Actin- and microtubule-based motors contribute to clathrin-independent endocytosis in yeast.

Most eukaryotic cells utilize clathrin-mediated endocytosis as well as multiple clathrin-independent pathways to internalize proteins and membranes. Although clathrin-mediated endocytosis has been studied extensively and many machinery proteins have been identified, clathrin-independent pathways remain poorly characterized by comparison. We previously identified the first known yeast clathrin-independent endocytic pathway, which relies on the actin-modulating GTPase Rho1, the formin Bni1 and unbranched actin filaments, but does not require the clathrin coat or core clathrin machinery proteins. In this study, we sought to better understand clathrin-independent endocytosis in yeast by exploring the role of myosins as actin-based motors, because actin is required for endocytosis in yeast. We find that Myo2, which transports secretory vesicles, organelles and microtubules along actin cables to sites of polarized growth, participates in clathrin-independent endocytosis. Unexpectedly, the ability of Myo2 to transport microtubule plus ends to the cell cortex appears to be required for its role in clathrin-independent endocytosis. In addition, dynein, dynactin, and proteins involved in cortical microtubule capture are also required. Thus, our results suggest that interplay between actin and microtubules contributes to clathrin-independent internalization in yeast.

Racial Differences in Patients Undergoing Anterior Cervical Discectomy and Fusion: A Multi-Site Study.

STUDY DESIGN: This was a retrospective chart review. OBJECTIVE: The objective of this study was to examine disparities within patients undergoing anterior cervical discectomy and fusion (ACDF) at a multi-site tertiary referral center with specific focus on factors related to length of stay (LOS). SUMMARY OF BACKGROUND DATA: There are previously described racial disparities in spinal surgery outcomes and quality metrics. METHODS: A total of 278 consecutive patients undergoing ACDF by 8 different surgeons over a 5-year period were identified retrospectively. Demographic data, including age at time of surgery, sex, smoking status, and self-identified race [White or African American (AA)], as well as surgical data and postoperative course were recorded. Preoperative health status was recorded, and comorbidities were scored by the Charlson Comorbidity Index. Univariable and multivariable linear regression models were employed to quantify the degree to which a patient's LOS was related to their self-identified race, demographics, and perioperative clinical data. RESULTS: Of the 278 patients who received an ACDF, 71.6% (199) self-identified as White and 28.4% (79) identified as AA. AA patients were more likely to have an ACDF due to myelopathy, while White patients were more likely to have an ACDF due to radiculopathy (P=0.001). AA patients had longer LOS by an average of half a day (P=0.001) and experienced a larger percentage of extended stays (P=0.002). AA patients experienced longer overall operation times on average (P=0.001) across all different levels of fusion. AA race was not an independent driver of LOS (ß=0.186; P=0.246). CONCLUSIONS: As hypothesized, and consistent with previous literature on racial surgical disparities, AA race was associated with increased LOS, increased operative times, and increased indication of myelopathy in this study. Additional research is necessary to evaluate the underlying social determinants of health and other factors that may contribute to this study's results. LEVEL OF EVIDENCE: Level III.

Opioid Prescriptions After Pediatric Orthopedic Surgery: Analyzing Rates of Unfilled Prescriptions.

BACKGROUND: The prevalence of nonmedical use of prescription opioids among American teenagers is staggering. Reducing the quantity of postoperative opioid prescriptions can help address this epidemic by decreasing the availability of opioids. As the fourth most common opioid prescribers, orthopaedic surgeons are primed to lead efforts to reverse this crisis. The purpose of this study was to determine patient factors associated with filling opioid prescriptions after pediatric orthopaedic surgery and to recommend potential methods to limit excess opioid prescriptions. METHODS: This retrospective cohort study included 1413 patients who were prescribed opioids upon discharge after an orthopaedic surgical procedure at a single urban children's hospital from 2017 to 2018. The state's Prescription Drug Monitoring Program was used to determine which patients filled their opioid prescriptions. Comparisons of demographic, clinical, and surgical factors were made between patients that filled their opioid prescription upon discharge and those that did not. Statistical analysis included λ2, Mann-Whitney U, and binary logistic regression for significant factors from bivariate analysis. RESULTS: Nine percent (n=127) of patients did not fill their opioid prescriptions after discharge. Those who filled and did not fill prescriptions were similar in terms of sex, ethnicity, race, insurance type, and age at surgery (P>0.05). Patients who did not fill their opioid prescriptions received significantly fewer opioids during their postoperative hospital course (5.3 vs. 7.7 mg oxycodone, P=0.01). Not receiving oral oxycodone during postoperative hospitalization (odds ratio=2.16, 95% confidence interval: 1.49-3.14) and undergoing upper extremity surgery (odds ratio=2.00, 95% confidence interval: 1.37-2.91) were independently associated with not filling opioid prescriptions after surgery in the multivariate analysis. CONCLUSIONS: This study identified a subset of pediatric orthopaedic surgery patients who were prescribed opioids upon discharge but did not fill those prescriptions. Factors that increased the likelihood that patients did not fill their prescriptions upon discharge included fewer postoperative hospital course opioids, no oral oxycodone, and surgery on the upper extremity. Future studies must be performed to help decrease the number of opioids prescribed unnecessarily. LEVEL OF EVIDENCE: Level III-prognostic cohort study.

