NPEPPS Is a Druggable Driver of Platinum Resistance.

There is an unmet need to improve the efficacy of platinum-based cancer chemotherapy, which is used in primary and metastatic settings in many cancer types. In bladder cancer, platinum-based chemotherapy leads to better outcomes in a subset of patients when used in the neoadjuvant setting or in combination with immunotherapy for advanced disease. Despite such promising results, extending the benefits of platinum drugs to a greater number of patients is highly desirable. Using the multiomic assessment of cisplatin-responsive and -resistant human bladder cancer cell lines and whole-genome CRISPR screens, we identified puromycin-sensitive aminopeptidase (NPEPPS) as a driver of cisplatin resistance. NPEPPS depletion sensitized resistant bladder cancer cells to cisplatin in vitro and in vivo. Conversely, overexpression of NPEPPS in sensitive cells increased cisplatin resistance. NPEPPS affected treatment response by regulating intracellular cisplatin concentrations. Patient-derived organoids (PDO) generated from bladder cancer samples before and after cisplatin-based treatment, and from patients who did not receive cisplatin, were evaluated for sensitivity to cisplatin, which was concordant with clinical response. In the PDOs, depletion or pharmacologic inhibition of NPEPPS increased cisplatin sensitivity, while NPEPPS overexpression conferred resistance. Our data present NPEPPS as a druggable driver of cisplatin resistance by regulating intracellular cisplatin concentrations. SIGNIFICANCE: Targeting NPEPPS, which induces cisplatin resistance by controlling intracellular drug concentrations, is a potential strategy to improve patient responses to platinum-based therapies and lower treatment-associated toxicities.

How and why do financial incentives contribute to helping people stop smoking? A realist review.

BACKGROUND: Tobacco smoking remains a key cause of preventable illness and death globally. In response, many countries provide extensive services to help people to stop smoking by offering a variety of effective behavioural and pharmacological therapies. However, many people who wish to stop smoking do not have access to or use stop smoking supports, and new modes of support, including the use of financial incentives, are needed to address this issue. A realist review of published international literature was undertaken to understand how, why, for whom, and in which circumstances financial incentives contribute to success in stopping smoking for general population groups and among pregnant women. METHODS: Systematic searches were undertaken from inception to February 2022 of five academic databases: MEDLINE (ovid), Embase.com, CIHAHL, Scopus and PsycINFO. Study selection was inclusive of all study designs. Twenty-two studies were included. Using Pawson and Tilley's iterative realist review approach, data collected were screened, selected, coded, analysed, and synthesised into a set of explanatory theoretical findings. RESULTS: Data were synthesised into six Context-Mechanism-Outcome Configurations and one overarching programme theory after iterative rounds of analysis, team discussion, and expert panel feedback. Our programme theory shows that financial incentives are particularly useful to help people stop smoking if they have a financial need, are pregnant or recently post-partum, have a high threshold for behaviour change, and/or respond well to external rewards. The incentives work through a number of mechanisms including the role their direct monetary value can play in a person's life and through a process of reinforcement where they can help build confidence and self-esteem. CONCLUSION: This is the first realist review to synthesise how, why, and for whom financial incentives work among those attempting to stop smoking, adding to the existing evidence demonstrating their efficacy. The findings will support the implementation of current knowledge into effective programmes which can enhance the impact of stop smoking care. PROSPERO REGISTRATION NUMBER: CRD42022298941.

The Molecular Twin artificial-intelligence platform integrates multi-omic data to predict outcomes for pancreatic adenocarcinoma patients.

Contemporary analyses focused on a limited number of clinical and molecular biomarkers have been unable to accurately predict clinical outcomes in pancreatic ductal adenocarcinoma. Here we describe a precision medicine platform known as the Molecular Twin consisting of advanced machine-learning models and use it to analyze a dataset of 6,363 clinical and multi-omic molecular features from patients with resected pancreatic ductal adenocarcinoma to accurately predict disease survival (DS). We show that a full multi-omic model predicts DS with the highest accuracy and that plasma protein is the top single-omic predictor of DS. A parsimonious model learning only 589 multi-omic features demonstrated similar predictive performance as the full multi-omic model. Our platform enables discovery of parsimonious biomarker panels and performance assessment of outcome prediction models learning from resource-intensive panels. This approach has considerable potential to impact clinical care and democratize precision cancer medicine worldwide.

