Sean Ryan

Single continuous lumen formation in the zebrafish gut is mediated by smoothened-dependent tissue remodeling

The formation of a single lumen during tubulogenesis is crucial for the development and function of many organs. Although 3D cell culture models have identified molecular mechanisms controlling lumen formation in vitro, their function during vertebrate organogenesis is poorly understood. Using light sheet microscopy and genetic approaches we have investigated single lumen formation in the zebrafish gut. Here we show that during gut development multiple lumens open and enlarge to generate a distinct intermediate, which consists of two adjacent unfused lumens separated by basolateral contacts. We observed that these lumens arise independently from each other along the length of the gut and do not share a continuous apical surface. Resolution of this intermediate into a single, continuous lumen requires the remodeling of contacts between adjacent lumens and subsequent lumen fusion. We show that lumen resolution, but not lumen opening, is impaired in smoothened (smo) mutants, indicating that fluid-driven lumen enlargement and resolution are two distinct processes. Furthermore, we show that smo mutants exhibit perturbations in the Rab11 trafficking pathway and demonstrate that Rab11-mediated trafficking is necessary for single lumen formation. Thus, lumen resolution is a distinct genetically controlled process crucial for single, continuous lumen formation in the zebrafish gut.

Rapid identification of kidney cyst mutations by whole exome sequencing in zebrafish

Forward genetic approaches in zebrafish have provided invaluable information about developmental processes. However, the relative difficulty of mapping and isolating mutations has limited the number of new genetic screens. Recent improvements in the annotation of the zebrafish genome coupled to a reduction in sequencing costs prompted the development of whole genome and RNA sequencing approaches for gene discovery. Here we describe a whole exome sequencing (WES) approach that allows rapid and cost-effective identification of mutations. We used our WES methodology to isolate four mutations that cause kidney cysts; we identified novel alleles in two ciliary genes as well as two novel mutants. The WES approach described here does not require specialized infrastructure or training and is therefore widely accessible. This methodology should thus help facilitate genetic screens and expedite the identification of mutants that can inform basic biological processes and the causality of genetic disorders in humans.

Basolateral EGF receptor sorting regulated by functionally distinct mechanisms in renal epithelial cells.

Proliferation of epithelial tissues is controlled by polarized distribution of signaling receptors including the EGF receptor (EGFR). In kidney, EGFRs are segregated from soluble ligands present in apical fluid of nephrons by selective targeting to basolateral membranes. We have shown previously that the epithelial‐specific clathrin adaptor AP1B mediates basolateral EGFR sorting in established epithelia. Here we show that protein kinase C (PKC)‐dependent phosphorylation of Thr654 regulates EGFR polarity as epithelial cells form new cell–cell junctional complexes. The AP1B‐dependent pathway does not override a PKC‐resistant T654A mutation, and conversely AP1B‐defective EGFRs sort basolaterally by a PKC‐dependent mechanism, in polarizing cells. Surprisingly, EGFR mutations that interfere with these different sorting pathways also produce very distinct phenotypes in three‐dimensional organotypic cultures. Thus EGFRs execute different functions depending on the basolateral sorting route. Many renal disorders have defects in cell polarity and the notion that apically mislocalized EGFRs promote proliferation is still an attractive model to explain many aspects of polycystic kidney disease. Our data suggest EGFR also integrates various aspects of polarity by switching between different basolateral sorting programs in developing epithelial cells. Fundamental knowledge of basic mechanisms governing EGFR sorting therefore provides new insights into pathogenesis and advances drug discovery for these renal disorders.

The Value of MRI in Distinguishing Subtypes of Lipomatous Extremity Tumors Needs Reassessment in the Era of MDM2 and CDK4 Testing

