolve species with the highest resolution, reveal that Redß forms an oligomer of 12 subunits in the absence of DNA, complexes ranging from 4 to 14 subunits on 38-mer ssDNA, and a much more distinct and stable complex of 11 subunits on 38-mer annealed duplex. We also measure the concentration of Redß in cells active for recombination and find it to range from 7 to 27 µM. Collectively, these data provide new insights into the dynamic nature of the complex on ssDNA, and the more stable and defined complex on annealed duplex.

Optimal Approach to Small Unilateral Testicular Nodules-Can We Reliably Predict Tumor Histology Based on Size?

INTRODUCTION: We evaluated primary testicular tumor characteristics associated with nongerm cell tumor histology and potential appropriateness for testis sparing surgery in selected patients from our institution. METHODS: We retrospectively reviewed medical records of patients undergoing surgery for testicular masses between 2003 and 2015. We included patients with unilateral testicular tumors, normal preoperative serum tumor markers and no preoperative evidence of metastatic spread. Demographic and clinical information were extracted. The primary outcome studied was tumor pathology, germ cell tumor histology vs nongerm cell histology. We compared patients in these cohorts based on the testicular tumor size. RESULTS: A total of 48 patients met study criteria, 18 (37.5%) of whom had a final pathology consistent with nongerm cell histology. In general, the median tumor size was less in the nongerm cell group (11 mm vs 27 mm, p=0.001). Tumor size less than 2 cm was associated with increased likelihood of nongerm cell histology (p=0.003) with 61.9% of those with tumors less than 2 cm harboring nongerm cell tumors and therefore likely appropriate for organ-sparing surgery. A receiver operating characteristic analysis demonstrated a maximum sensitivity and specificity for selecting masses with normal tumor markers as having nongerm cell histology at a size cutoff of 18 mm. CONCLUSIONS: It appears that a majority of patients with localized small testicular masses and nonelevated tumor markers will have nongerm cell histology, which makes them potentially eligible for testicular sparing surgery at centers with expertise in intraoperative frozen section analysis.

A decision tree to guide long term venous access placement in children and adolescents undergoing surgery for renal tumors.

BACKGROUND/PURPOSE: While many children with renal tumors require long term venous access (VA) for adjuvant chemotherapy, certainly not all do. This study develops and tests a VA decision tree (DT) to direct the placement of VA in patients with renal tumors. METHODS: Utilizing data readily available at surgery a VADT was developed. The VADT was tested retrospectively by 2 independent reviewers on a historic cohort. The ability of the VADT to appropriately select which patients would benefit from VA placement was tested. RESULTS: 160 patients underwent renal tumor surgery between 2005 and 2018. 70 (43.8%) patients met study criteria with median age of 45.1 months (range 1.1-224); 73% required VA. Using the VADT, VA placement was "needed" in 67.1% of patients and "deferred" in 32.9%. Interrater reliability was very high (kappa = 0.97, 95% CI 0.91-1, p < 0.001). The sensitivity and specificity of the VADT to correctly decide on VA placement were 0.92 (0.8-0.98) and 1 (0.79-1). Using the VADT, no patient would have undergone unnecessary VA placement. In reality, 4.3% of patients had an unnecessary VA placed which required a subsequent removal. CONCLUSIONS: These preliminary data support the continued study of this VADT to guide intraoperative decisions regarding VA placement in patients with renal tumors. LEVEL OF EVIDENCE: III - Study of diagnostic test.

Differential neurodegenerative phenotypes are associated with heterogeneous voiding dysfunction in a coronavirus-induced model of multiple sclerosis.

Patients with multiple sclerosis (MS) develop a variety of lower urinary tract symptoms (LUTS). We previously characterized a murine model of neurogenic bladder dysfunction induced by a neurotropic strain of a coronavirus. In the present study, we further study the role of long-lasting neurodegeneration on the development of neurogenic bladder dysfunction in mice with corona-virus induced encephalitis (CIE). Long-term follow up study revealed three phenotypes of neurodegenerative symptom development: recovery (REC group), chronic progression (C-PRO group) and chronic disease with relapsing-remitting episodes (C-RELAP group). The levels of IL-1ß in REC group, IL-10 in C-RELAP group, and IL-1ß, IL-6, IL-10 and TNF-α in C-PRO group were diminished in the brain. The levels of TNF-α in REC group and INF-γ, IL-2, TGF-ß and TNF-α in the C-PRO group were also diminished in the urinary bladder. Mice in C-RELAP group showed a delayed recovery of voiding function. In vitro contractility studies determined a decreased basal detrusor tone and reduced amplitude of nerve-mediated contractions in C-RELAP group, whereas C-PRO group had elevated muscle-mediated contractions. In conclusion, mice with CIE developed three phenotypes of neurologic impairment mimicking different types of MS progression in humans and showed differential mechanisms driving neurogenic bladder dysfunction.