A Noninvasive Circulating Signature of Combined Right Ventricular Pressure and Volume Overload in Tetralogy of Fallot/Pulmonary Atresia/Major Aortopulmonary Collateral Arteries.

Background: Despite surgical advances, children with tetralogy of Fallot/pulmonary atresia/major aortopulmonary collaterals (TOF/PA/MAPCAs) are subject to chronic right ventricular (RV) pressure and volume overload. Current diagnostic tools do not identify adverse myocardial remodeling and cannot predict progression to RV failure. We sought to identify a noninvasive, circulating signature of the systemic response to right heart stress to follow disease progression. Methods: Longitudinal data were collected from patients with TOF/PA/MAPCAs (N = 5) at the time of (1) early RV pressure overload and (2) late RV pressure and volume overload. Plasma protein and microRNA expression were evaluated using high-throughput data-independent mass spectroscopy and Agilent miR Microarray, respectively. Results: At the time of early RV pressure overload, median patient age was 0.34 years (0.02-9.37), with systemic RV pressures, moderate-severe hypertrophy, and preserved systolic function. Late RV pressure and volume overload occurred at a median age of 4.08 years (1.51-10.83), with moderate RV hypertrophy and dilation, and low normal RV function; 277 proteins were significantly dysregulated (log2FC ≥0.6/≤-0.6, FDR≤0.05), predicting downregulation in lipid transport (apolipoproteins), fibrinolytic system, and extracellular matrix structural proteins (talin 1, profilin 1); and upregulation in the respiratory burst. Increasing RV size and decreasing RV function correlated with decreasing structural protein expression. Similarly, miR expression predicted downregulation of extracellular matrix-receptor interactions and upregulation in collagen synthesis. Conclusion: To our knowledge, we show for the first time a noninvasive protein and miR signature reflecting the systemic response to adverse RV myocardial remodeling in TOF/PA/MAPCAs which could be used to follow disease progression.

MicroRNA and Protein Cargos of Human Limbal Epithelial Cell-Derived Exosomes and Their Regulatory Roles in Limbal Stromal Cells of Diabetic and Non-Diabetic Corneas.

Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LECs). Presently, we quantify the miRNA and proteome profiles of human LEC-derived Exos and their regulatory roles in N- and DM-LSC. We revealed some miRNA and protein differences in DM vs. N-LEC-derived Exos' cargos, including proteins involved in Exo biogenesis and packaging that may affect Exo production and ultimately cellular crosstalk and corneal function. Treatment by N-Exos, but not by DM-Exos, enhanced wound healing in cultured N-LSCs and increased proliferation rates in N and DM LSCs vs. corresponding untreated (control) cells. N-Exos-treated LSCs reduced the keratocyte markers ALDH3A1 and lumican and increased the MSC markers CD73, CD90, and CD105 vs. control LSCs. These being opposite to the changes quantified in wounded LSCs. Overall, N-LEC Exos have a more pronounced effect on LSC wound healing, proliferation, and stem cell marker expression than DM-LEC Exos. This suggests that regulatory miRNA and protein cargo differences in DM- vs. N-LEC-derived Exos could contribute to the disease state.

17 ß-Estradiol Impedes Aortic Root Dilation and Rupture in Male Marfan Mice.