Introduction Extremity lipomas and well-differentiated liposarcomas (WDLs) are difficult to distinguish on MR imaging. We sought to evaluate the accuracy of MRI interpretation using MDM2 amplification, via fluorescence in-situ hybridization (FISH), as the gold standard for pathologic diagnosis. Furthermore, we aimed to investigate the utility of a diagnostic formula proposed in the literature. Methods We retrospectively collected 49 patients with lipomas or WDLs utilizing MDM2 for pathologic diagnosis. Four expert readers interpreted each patients MRI independently and provided a diagnosis. Additionally, a formula based on imaging characteristics (i.e. tumor depth, diameter, presence of septa, and internal cystic change) was used to predict the pathologic diagnosis. The accuracy and reliability of imaging-based diagnoses were then analyzed in comparison to the MDM2 pathologic diagnoses. Results The accuracy of MRI readers was 73.5% (95% CI 61–86%) with substantial interobserver agreement (κ=0.7022). The formula had an accuracy of 71%, which was not significantly different from the readers (p=0.71). The formula and expert observers had similar sensitivity (83% versus 83%) and specificity (64.5% versus 67.7%; p=0.659) for detecting WDLs. Conclusion The accuracy of both our readers and the formula suggests that MRI remains unreliable for distinguishing between lipoma and WDLs.

Albumin Versus American Society of Anesthesiologists Score: Which Is More Predictive of Complications Following Total Joint Arthroplasty?

Malnutrition is not uncommon in arthroplasty patients, and hypoalbuminemia has been shown to be predictive of postoperative complications. The authors sought to compare albumin concentration with a global assessment of physical health, the American Society of Anesthesiologists (ASA) score, to further discriminate the importance of albumin in predicting postoperative complications. A cohort of 128,412 patients undergoing total hip arthroplasty (THA) and total knee arthroplasty (TKA) were identified from 2005 to 2015 through use of the American College of Surgeons National Surgical Quality Improvement Program database. Patients were divided based on surgery performed, albumin concentration (with <3.5 g/dL defining hypoalbuminemia), and ASA score (≤2 vs >2). Postoperative complications were evaluated through multivariable regression analyses adjusted for age, sex, body mass index, and current smoking status. The study population included 48,751 THA and 79,661 TKA patients. On multivariable analyses, both hypoalbuminemia and ASA score were significant (P<.05) predictors of complications such as death, superficial infection, pneumonia, renal insufficiency, reintubation, transfusion, readmission, and reoperation. Furthermore, hypoalbuminemia more robustly predicted deep infection for THA patients, as well as superficial infection for TKA patients. American Society of Anesthesiologists score was otherwise predictive of more postoperative complications than hypoalbuminemia within the TKA group and provided similar predictability within the THA group. Albumin is associated with complications following THA and TKA, and if used in conjunction with ASA score, albumin contributes to enhanced risk stratification. The authors recommend that a preoperative albumin concentration be obtained and efforts made to modify this risk factor prior to elective arthroplasty. [Orthopedics. 2018; 41(6):354-362.].

Preoperative Hypoalbuminemia Predicts Poor Short-term Outcomes for Hip Fracture Surgery

Hip fractures are common in elderly patients, and which surgical modality to pursue is often debated. Malnutrition, which cannot be corrected preoperatively in this population, is often not considered. Therefore, the authors sought to investigate the association between hypoalbuminemia and postoperative outcomes based on surgical intervention. Patients undergoing arthroplasty (hemiarthroplasty or total hip arthroplasty), open reduction and internal fixation, and intramedullary nailing placement for treatment of hip fractures were identified in the American College of Surgeons National Surgical Quality Improvement Program database. Patients were stratified by preoperative albumin level, with less than 3.5 g/dL indicating hypoalbuminemia. Albumins association with postoperative complications was evaluated with multivariate logistic regression controlling for patient age, body mass index, American Society of Anesthesiologists score, and functional independence. A total of 20,278 patients with hip fractures and available albumin levels were included. Multivariate analysis revealed hypoalbuminemia was predictive of readmission, reintubation, mortality, and length of stay for all surgeries performed. When analyzing across surgical modalities, unique complications were identified for patients with hypoalbuminemia undergoing open reduction and internal fixation/prosthetic replacement (reoperation, P<.001) and arthroplasty (any infection, P=.028) compared with other treatment options. Hypoalbuminemia can predict postoperative complications for patients with hip fractures and should be considered preoperatively to guide surgical decision making in equivocal cases where multiple modalities may be used based on fracture pattern. This study supports that, compared with other interventions, intramedullary nailing is associated with fewer postoperative complications in patients with hypoalbuminemia. [Orthopedics. 2018; 41(6):e789-e796.].