Structure and mechanism of the Red recombination system of bacteriophage λ.

While much of this volume focuses on mammalian DNA repair systems that are directly involved in genome stability and cancer, it is important to still be mindful of model systems from prokaryotes. Herein we review the Red recombination system of bacteriophage λ, which consists of an exonuclease for resecting dsDNA ends, and a single-strand annealing protein (SSAP) for binding the resulting 3'-overhang and annealing it to a complementary strand. The genetics and biochemistry of Red have been studied for over 50 years, in work that has laid much of the foundation for understanding DNA recombination in higher eukaryotes. In fact, the Red exonuclease (λ exo) is homologous to Dna2, a nuclease involved in DNA end-resection in eukaryotes, and the Red annealing protein (Redß) is homologous to Rad52, the primary SSAP in eukaryotes. While eukaryotic recombination involves an elaborate network of proteins that is still being unraveled, the phage systems are comparatively simple and streamlined, yet still encompass the fundamental features of recombination, namely DNA end-resection, homologous pairing (annealing), and a coupling between them. Moreover, the Red system has been exploited in powerful methods for bacterial genome engineering that are important for functional genomics and systems biology. However, several mechanistic aspects of Red, particularly the action of the annealing protein, remain poorly understood. This review will focus on the proteins of the Red recombination system, with particular attention to structural and mechanistic aspects, and how the lessons learned can be applied to eukaryotic systems.

Crystal structure of the Redß C-terminal domain in complex with λ Exonuclease reveals an unexpected homology with λ Orf and an interaction with Escherichia coli single stranded DNA binding protein.

Bacteriophage λ encodes a DNA recombination system that includes a 5'-3' exonuclease (λ Exo) and a single strand annealing protein (Redß). The two proteins form a complex that is thought to mediate loading of Redß directly onto the single-stranded 3'-overhang generated by λ Exo. Here, we present a 2.3 Å crystal structure of the λ Exo trimer bound to three copies of the Redß C-terminal domain (CTD). Mutation of residues at the hydrophobic core of the interface disrupts complex formation in vitro and impairs recombination in vivo. The Redß CTD forms a three-helix bundle with unexpected structural homology to phage λ Orf, a protein that binds to E. coli single-stranded DNA binding protein (SSB) to function as a recombination mediator. Based on this relationship, we found that Redß binds to full-length SSB, and to a peptide corresponding to its nine C-terminal residues, in an interaction that requires the CTD. These results suggest a dual role of the CTD, first in binding to λ Exo to facilitate loading of Redß directly onto the initial single-stranded DNA (ssDNA) at a 3'-overhang, and second in binding to SSB to facilitate annealing of the overhang to SSB-coated ssDNA at the replication fork.

A Study of Adolescent Female Genitalia: What is Normal?

STUDY OBJECTIVE: Female adolescents often present to health care providers with concerns about the appearance of their external genitalia. These patients might experience significant distress about their genital appearance and might request surgery to correct a perceived abnormality. Accurate descriptions of normal adolescent female genital anatomy are lacking in the literature. The purpose of this study was to examine a small sample of normal female adolescents to obtain measurements and descriptors of the external genital structures, with a focus on the size and morphology of the labia minora. DESIGN, SETTING, PARTICIPANTS, INTERVENTIONS, AND MAIN OUTCOME MEASURES: Participants were female adolescent patients, ages 10-19 years, who underwent routine surgical procedures in the operating room. RESULTS: Forty-four patients were examined. The mean age was 14.4 years (range 10-19 years). Mean height was 159.6 cm and mean weight was 60.8 kg. Most were non-Hispanic ethnicity (n = 32/44; 72%) and were Caucasian race (n = 38/44; 86%). Right and left labia minora lengths were different in n = 19/44 patients (43%). Right and left labia minora widths also differed, in stretched (n = 20/33; 61%) and unstretched (n = 24/44; 55%) labia, with a difference ranging from 1 to 22 mm. There was no correlation between size and shape of labia minora and patient age, height, weight, or race. CONCLUSION: Wide variability exists in female adolescent genital anatomy with no established normal range. This study provides a resource for physicians who care for adolescent girls, who need normative data to describe female genital anatomy. We propose that the role of labiaplasty in adolescents should be considered with extreme caution because of the wide range in size and morphology and paucity of data in this population.