Marfan syndrome causes a hereditary form of thoracic aortic aneurysms with worse outcomes in male compared to female patients. In this study, we examine the effects of 17 ß-estradiol on aortic dilation and rupture in a Marfan mouse model. Marfan male mice were administered 17 ß-estradiol, and the growth in the aortic root, along with the risk of aortic rupture, was measured. Transcriptomic profiling was used to identify enriched pathways from 17 ß-estradiol treatments. Aortic smooth muscle cells were then treated with cytokines to validate functional mechanisms. We show that 17 ß-estradiol decreased the size and rate of aortic root dilation and improved survival from rupture. The Marfan transcriptome was enriched in inflammatory genes, and the addition of 17 ß-estradiol modulated a set of genes that function through TNFα mediated NF-κB signaling. In addition, 17 ß-estradiol suppressed the induction of these TNFα induced genes in aortic smooth muscle cells in vitro in an NF-κB dependent manner, and 17 ß-estradiol decreased the formation of adventitial inflammatory foci in aortic roots in vivo. In conclusion, 17 ß-estradiol protects against the dilation and rupture of aortic roots in Marfan male mice through the inhibition of TNFα-NF-κB signaling.

Alone Together: Is Strain Experienced Concurrently by Members of Operating Room Teams?: An Event-based Study.

Objective: To identify which strain episodes are concurrently reported by several team members; to identify triggers of strain experienced by operating room (OR) team members during the intraoperative phase. Summary: OR teams are confronted with many sources of strain. However, most studies investigate strain on a general, rather than an event-based level, which does not allow to determine if strain episodes are experienced concurrently by different team members. Methods: We conducted an event-based, observational study, at an academic medical center in North America and included 113 operations performed in 5 surgical departments (general, vascular, pediatric, gynecology, and trauma/acute care). Strain episodes were assessed with a guided-recall method. Immediately after operations, participants mentally recalled the operation, described the strain episodes experienced and their content. Results: Based on 731 guided recalls, 461 strain episodes were reported; these refer to 312 unique strain episodes. Overall, 75% of strain episodes were experienced by a single team member only. Among different categories of unique strain episodes, those triggered by task complexity, issues with material, or others' behaviors were typically experienced by 1 team member only. However, acute patient issues (n = 167) and observations of others' strain (n = 12) (respectively, 58.5%; P < 0.001 and 83.3%; P < 0.001) were often experienced by 2 or more team members. Conclusions and relevance: OR team members are likely to experience strain alone, unless patient safety is at stake. This may jeopardize the building of a shared understanding among OR team members.

A "sludge audit" for health system colorectal cancer screening services.

OBJECTIVES: "Sludge," or the frictions or administrative burdens that make it difficult for people to attain what they want or need, is an unexplored health care delivery factor that may contribute to deficiencies in colorectal cancer (CRC) screening. We piloted a method to identify and quantify sludge in a southeastern US health system's delivery of CRC screening services. STUDY DESIGN: Mixed methods sludge audit. METHODS: We collected and analyzed quantitative (insurance claims, electronic health record, and administrative files) and qualitative (stakeholder interviews and process observations) data associated with CRC screening for instances of sludge. Because they contribute to sludge and reduce system capacity for high-value screening, we also evaluated low-value CRC screening processes. RESULTS: Although specific results were likely amplified by effects of the COVID-19 pandemic, the sludge audit revealed important areas for improvement. A 60.4% screening rate was observed. Approximately half of screening orders were not completed. The following categories of sludge were identified: communication, time, technology, administrative tasks, paperwork, and low-value care. For example, wait times for screening colonoscopy were substantial, duplicate orders were common, and some results were not accessible in the electronic health record. Of completed screenings, 32% were low-value and 38% were associated with low-value preoperative testing. There was evidence of a differential negative impact of sludge to vulnerable patients. CONCLUSIONS: Our sludge audit method identified and quantified multiple instances of sludge in a health system's CRC screening processes. Sludge audits can help organizations to systematically evaluate and reduce sludge for more effective and equitable CRC screening.

Use of CT and MR imaging in radiation therapy planning of imaging-diagnosed canine intracranial meningioma achieves better tumor coverage than CT alone.

