Extracellular vesicles from retinal pigment epithelial cells expressing R345W-Fibulin-3 induce epithelial-mesenchymal transition in recipient cells.

We have shown previously that expression of R345W-Fibulin-3 induces epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. The purpose of the current study was to determine if extracellular vesicles (EVs) derived from RPE cells expressing R345W-Fibulin-3 mutation are sufficient to induce EMT in recipient cells. ARPE-19 cells were infected with luciferase-tagged wild-type (WT)- Fibulin-3 or luciferase-tagged R345W-Fibulin-3 (R345W) using lentiviruses. EVs were isolated from the media by ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy and cryogenic electron microscopy were performed to study the morphology of the EVs. The size distribution of EVs were determined by nanoparticle tracking analysis (NTA). EV cargo was analysed using LC-MS/MS based proteomics. EV-associated transforming growth factor beta 1 (TGFß1) protein was measured by enzyme-linked immunosorbent assay. The capacity of EVs to stimulate RPE migration was evaluated by treating recipient cells with WT- or R345W-EVs. The role of EV-bound TGFß was determined by pre-incubation of EVs with a pan-TGFß blocking antibody or IgG control. EM imaging revealed spherical vesicles with two subpopulations of EVs: a group with diameters around 30 nm and a group with diameters over 100 nm, confirmed by NTA analysis. Pathway analysis revealed that members of the sonic hedgehog pathway were less abundant in R345W- EVs, while EMT drivers were enriched. Additionally, R345W-EVs had higher concentrations of TGFß1 compared to control. Critically, treatment with R345W-EVs was sufficient to increase EMT marker expression, as well as cell migration in recipient cells. This EV-increased cell migration was significantly inhibited by pre-incubation of EVs with pan-TGFß-neutralising antibody. In conclusion, the expression of R345W-Fibulin-3 alters the size and cargo of EVs, which are sufficient to enhance the rate of cell migration in a TGFß dependent manner. These results suggest that EV-bound TGFß plays a critical role in the induction of EMT in RPE cells.

Comparing Virtual and In-Person Implementation of a School-Based Sexual Health Promotion Program in High Schools with Large Latino Populations.

Many sexual health programs transitioned to virtual implementation during the COVID-19 pandemic. Despite its devastation, the pandemic provided an opportunity to learn about virtual compared to in-person implementation of a sexual health promotion program-El Camino. This study assessed differences in program attendance, engagement, quality, and student ratings for virtual versus in-person implementation of El Camino as part of a rigorous evaluation in high schools with high Latino populations in Maryland. Drawing on positive youth development practices, El Camino helps participants identify personal goals and learn about sexual reproductive health and healthy relationships. This mixed-methods study incorporates data from performance measures, baseline and post-intervention participant surveys, observations, monthly implementation reports, and debriefs with facilitators to describe and compare virtual and in-person program implementation. At baseline, participants were an average of 16.2 years old; between 8 and 12th grade; 61% female; 79% Hispanic, Latino, or of Spanish origin; and 54% spoke mostly Spanish at home. Recruitment and retention of students outside of school classes were challenging for both forms of implementation. However, attendance was higher during in-person implementation and in schools where the organization implementing El Camino had a strong presence before the pandemic. Findings indicate high fidelity, excellent quality ratings, and positive student perceptions of the program and facilitators in both the virtual and in-person cohorts, which suggest that both forms of implementation were comparable and furthermore highlight the strength of the virtual adaptation of the El Camino program.

Association of genetic variants and survival in patients with acute myeloid leukemia in rural Appalachia.