Reliability of intraoperative frozen section for the diagnosis of renal tumors suspicious for malignancy in children and adolescents.

BACKGROUND: The ability of intraoperative frozen section (IFS) to reliably diagnose renal tumors in children and adolescents is largely unknown. The objective of our study is to evaluate the ability of IFS to establish a histologic diagnosis for renal tumors in this population. METHODS: We reviewed our experience with patients who underwent IFS at the time of surgery for a renal tumor suspicious for malignancy from 2005 to 2015. The IFS was compared to the final pathology (FP). Data on concordance and reliability were analyzed. RESULTS: One hundred thirty patients underwent surgical interventions for a renal tumor suspicious for malignancy, and 32 (25%) patients underwent IFS. Median turnaround time for IFS was 20 min (range 13-44). The histologic IFS diagnosis correlated with FP in 26 (81.2%) cases was discrepant in three (9.4%) cases, and IFS was deferred to FP in three (9.4%) cases (kappa 0.71, 95% confidence interval [CI]: 0.52-0.899, P < 0.001). The IFS correctly distinguished between Wilms tumor and non-Wilms tumor in 30 (94%) cases (kappa 0.874, 95% CI: 0.705-1, P < 0.001). A total of 17 of 19 (89.5%) Wilms tumors were correctly diagnosed by IFS, yielding a sensitivity of 0.89 (95% CI: 0.67-0.99) and a specificity of 1 (95% CI: 0.75-1). CONCLUSION: IFS is a reliable tool to establish a histologic diagnosis and to differentiate between Wilms and non-Wilms tumors in children and adolescents with renal tumors. The use of IFS should be encouraged in cases in which obtaining a diagnosis will provide guidance for important "real-time" medical decision making, specifically additional adjunctive surgical procedures.

Large Cystic Metanephric Adenoma in a 15-Year-Old Girl.

Metanephric adenoma is a rare pediatric renal tumor, generally considered to be benign. It can be difficult to distinguish from Wilms tumor and renal cell carcinoma based on imaging alone, and even may be difficult on histopathologic analysis. We present a case of a large cystic metanephric adenoma managed with surgical resection. This case highlights the difficulty in managing cystic renal lesions in children and adolescents as there is a paucity of data on the radiologic and pathologic correlation in such patients.

Long-term urological outcomes in cloacal anomalies.

Cloacal anomalies are the most complex and severe form of congenital anorectal malformations (ARM) and urogenital malformations, and it has been well documented that increased severity of ARM leads to worse outcomes. While short-term data on persistent cloaca are available, a paucity of data on long-term outcomes exists, largely because of a lack of uniform terminology, inclusion with other ARM and evolution of the operative technique. On comprehensive review of the published literature on long-term urological outcomes in patients with cloacal anomalies, we found a significant risk of chronic kidney disease and incontinence, however, with improvements in surgical technique, outcomes have improved. Continence often requires intermittent catheterization and in some cases, bladder augmentation. The complexity of cloacal malformations and associated anomalies make long-term multidisciplinary follow-up imperative.

Do Referral Patterns in Adolescents and Young Adults with Testicular Cancer Impact Oncologic Outcomes?

PURPOSE: Oncologic outcomes in advanced testicular cancer (TC) depend on appropriate and timely care. Often this care is referred to tertiary academic medical centers (AMCs). The aim of this study was to compare oncologic outcomes of adolescent and young adult (AYA) patients with TC treated from the outset at an AMC to those whose care was initiated elsewhere with subsequent referral. METHODS: An institutional TC database was reviewed, and those AYA patients initiating TC care either inside or outside an AMC were compared. Patients were classified as initiating care outside if they had any non-orchiectomy surgery, chemotherapy, or radiotherapy for TC outside an AMC. RESULTS: A total of 183 patients were reviewed, of whom 59 initiated TC care outside and 124 were managed initially at an AMC. Patients initiating care outside were more likely to have non-seminoma histology and more often presented with metastatic disease (Stage II [30.5%] or III [35.6%] vs. Stage II [19.4%] or III [19.4%]; p = 0.007). Lower 3-year event-free survival (EFS) was observed in those initiating treatment outside an AMC (60.6% vs. 78.7%; p = 0.027). However, on multivariate analysis adjusting for stage and histology, the location of initiating TC care was no longer significant (hazard ratio = 1.5, 95% confidence interval 0.8-2.9). CONCLUSION: AYA patients initially treated for TC in the community and subsequently referred to an AMC were initially observed to experience worse EFS than those who were managed at an AMC from the outset. However, on multivariate analysis, these findings were largely explained by referral bias, where AYA patients with advanced disease were more likely to be referred to AMCs.