The aim of this retrospective, secondary analysis study was to quantify the dosimetric impact of the lack of interobserver agreement on gross tumor volume (GTV) delineation for canine meningioma. This study used a previously reported population of 13 dogs with GTVs contoured on CT alone and on registered CT-MR by 18 radiation oncologists. The "true" GTV was generated for each dog using a simultaneous truth and performance-level estimation algorithm, and "true" brain was defined as the whole brain minus true GTV. Treatment plans were generated for each dog and observer combination, using criteria applied to the observer's GTV and brain contours. Plans were then categorized as a pass (met all planning criteria for true GTV and true brain) or fail. A mixed-effects linear regression was performed to examine differences in metrics between CT and CT-MR plans and mixed-effects logistic regression was performed to examine differences in percentages of pass/fail between CT and CT-MRI plans. The mean percent coverage of true GTV by prescribed dose was higher for CT-MR plans than for CT plans (mean difference 5.9%; 95% CI, 3.7-8.0; P < 0.001). There was no difference in the mean volume of true brain receiving ≥24 Gy and in maximum true brain dose between CT plans and CT-MR plans (P ≥ 0.198). CT-MR plans were significantly more likely to pass the criteria for true GTV and true brain than CT plans (OR 1.75; 95% CI, 1.02-3.01; P = 0.044). This study demonstrated significant dosimetric impact when GTV contouring was performed on CT alone compared with CT-MR.

Proteomics of novel induced pluripotent stem cell-derived vascular endothelial cells reveal extensive similarity with an immortalized human endothelial cell line.

The vascular endothelium constitutes the inner lining of the blood vessel, and malfunction and injuries of the endothelium can cause cardiovascular diseases as well as other diseases including stroke, tumor growth, and chronic kidney failure. Generation of effective sources to replace injured endothelial cells (ECs) could have significant clinical impact, and somatic cell sources like peripheral or cord blood cannot credibly supply enough endothelial cell progenitors for multitude of treatments. Pluripotent stem cells are a promising source for a reliable EC supply, which have the potential to restore tissue function and treat vascular diseases. We have developed methods to differentiate induced pluripotent stem cells (iPSCs) efficiently and robustly across multiple iPSC lines into nontissue-specific pan vascular ECs (iECs) with high purity. These iECs present with canonical endothelial cell markers and exhibit measures of endothelial cell functionality with the uptake of Dil fluorescent dye-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) and tube formation. Using proteomic analysis, we revealed that the iECs are more proteomically similar to established human umbilical vein ECs (HUVECs) than to iPSCs. Posttranslational modifications (PTMs) were most shared between HUVECs and iECs, and potential targets for increasing the proteomic similarity of iECs to HUVECs were identified. Here we demonstrate an efficient robust method to differentiate iPSCs into functional ECs, and for the first time provide a comprehensive protein expression profile of iECs, which indicates their similarities with a widely used immortalized HUVECs, allowing for further mechanistic studies of EC development, signaling, and metabolism for future regenerative applications.NEW & NOTEWORTHY We have developed methods to differentiate induced pluripotent stem cells (iPSCs) across multiple iPSC lines into nontissue-specific pan vascular ECs (iECs) and demonstrated the proteomic similarity of these cells to a widely used endothelial cell line (HUVECs). We also identified posttranslational modifications and targets for increasing the proteomic similarity of iECs to HUVECs. In the future, iECs can be used to study EC development, signaling, and metabolism for future regenerative applications.

High-Throughput Single-Cell Proteomic Analysis of Organ-Derived Heterogeneous Cell Populations by Nanoflow Dual-Trap Single-Column Liquid Chromatography.

Identification and proteomic characterization of rare cell types within complex organ-derived cell mixtures is best accomplished by label-free quantitative mass spectrometry. High throughput is required to rapidly survey hundreds to thousands of individual cells to adequately represent rare populations. Here we present parallelized nanoflow dual-trap single-column liquid chromatography (nanoDTSC) operating at 15 min of total run time per cell with peptides quantified over 11.5 min using standard commercial components, thus offering an accessible and efficient LC solution to analyze 96 single cells per day. At this throughput, nanoDTSC quantified over 1000 proteins in individual cardiomyocytes and heterogeneous populations of single cells from the aorta.