Soft Landing technique as a possible prevention strategy for proximal junctional failure following adult spinal deformity surgery.

BACKGROUND: This cross sectional study describes a "Soft Landing" strategy utilizing hooks for minimizing proximal junctional kyphosis (PJK) and proximal junctional failure (PJF). The technique creates a gradual transition from a rigid segmental construct to unilateral hooks at the upper instrumented level and preservation of the soft tissue attachments on the contralateral side of the hooks. Authors devise a novel classification system for better grading of PJK severity. METHODS: Thirty-nine consecutive adult spinal deformity (ASD) patients at a single institution received the "Soft Landing" technique. The proximal junctional angle was measured preoperatively and at last follow-up using standing 36-inch spinal radiographs. Changes in proximal junctional angle and rates of PJK and PJF were measured and used to create a novel classification system for evaluating and categorizing ASD patients postoperatively. RESULTS: The mean age of the cohort was 61.4 years, and 90% of patients were women. Average follow up was 2.2 years. The mean change in proximal junctional angle was 8° (SD 7.4°) with the majority of patients (53%) experiencing less than 10° and only 1 patients with proximal junctional angle over 20°. Four patients (10%) needed additional surgery for proximal extension of the uppermost instrumented vertebra (UIV) secondary to PJF. CONCLUSIONS: Soft Landing technique is a possibly effective treatment strategy to prevent PJK and PJF following ASD that requires further evaluation. The described classification system provides management framework for better grading of PJK. The "Soft Landing" technique warrants further comparison to other techniques currently used to prevent both PJK and failure.

Refilling Opioid Prescriptions After Pediatric Orthopaedic Surgery: An Analysis of Incidence and Risk Factors.

BACKGROUND: Understanding which pediatric patients seek opioid refills is crucial as prescription opioid use in childhood is associated with an increased risk of future opioid misuse. Orthopaedic surgeons are optimally positioned to lead the charge in addressing the opioid epidemic. The aim of this study was to describe the incidence of and risk factors associated with requiring opioid refills after pediatric orthopaedic surgery in children. METHODS: This retrospective case-control study included 1413 patients aged 0 to 18 years that underwent orthopaedic surgery at a single tertiary care children's hospital and were prescribed opioids at discharge. Using the state Prescription Drug Monitoring Program (PDMP) database, we determined which patients filled additional opioid prescriptions within 6 months following an orthopaedic procedure. Comparisons were made between patients that sought additional opioids and those that did not use bivariate analysis and binomial logistic regression. RESULTS: In total, 31 (2.2%) patients sought additional opioid prescriptions a median 41 days postoperatively (range, 2 to 184). Nearly half of these patients obtained refills from providers outside of our institution, suggesting that previous reports using hospital records may underestimate its prevalence. Factors associated with requiring opioid refills included receiving hydromorphone [odds ratio (OR)=3.04, P=0.04] or methadone (OR=38.14, P<0.01) while inpatient, surgery on the axial skeleton (OR=5.42, P=0.01) or lower extremity (OR=2.49, P=0.04), and nonfracture surgery (OR=3.27, P=0.01). Patients who obtained additional opioids received significantly more opioids during their inpatient recovery (32.9 vs. 11.1 morphine equivalents, P<0.01). CONCLUSIONS: Approximately 2% of children and families obtain additional opioids within 6 months of orthopaedic surgery. The volume of opioids during inpatient hospitalization may predict the need for opioid prescription refills after discharge. Clinicians should maximize efforts to achieve pain control with multimodal analgesia and opioid alternatives, and use caution when administering high-dose opioids during postoperative hospitalization. LEVEL OF EVIDENCE: Level III-prognostic.

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast.

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.