BACKGROUND: Previous population health studies examining adults with acute myeloid leukemia (AML); however many of these, such as the Cancer Genome Atlas, are derived from databases collected by large urban centers. Due to its unique industry and environmental exposures, we hypothesized the West Virginia Appalachian population may have different mutational trends and clinical outcomes. AIMS: To address the concern of under-representation of rural minorities in cancer genomic databases, we performed exploratory whole exome sequencing in patients with newly diagnosed AML in rural Appalachia. METHODS & RESULTS: Correlations between genetic variants and clinical outcome variables were examined via retrospective chart review. A total of 26 patients were identified and whole exome sequencing was performed. Median age was 68 years old. Twenty-one patients had de novo AML (84%). As per European LeukemiaNet (ELN) criteria, 8 patients were favorable (32%), 12 were intermediate (48%), and 5 were adverse risk (20%). Eight patients proceeded to transplant. The median progression-free survival and overall survival were 16.5 months and 26.6 months, respectively. We noted an increased tumor mutation burden and a higher frequency of specific known driver mutations when compared to The Cancer Genome Atlas database; we also found novel mutations in MUC3A, MUC5AC, HCAR3, ORT2B, and PABPC. Survival outcomes were slightly lower than national average and BCOR mutation correlated with inferior outcomes. CONCLUSION: Our findings provide novel insight into detrimental mutations in AML in a rural, underrepresented population. We discovered several novel mutations and higher frequency of some known driver mutations, which will help us identify therapeutic targets to improve patient outcomes.

A validated analysis pipeline for mass spectrometry-based vitreous proteomics: new insights into proliferative diabetic retinopathy.

BACKGROUND: Vitreous is an accessible, information-rich biofluid that has recently been studied as a source of retinal disease-related proteins and pathways. However, the number of samples required to confidently identify perturbed pathways remains unknown. In order to confidently identify these pathways, power analysis must be performed to determine the number of samples required, and sample preparation and analysis must be rigorously defined. METHODS: Control (n = 27) and proliferative diabetic retinopathy (n = 23) vitreous samples were treated as biologically distinct individuals or pooled together and aliquoted into technical replicates. Quantitative mass spectrometry with tandem mass tag labeling was used to identify proteins in individual or pooled control samples to determine technical and biological variability. To determine effect size and perform power analysis, control and proliferative diabetic retinopathy samples were analyzed across four 10-plexes. Pooled samples were used to normalize the data across plexes and generate a single data matrix for downstream analysis. RESULTS: The total number of unique proteins identified was 1152 in experiment 1, 989 of which were measured in all samples. In experiment 2, 1191 proteins were identified, 727 of which were measured across all samples in all plexes. Data are available via ProteomeXchange with identifier PXD025986. Spearman correlations of protein abundance estimations revealed minimal technical (0.99-1.00) and biological (0.94-0.98) variability. Each plex contained two unique pooled samples: one for normalizing across each 10-plex, and one to internally validate the normalization algorithm. Spearman correlation of the validation pool following normalization was 0.86-0.90. Principal component analysis revealed stratification of samples by disease and not by plex. Subsequent differential expression and pathway analyses demonstrated significant activation of metabolic pathways and inhibition of neuroprotective pathways in proliferative diabetic retinopathy samples relative to controls. CONCLUSIONS: This study demonstrates a feasible, rigorous, and scalable method that can be applied to future proteomic studies of vitreous and identifies previously unrecognized metabolic pathways that advance understanding of diabetic retinopathy.

Juvenile nasopharyngeal angiofibroma: National practice patterns and resource utilization via HCUP KID.

BACKGROUND: Juvenile nasopharyngeal angiofibroma (JNA) is a locally aggressive benign vascular tumor that typically afflicts young adolescent males. Historically removed via open approaches, these tumors are now being removed endoscopically. As the modern healthcare setting emphasizes value, efficient utilization of resources may lead to decreased cost while maintaining or improving patient outcomes. OBJECTIVE: The objective of this study was to investigate how perioperative management of juvenile nasopharyngeal angiofibromas (JNAs) influence overall cost. We specifically investigate the effect of approach type (open, endoscopic, or combined) with regards to cost and length of stay. We also delineated practice patterns, analyzed safety profiles, and characterize clinical outcomes. METHODS: The 2016 Healthcare Cost and Utilization Project Kids' Inpatient Database (HCUP-KID) was queried to identify males aged <21 years with an ICD-10-CM diagnosis code of D10.6 (benign neoplasm of nasopharynx) and ICD-10-PCS codes to determine whether an open, endoscopic or combined approach was performed. Univariate statistical analysis and multivariable logistic regression were performed to examine the effects of demographics, patient characteristics, procedure type, and complications on length of stay (LOS) and cost. RESULTS: A total of 89 male patients were analyzed with a mean age of 14.8 years (range 8-20 years). Mean LOS was 3.4 days. Mean total charges were $128,780. Comparing open (n = 16), endoscopic (n = 65), and combined (n = 8) approaches, there was a significant difference in the need for fresh frozen plasma (p = 0.02) and packed red blood cell (pRBC) (p = 0.03) transfusion but no difference in preoperative embolization (p > 0.05) between approach types. LOS was associated with age (p = 0.02), pRBC transfusion (p = 0.04) and septal deviation (p = 0.03). Charges varied with LOS (p < 0.001) on linear regression analysis but not with other variables in this dataset. CONCLUSION: Approach type for JNA appears to be unrelated to LOS or charges in this multi-site, population-based analysis. However, septal deviation, pRBC transfusion, and young age are associated with increased LOS in patients undergoing JNA resection.