Plasma Metabolomics Predicts Chemotherapy Response in Advanced Pancreatic Cancer.

Pancreatic cancer (PC) is one of the deadliest cancers. Developing biomarkers for chemotherapeutic response prediction is crucial for improving the dismal prognosis of advanced-PC patients (pts). To evaluate the potential of plasma metabolites as predictors of the response to chemotherapy for PC patients, we analyzed plasma metabolites using high-performance liquid chromatography-mass spectrometry from 31 cachectic, advanced-PC subjects enrolled into the PANCAX-1 (NCT02400398) prospective trial to receive a jejunal tube peptide-based diet for 12 weeks and who were planned for palliative chemotherapy. Overall, there were statistically significant differences in the levels of intermediates of multiple metabolic pathways in pts with a partial response (PR)/stable disease (SD) vs. progressive disease (PD) to chemotherapy. When stratified by the chemotherapy regimen, PD after 5-fluorouracil-based chemotherapy (e.g., FOLFIRINOX) was associated with decreased levels of amino acids (AAs). For gemcitabine-based chemotherapy (e.g., gemcitabine/nab-paclitaxel), PD was associated with increased levels of intermediates of glycolysis, the TCA cycle, nucleoside synthesis, and bile acid metabolism. These results demonstrate the feasibility of plasma metabolomics in a prospective cohort of advanced-PC patients for assessing the effect of enteral feeding as their primary source of nutrition. Metabolic signatures unique to FOLFIRINOX or gemcitabine/nab-paclitaxel may be predictive of a patient's response and warrant further study.

Contouring in the optic plane improves the accuracy of computed tomography-based segmentation of the optic pathway.

Canine optic pathway structures are often contoured on CT images, despite the difficulty of visualizing the optic pathway with CT using standard planes. The purpose of this prospective, analytical, diagnostic accuracy study was to examine the accuracy of optic pathway contouring by veterinary radiation oncologists (ROs) before and after training on optic plane contouring. Optic pathway contours used as the gold standard for comparison were created based on expert consensus from registered CT and MRI for eight dogs. Twenty-one ROs contoured the optic pathway on CT using their preferred method, and again following atlas and video training demonstrating contouring on the optic plane. The Dice similarity coefficient (DSC) was used to assess contour accuracy. A multilevel mixed model with random effects to account for repeated measures was used to examine DSC differences. The median DSC (5th and 95th percentile) before and after training was 0.31 (0.06, 0.48) and 0.41 (0.18, 0.53), respectively. The mean DSC was significantly higher after training compared with before training (mean difference = 0.10; 95% CI, 0.08-0.12; P < 0.001) across all observers and patients. DSC values were comparable to those reported (0.4-0.5) for segmentation of the optic chiasm and nerves in human patients. Contour accuracy improved after training but remained low, potentially due to the small optic pathway volumes. When registered CT-MRI images are not available, our study supports routine addition of an optic plane with specific window settings to improve segmentation accuracy in mesaticephalic dogs ≥11 kg.

Optimization of Large-Scale Adeno-Associated Virus (AAV) Production.

Genetic manipulation in vivo is a critical method for mechanistically understanding gene function in disease and physiological processes. To facilitate this, embryonic transgenesis in popular animal models like mice has been developed. Compared to the longer, expensive methods of transgenesis, viral vectors, such as adeno-associated virus (AAV), have grown increasingly in popularity due to their relatively low cost and ease of production, translating to an overall greater versatility as a biological tool. In this article, we describe protocols for AAV production and purification for efficient transduction in vivo. Importantly, our method differs from others in application of a streamlined, more cost-effective approach. From this method, as many as 2 × 1013 genome-containing viral particles (vp), or 200 units, can be produced within 3 to 4 weeks, with a minimal cost of $1800 to $2000 for supplies and reagents and <15 hr of personnel time per week. A unit here is defined as 1 × 1011 vp, our standard dose of AAV per animal, injected via tail vein. Therefore, our method provides production and purification of AAV in quantities capable of transducing up to 200 animals. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: AAV production Basic Protocol 2: AAV purification.