Conversion vs. Primary Total Hip Arthroplasty: Increased Cost of Care and Peri-operative Complications

BACKGROUND With the increasing incidence of hip fractures and hip preservation surgeries, there has been a concomitant rise in the number of conversion total hip arthroplasties (THAs) performed. Prior studies have shown higher complication rates in conversion THA. However, there is a paucity of data showing differences in cost between these 2 procedures. Currently, the Center for Medicare and Medicaid Services bundles primary and conversion THA in the same Medicare Severity-Diagnosis Related Group for hospital reimbursement. More evidence is needed to support the reclassification of conversion THA. METHODS The cohort provided by the institutional database included 163 conversion THAs between January 1, 2012 and December 31, 2015. Intraoperative complications, estimated blood loss, operative time, postoperative complications, and perioperative cost data were analyzed for 163 primary THA patients matched to the conversion THA cohort. RESULTS Compared with primary THA, conversion THA had significantly (P < .05) greater cost for direct labor, other direct costs, intermediate nursing services, other diagnostic/therapy, surgery services, physical/occupational/speech therapy, radiology, laboratories, blood, medical/surgical supply, and total direct costs. In addition, the conversion THA group had significantly greater operative times, estimated blood loss, length of stay, intraoperative complications, and postoperative complications. CONCLUSION Conversion THA, as compared with primary THA, is associated with greater costs (approximately 19% greater), increased surgical times, and perioperative complications. To prevent these additional expenses from creating patient selection bias and a barrier to care, the conversion THA Medicare Severity-Diagnosis Related Group should be reclassified, or modifiers created.

Treatment of Prosthetic Joint Infection: Established Patient Relationships May Impact Medical Decision-Making

BACKGROUND Choosing the intervention for prosthetic joint infections, whether debridement, antibiotics, and implant retention (DAIR), or explant and antibiotic spacer placement, is multifactorial. One characteristic that may influence this decision is a previously established relationship with the patient. We hypothesized that patients receiving their arthroplasty at an outside institution and presenting with infection would be more likely to undergo removal of their implant without an attempt at DAIR compared to patients who underwent primary arthroplasty at the investigating institution. METHODS The institutional database was queried for primary total hip and knee arthroplasty infections. Manual review of medical records was performed, excluding patients who did not meet the Musculoskeletal Infection Society definition of infection. Patient demographics, medical comorbidities, presenting infection characteristics, and surgical intervention were collected. Multivariable analysis was performed to determine the independent predictors of treatment. RESULTS A total of 270 patients were included for analysis. McPherson score (P < .001) and duration of symptoms (P < .001) were associated with subsequent treatment. Additionally, when controlling for age, gender, symptom duration category, procedure, McPherson score, and American Society of Anesthesiologists category, patients with index procedures at outside hospitals were more likely to undergo implant removal (odds ratio, 36.30; 95% confidence interval, 8.16-161.51; P < .001). CONCLUSION Patients receiving their primary arthroplasty at an outside hospital and presenting with infection are more likely to undergo removal of hardware as their initial treatment. To avoid treatment bias, institutional protocols should be implemented to guide the shared decision-making process.

Skilled Nursing Facilities After Total Knee Arthroplasty: The Time for Selective Partnerships Is Now!

BACKGROUND Bundled payment initiatives for total knee arthroplasty (TKA) patients are dramatically impacted by discharges to skilled nursing facilities (SNFs), making target prices set by the Center for Medicare and Medicaid Services difficult to achieve. However, we hypothesized that a granular examination of SNF discharges would reveal that some may disproportionately increase costs compared to others. METHODS The institutional database was retrospectively queried for primary TKA patients under bundled payment initiatives. The 4 most common SNFs utilized by our patient population (A, B, C, and D) were investigated for length of stay, cost of care, and whether the overall target price for the episode of care (EOC) was reached. RESULTS In total, 1223 TKA patients were analyzed, with 378 (30.9%) discharged to an SNF and 246 patients selecting one of the 4 most common SNFs (A: 198, B: 21, C: 15, D: 12). Each SNF represented a significant fiscal portion of the total EOC; however, SNF D had significantly longer length of stay (21 vs 13 days, P < .001) and cost of care (

Chronic Osteomyelitis of the Distal Femur Treated with Resection and Delayed Endoprosthetic Reconstruction: A Report of Three Cases

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