Proteomic Analyses of Vitreous in Proliferative Diabetic Retinopathy: Prior Studies and Future Outlook.

Vitreous fluid is becoming an increasingly popular medium for the study of retinal disease. Numerous studies have demonstrated that proteomic analysis of the vitreous from patients with proliferative diabetic retinopathy yields valuable molecular information regarding known and novel proteins and pathways involved in this disease. However, there is no standardized methodology for vitreous proteomic studies. Here, we share a suggested protocol for such studies and outline the various experimental and analytic methods that are currently available. We also review prior mass spectrometry-based proteomic studies of the vitreous from patients with proliferative diabetic retinopathy, discuss common pitfalls of these studies, and propose next steps for moving the field forward.

Adult Palatopharyngoplasty: Trends in Morbidity and Mortality from the NSQIP Database.

OBJECTIVES: Describe the postop morbidity of adults undergoing palatopharyngoplasty (PPP). METHOD: Adults who underwent PPP were studied using ACS-NSQIP (American College of Surgeons National Surgical Quality Improvement Program) database (2016-2017) via CPT code 42145. Analyzed outcomes included length of stay (LOS), readmission, reoperation, and postop complications. Predictive variables were age, gender, BMI, comorbidities. RESULTS: A total of 1081 patients (73.7% male, mean age 42.0 years, range 18-79 years) were included. 95 (8.8%) were diabetic, 183 (16.9%) were smokers, 30 (2.8%) had preoperative dyspnea. 328 (30.3%) took medicine for hypertension. Concurrent procedures occurred in 646 (59.76%), 357 (33.02%) had nasal procedures, 320 (29.60%) had tonsil procedures, 66 (6.11%) had tongue procedures. Within 30 days postop, there were two (0.19%) mortalities. Complications included six wound infections, two dehiscences, four with pneumonia, two pulmonary embolisms, three myocardial infarctions, one DVT, three sepsis, one UTI, one who required CPR, and two who were ventilated for >48 hours. Five required reintubation. A total of 41 (3.79%) returned to OR for a related reason, at least 27 (65.90%) for bleeding. LOS ranged from 0 to 15 days, median 1 day. Overall 38 (3.52%) were readmitted for a related reason, 12 (31.58%) for bleeding and three (7.89%) for pain. Using a significance level of 0.002 (Bonferroni correction), LOS varied with presence of any concurrent procedure, BMI, and estimated probability of mortality and morbidity indices; readmission and reoperation had no significantly associated variables. CONCLUSION: PPP is associated with low frequency but significant morbidity and mortality.

Standardization and Reduction of Narcotics After Pediatric Tonsillectomy.

OBJECTIVES: (1) To measure caregiver satisfaction with a nonstandardized postoperative pain regimen after pediatric tonsillectomy. (2) To implement a quality improvement project (QIP) to reduce the number and volume of narcotics prescribed and to describe the effect on caregiver satisfaction. METHODS: A prospective cohort study at a tertiary children's hospital examined postoperative narcotics prescribed to children following adenotonsillectomy. A QIP was implemented 3 months into the observation, with the goal to standardize nonnarcotic analgesics and reduce the volume of narcotics prescribed. Caregivers were called 2 to 3 weeks postoperatively to assess pain control and caregiver satisfaction. RESULTS: Over an 8-month period, 118 patients were recruited (66 before the QIP, 52 after induction). Prior to the QIP, 47% of patients were prescribed postoperative narcotics, as opposed to 27% after the QIP (P < .05). There was a significant reduction in the volume of narcotics prescribed before (mean ± SD, 300 ± 150 mL) versus after (180 ± 111 mL) the initiative (P < .05). The per-kilogram dose did not change over the study time frame. On a 5-point Likert scale, there was no difference in the caregivers' satisfaction regarding pain control before (4.37 ± 0.85) versus after (4.35 ± 1.0) the project started. DISCUSSION: A system shift was identified with the establishment of a posttonsillectomy pain control protocol associated with a reduction in prescribed narcotics without a significant change in caregiver satisfaction. IMPLICATIONS FOR PRACTICE: Implementing a standardized plan for the use of nonnarcotic medications was associated with reduced frequency and volume of narcotics prescribed. Future work will further standardize our postoperative pain regimen.