Public attitudes to implementing financial incentives in stopsmoking services in Ireland.

INTRODUCTION: Financial incentives improve stop-smoking service outcomes. Views on acceptability can influence implementation success. To inform implementation planning in Ireland, public attitudes on financial incentives to stop smoking were measured. METHODS: A cross-sectional telephone survey was administered to 1000 people in Ireland aged ≥15 years in 2022, sampled through random digit dialing. The questionnaire included items on support for financial incentives under different conditions. Prevalence of support was calculated with 95% Confidence Intervals (CIs) and multiple logistic regression identified associated factors using adjusted odds ratios (AORs) with 95% CIs. RESULTS: Almost half (47.0%, 95% CI: 43.9-50.1) of the participants supported at least one type of financial incentive to stop smoking, with support more prevalent for shopping vouchers (43.3%, 95% CI: 40.3-46.5) than cash payments (32.1%, 95% CI: 29.2-35.0). Support was similar for universal and income-restricted schemes. Of those who supported financial incentives, the majority (60.6%) believed the maximum amount given on proof of stopping smoking should be under €250 (median=100, range: 1-7000). Compared to their counterparts, those of lower education level (AOR=1.49; 95% CI: 1.10-2.03, p=0.010) and tobacco/e-cigarette users (AOR=1.43; 95% CI: 1.02-2.03, p=0.041) were significantly more likely to support either financial incentive type, as were younger people. CONCLUSIONS: While views on financial incentives to stop smoking in Ireland were mixed, the intervention is more acceptable in groups experiencing the heaviest burden of smoking-related harm and most capacity to benefit. Engagement and communication must be integral to planning for successful implementation to improve stop-smoking service outcomes.

Risk factors for appendicular osteosarcoma occurrence in large and giant breed dogs in western Canada.

Objective: Risk factors for the development of canine appendicular osteosarcoma (OSA) have been investigated in numerous studies, but with contradictory results. The aim of this study was to analyze weight, age, breed, sex, neuter status, body condition score, and previous lameness in a population of large and giant breed dogs in western Canada with and without appendicular OSA. Animals and procedure: Medical records of 227 large or giant breed dogs diagnosed with appendicular OSA were compared to records from a control population of 454 large and giant breed dogs from the years 2000 to 2020. Results: Gonadectomized dogs, body condition score (BCS), and a history of lameness condition(s) (other than OSA) were associated with increased odds for presentation with OSA. Breeds shown to have increased odds for appendicular OSA occurrence included Rottweilers and Great Danes relative to Labrador retrievers. Conclusion and clinical relevance: Obesity and lameness appear to be independently associated with appendicular osteosarcoma. This study demonstrated that spayed females had the greatest risk compared to other sex and neuter status combinations; further investigation of these factors would be beneficial.

Facteurs de risque d'apparition d'ostéosarcome appendiculaire chez les chiens de grandes races et de races géantes dans l'Ouest canadien. Objectif: Les facteurs de risque de développement de l'ostéosarcome (OSA) appendiculaire canin ont été étudiés dans de nombreuses études, mais avec des résultats contradictoires. Le but de cette étude était d'analyser le poids, l'âge, la race, le sexe, la stérilisation, le score d'état corporel et les boiteries antérieures dans une population de chiens de grande race et de race géante de l'Ouest canadien avec et sans OSA appendiculaire. Animaux et procédure: Les dossiers médicaux de 227 chiens de grande race ou de race géante diagnostiqués avec l'OSA appendiculaire ont été comparés aux dossiers d'une population témoin de 454 chiens de grande race et de race géante des années 2000 à 2020. Résultats: Les chiens gonadectomisés, le score d'état corporel (BCS) et des antécédents de condition(s) de boiterie (autres que l'OSA) étaient associés à une probabilité accrue de présentation d'OSA. Les races dont le risque d'apparition d'OSA appendiculaire était plus élevé comprenaient les Rottweilers et les Grands Danois par rapport aux Labrador retrievers. Conclusion et pertinence clinique: L'obésité et la boiterie semblent être indépendamment associées à l'ostéosarcome appendiculaire. Cette étude a démontré que les femelles stérilisées présentaient le plus grand risque par rapport aux autres combinaisons de sexe et de statut neutre, une enquête plus approfondie sur ces facteurs serait bénéfique.(Traduit par Dr Serge Messier).