Current urologic care for testicular germ cell tumors in pediatric and adolescent patients.

Testicular germ cell tumors make up 0.5% of pediatric malignancies, and 14% of adolescent malignancies. Young boys have primarily pure teratoma and pure yolk sac histologies; however, adolescent histology is mostly mixed nonseminomatous germ cell tumor. Surgical excision of the primary tumor is the crux of treatment. Chemotherapy, retroperitoneal lymph node dissection, and targeted treatment of distant metastases make even widely disseminated disease treatable. Since the discovery of platinum-based chemotherapy, testicular germ cell tumors are a highly curable disease. However, adolescents remain the group with the highest mortality. Focus has expanded beyond survival to emphasize quality of life issues when optimizing treatment algorithms.

Variations in the circle of Willis in the New Zealand white rabbit.

PURPOSE: The New Zealand White rabbit (NZWR) serves an important role as an experimental model for vascular research, specifically in the area of stroke. Here the authors document vascular variations in the circle of Willis (COW). MATERIALS AND METHODS: Subselective internal carotid digital subtraction angiography was performed in 100 NZWRs. RESULTS: Important variations include hypoplasia in 36%, duplication of the middle cerebral artery in 29%, asymmetries of the posterior region in 19%, and multiple variations in 28%. The complete classical symmetric COW without significant variation is present in fewer than 30% of animals. CONCLUSIONS: With recognition of the variations, the NZWR becomes an improved research model.

Comparative efficacy of three commercial PCV2 vaccines in conventionally reared pigs.

The aim of this study was to compare the efficacy of three commercial porcine circovirus type 2 (PCV2) vaccines. Twenty 3-week old pigs were injected with one of three different vaccines, A, B, C, or were unvaccinated controls. Pigs in group A were given a second dose at 6 weeks of age according to the manufacturer's protocol. Antibody titers in all pigs declined gradually, but increased sharply by 22 weeks of age. Viremia in the three vaccinated groups at 22 weeks of age was significantly less than in the control group. The average daily gains of pigs in groups A-C, and in controls were 0.81 ± 0.03, 0.80 ± 0.05, 0.78 ± 0.04, and 0.74 ± 0.05 kg, respectively. It was concluded that PCV2 vaccine reduced viremia and improved weight gain, but differences in weight gain among vaccinated groups were not statistically significant.

Seasonal variation in physiological fitness of a semiprofessional soccer team.

The purpose of this study was to investigate seasonal variations in physiological fitness of semiprofessional soccer players over a 12-month period. Thirteen male players were tested 5 times over a 12-month period using bioelectrical impedance, a 20-m multistage fitness test, countermovement standing vertical jump, 15-m sprint test, Illinois agility test, and sit and reach test. Significant deconditioning was apparent in all fitness variables from end of season one season to prepreseason training of the next season. Aerobic fitness, vertical jump, percent body fat, agility, and sprint performance improved from prepreseason to midseason. Significant decreases in aerobic fitness and the cessation of significant increases in vertical jump, sprint, and agility performance were shown from midseason onward. No differences between the fitness components at the end of season one and the end of season two were identified. The deconditioning apparent in all fitness parameters during the off season, together with progressive improvement in most from postpreseason to midseason would support these parameters as sport-specific fitness requirements. Such improvements suggest that the short-term demands of playing and training in the first half of the season develop fitness and these trends are similar to those for professional players. Body fat was also shown to be detrimental to sprint performance throughout the 12-month period. Further research is needed to identify if the plateau in fitness from midseason is the result of attaining the required level of fitness, fatigue, allied training, or even relative success. Enhancing off-season training may enable yearly fitness increases by at least maintaining fitness levels for the next year's preseason.