Embolization of a Superior Thyroid Artery Hemorrhage after Fine-Needle Aspiration Biopsy of a Thyroid Nodule.

Fine-needle aspiration biopsy (FNAB) is a procedure completed thousands of times daily across the world as an efficacious and safe way to evaluate thyroid nodules. Complications of an FNAB typically range from patient intolerance to small intrathyroidal hematomas. In rare situations, an FNA may result in significant bleeding leading to airway compromise or significant blood loss. In this case report, a patient underwent an FNAB and developed an arterial bleed leading to an intrathyroidal hematoma and airway compromise requiring intubation. This case report is unique in that it identifies the source of bleeding, exemplifies the complications of a large intrathyroidal hematoma, and describes subsequent treatment of both the arterial bleed and the hematoma.

Bevacizumab as adjuvant therapy for recurrent respiratory papillomatosis in an infant.

Recurrent Respiratory Papillomatosis (RRP) is a benign disease of the airway that can result in symptoms ranging from mild dysphonia to respiratory distress to respiratory failure related to colonization of the lung parenchyma. It is a disease that typically begins in childhood and can require treatment indefinitely, though may remit in adolescence. Although treatment includes surgical management, certain cases require adjuvant therapy. Many therapies have been tried over the years with varying levels of success. Our patient presented as a 7 month old male in respiratory distress, who was subsequently diagnosed with RRP. He has required a tracheostomy, ventilation, and frequent surgical debridement for the last 20 months. Due to the aggressiveness of his disease, he was started on systemic bevacizumab (Avastin) in an effort to stabilize his lesions. Though early effect seemed apparent based on Derkay scores, systemic bevacizumab has failed to elicit clinical improvement in our patient. He remains with a tracheostomy receiving bevacizumab 10 mg/kg as an intermittent infusion approximately every 4 weeks along with surgical debridement with little to no clinical improvement thus far. In light of the recent successes with bevacizumab therapy, this case highlights the difficulty in finding new treatment for this disease.

Intra-tumor genetic heterogeneity in rectal cancer.

Colorectal cancer arises in part from the cumulative effects of multiple gene lesions. Recent studies in selected cancer types have revealed significant intra-tumor genetic heterogeneity and highlighted its potential role in disease progression and resistance to therapy. We hypothesized the existence of significant intra-tumor genetic heterogeneity in rectal cancers involving variations in localized somatic mutations and copy number abnormalities. Two or three spatially disparate regions from each of six rectal tumors were dissected and subjected to the next-generation whole-exome DNA sequencing, Oncoscan SNP arrays, and targeted confirmatory sequencing and analysis. The resulting data were integrated to define subclones using SciClone. Mutant-allele tumor heterogeneity (MATH) scores, mutant allele frequency correlation, and mutation percent concordance were calculated, and copy number analysis including measurement of correlation between samples was performed. Somatic mutations profiles in individual cancers were similar to prior studies, with some variants found in previously reported significantly mutated genes and many patient-specific mutations in each tumor. Significant intra-tumor heterogeneity was identified in the spatially disparate regions of individual cancers. All tumors had some heterogeneity but the degree of heterogeneity was quite variable in the samples studied. We found that 67-97% of exonic somatic mutations were shared among all regions of an individual's tumor. The SciClone computational method identified 2-8 shared and unshared subclones in the spatially disparate areas in each tumor. MATH scores ranged from 7 to 41. Allele frequency correlation scores ranged from R(2)=0.69-0.96. Measurements of correlation between samples for copy number changes varied from R(2)=0.74-0.93. All tumors had some heterogeneity, but the degree was highly variable in the samples studied. The occurrence of significant intra-tumor heterogeneity may allow selected tumors to have a genetic reservoir to draw from in their evolutionary response to therapy and other challenges.