Evaluation of conjunctival graft procedures and factors that lead to graft complications in canine cases.

PURPOSE: To describe the complications of conjunctival graft surgery occurring in cases at a referral ophthalmic service and evaluate factors that lead to occurrence of complications in canine cases. METHODS: A retrospective case-control study was completed using data from the Veterinary Medical Center at the Western College of Veterinary Medicine, Saskatoon, Canada, between May 2015 and March 2020. Case records from dogs that underwent conjunctival pedicle graft surgery and subsequently either did or did not develop a conjunctival graft complication were reviewed. RESULTS: One hundred and six dogs undergoing conjunctival graft surgeries were identified. Sixteen conjunctival graft complications occurred, of which, eight led to negative outcomes. Univariable analysis comparing canine eyes that developed complications to control eyes revealed potential (p ≤ .05) differences between the groups in post-operative fluoroquinolone use, Streptococcus canis isolation, intraoperative intravenous cefazolin use, corneal stromal white cell infiltrate, and mean ulcer diameter. The use of intraoperative cefazolin could not be effectively evaluated with available data; however, no dogs (n = 22) receiving intraoperative cefazolin developed complications. Multivariable analysis revealed that dogs that were treated with a second-generation fluoroquinolone and that had S. canis isolated had higher odds of experiencing complications than dogs that were not treated with a second-generation fluoroquinolone and that were S. canis negative (Odds ratio = 64.7 [95% CI 6.3-669], p < .0001). CONCLUSIONS: Streptococcus species played a role in conjunctival graft complications in our study. Empiric selection of second-generation fluoroquinolone monotherapy may need reconsideration given the frequent isolation of Streptococcus spp. from canine ulcers. The use of intraoperative cefazolin may be associated with a lower complication rate.

Scene-dependent, feedforward eye gaze metrics can differentiate technical skill levels of trainees in laparoscopic surgery.

INTRODUCTION: In laparoscopic surgery, looking in the target areas is an indicator of proficiency. However, gaze behaviors revealing feedforward control (i.e., looking ahead) and their importance have been under-investigated in surgery. This study aims to establish the sensitivity and relative importance of different scene-dependent gaze and motion metrics for estimating trainee proficiency levels in surgical skills. METHODS: Medical students performed the Fundamentals of Laparoscopic Surgery peg transfer task while recording their gaze on the monitor and tool activities inside the trainer box. Using computer vision and fixation algorithms, five scene-dependent gaze metrics and one tool speed metric were computed for 499 practice trials. Cluster analysis on the six metrics was used to group the trials into different clusters/proficiency levels, and ANOVAs were conducted to test differences between proficiency levels. A Random Forest model was trained to study metric importance at predicting proficiency levels. RESULTS: Three clusters were identified, corresponding to three proficiency levels. The correspondence between the clusters and proficiency levels was confirmed by differences between completion times (F2,488 = 38.94, p < .001). Further, ANOVAs revealed significant differences between the three levels for all six metrics. The Random Forest model predicted proficiency level with 99% out-of-bag accuracy and revealed that scene-dependent gaze metrics reflecting feedforward behaviors were more important for prediction than the ones reflecting feedback behaviors. CONCLUSION: Scene-dependent gaze metrics revealed skill levels of trainees more precisely than between experts and novices as suggested in the literature. Further, feedforward gaze metrics appeared to be more important than feedback ones at predicting proficiency.

How and why do financial incentives contribute to helping people stop smoking? A realist review protocol.

INTRODUCTION: Smoking is harmful to human health and programmes to help people stop smoking are key public health efforts that improve individual and population health outcomes. Research shows that financial incentives improve the success of stop smoking programmes. However, a better understanding of how they work is needed to better inform policy and to support building capability for implementation.The aims of this study: (1) To review the international literature to understand: How, why, in what circumstances and for whom financial incentives improve the success of stop smoking interventions among general population groups and among pregnant women. (2) To provide recommendations for how to best use financial incentives in efforts to promote smoking cessation. METHODS AND ANALYSIS: A realist review of published international literature will be undertaken to understand how, why, for whom and in which circumstances financial incentives contribute to success in stopping smoking for general population groups and among pregnant women. Systematic searches were undertaken on 16 February 2022 of five academic databases: MEDLINE (ovid), Embase.com, CIHAHL, Scopus and PsycINFO. Iterative searching using citation tracking and of grey literature will be undertaken as needed. Using Pawson and Tilley's iterative realist review approach, data collected will be screened, selected, coded, analysed and synthesised into a set of explanatory theoretical findings. ETHICS AND DISSEMINATION: Ethical approval is not required for this review as data sources to be included are previously published. The study will provide important findings for policy-makers and health system leaders to guide the development of stop smoking services which use incentives, for example, as part of the Health Service Executive's Tobacco Free Programme in Ireland. Understanding how contextual factors impact implementation and programmatic success is key to developing a more effective public health approach to stop smoking. Our dissemination strategy will be developed with our stakeholders. PROSPERO REGISTRATION NUMBER: CRD42022298941.

Myofilament Phosphorylation in Stem Cell Treated Diastolic Heart Failure.

RATIONALE: Phosphorylation of sarcomeric proteins has been implicated in heart failure with preserved ejection fraction (HFpEF); such changes may contribute to diastolic dysfunction by altering contractility, cardiac stiffness, Ca2+-sensitivity, and mechanosensing. Treatment with cardiosphere-derived cells (CDCs) restores normal diastolic function, attenuates fibrosis and inflammation, and improves survival in a rat HFpEF model. OBJECTIVE: Phosphorylation changes that underlie HFpEF and those reversed by CDC therapy, with a focus on the sarcomeric subproteome were analyzed. METHODS AND RESULTS: Dahl salt-sensitive rats fed a high-salt diet, with echocardiographically verified diastolic dysfunction, were randomly assigned to either intracoronary CDCs or placebo. Dahl salt-sensitive rats receiving low salt diet served as controls. Protein and phosphorylated Ser, Thr, and Tyr residues from left ventricular tissue were quantified by mass spectrometry. HFpEF hearts exhibited extensive hyperphosphorylation with 98% of the 529 significantly changed phospho-sites increased compared with control. Of those, 39% were located within the sarcomeric subproteome, with a large group of proteins located or associated with the Z-disk. CDC treatment partially reverted the hyperphosphorylation, with 85% of the significantly altered 76 residues hypophosphorylated. Bioinformatic upstream analysis of the differentially phosphorylated protein residues revealed PKC as the dominant putative regulatory kinase. PKC isoform analysis indicated increases in PKC α, ß, and δ concentration, whereas CDC treatment led to a reversion of PKCß. Use of PKC isoform specific inhibition and overexpression of various PKC isoforms strongly suggests that PKCß is the dominant kinase involved in hyperphosphorylation in HFpEF and is altered with CDC treatment. CONCLUSIONS: Increased protein phosphorylation at the Z-disk is associated with diastolic dysfunction, with PKC isoforms driving most quantified phosphorylation changes. Because CDCs reverse the key abnormalities in HFpEF and selectively reverse PKCß upregulation, PKCß merits being classified as a potential therapeutic target in HFpEF, a disease notoriously refractory to medical intervention.