WITHDRAWN: Oncogenic KRAS G12D extrinsically induces an immunosuppressive microenvironment in lung adenocarcinoma.

This manuscript has been withdrawn by the authors due to a dispute over co-first authorship that is currently being arbitrated by the medical school at our institution. Therefore, the authors do not wish this work to be cited as reference for the project. Upon completion of the arbitration process, we will take steps to revert the current withdrawn status. If you have any questions, please contact the corresponding author.

Modeling Molecular Pathogenesis of Idiopathic Pulmonary Fibrosis-Associated Lung Cancer in Mice.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive, often fatal loss of lung function due to overactive collagen production and tissue scarring. Patients with IPF have a sevenfold-increased risk of developing lung cancer. The COVID-19 pandemic has increased the number of patients with lung diseases, and infection can worsen prognoses for those with chronic lung diseases and disease-associated cancer. Understanding the molecular pathogenesis of IPF-associated lung cancer is imperative for identifying diagnostic biomarkers and targeted therapies that will facilitate prevention of IPF and progression to lung cancer. To understand how IPF-associated fibroblast activation, matrix remodeling, epithelial-to-mesenchymal transition (EMT), and immune modulation influences lung cancer predisposition, we developed a mouse model to recapitulate the molecular pathogenesis of pulmonary fibrosis-associated lung cancer using the bleomycin and Lewis lung carcinoma models. We demonstrate that development of pulmonary fibrosis-associated lung cancer is likely linked to increased abundance of tumor-associated macrophages and a unique gene signature that supports an immune-suppressive microenvironment through secreted factors. Not surprisingly, preexisting fibrosis provides a pre-metastatic niche and results in augmented tumor growth, and tumors associated with bleomycin-induced fibrosis are characterized by a dramatic loss of cytokeratin expression, indicative of EMT. IMPLICATIONS: This characterization of tumors associated with lung diseases provides new therapeutic targets that may aid in the development of treatment paradigms for lung cancer patients with preexisting pulmonary diseases.

Histone H3 K27M-mediated regulation of cancer cell stemness and differentiation in diffuse midline glioma.

Therapeutic resistance remains a major obstacle to preventing progression of H3K27M-altered Diffuse Midline Glioma (DMG). Resistance is driven in part by ALDH-positive cancer stem cells (CSC), with high ALDH1A3 expression observed in H3K27M-mutant DMG biopsies. We hypothesized that ALDH-mediated stemness and resistance may in part be driven by the oncohistone itself. Upon deletion of H3K27M, ALDH1A3 expression decreased dramatically and was accompanied by a gain in astrocytic marker expression and a loss of neurosphere forming potential, indicative of differentiation. Here we show that the oncohistone regulates histone acetylation through ALDH1A3 in a Wnt-dependent manner and that loss of H3K27M expression results in sensitization of DMGs to radiotherapy. The observed elevated Wnt signaling in H3K27M-altered DMG likely stems from a dramatic suppression of mRNA and protein expression of the Wnt inhibitor EYA4 driven by the oncohistone. Thus, our findings identify EYA4 as a bona fide tumor suppressor in DMG that upon suppression, results in aberrant Wnt signaling to orchestrate stemness and differentiation. Future studies will explore whether overexpression of EYA4 in DMG can impede growth and invasion. In summary, we have gained mechanistic insight into H3K27M-mediated regulation of cancer stemness and differentiation, which provides rationale for exploring new therapeutic targets for DMG.

Reducing Caregiver Hunger During Pediatric Hospitalization.

BACKGROUND AND OBJECTIVES: Pediatric hospitalizations are costly, stressful events for families. Many caregivers, especially those with lower incomes, struggle to afford food while their child is hospitalized. We sought to decrease the mean percentage of caregivers of Medicaid-insured and uninsured children who reported being hungry during their child's hospitalization from 86% to <24%. METHODS: Our quality improvement efforts took place on a 41-bed inpatient unit at our large, urban academic hospital. Our multidisciplinary team included physicians, nurses, social workers, and food services leadership. Our primary outcome measure was caregiver-reported hunger; we asked caregivers near to the time of discharge if they experienced hunger during their child's hospitalization. Plan-do-study-act cycles addressed key drivers: awareness of how to obtain food, safe environment for families to seek help, and access to affordable food. An annotated statistical process control chart tracked our outcome over time. Data collection was interrupted because of the COVID-19 pandemic; we used that time to advocate for hospital-funded support for optimal and sustainable changes to caregiver meal access. RESULTS: We decreased caregiver hunger from 86% to 15.5%. A temporary test of change, 2 meal vouchers per caregiver per day, resulted in a special cause decrease in the percentage of caregivers reporting hunger. Permanent hospital funding was secured to provide cards to purchase 2 meals per caregiver per hospital day, resulting in a sustained decrease in rates of caregiver hunger. CONCLUSIONS: We decreased caregivers' hunger during their child's hospitalization. Through a data-driven quality improvement effort, we implemented a sustainable change allowing families to access enough food.

How Does TIMELESS Training Impact Resident Motivation for Learning, Assessment, and Feedback? Evaluating a Competency-Based Time-Variable Training Pilot.

PURPOSE: As competency-based medical education has become the predominant graduate medical education training model, interest in time-variable training has grown. Despite multiple competency-based time-variable training (CBTVT) pilots ongoing in the United States, little is known about how this training approach impacts learners. The authors aim to explore how their CBTVT pilot program impacted resident motivation for learning, assessment, and feedback. METHOD: The authors performed a qualitative educational case study on the Transitioning in Internal Medicine Education Leveraging Entrustment Scores Synthesis (TIMELESS) program at the University of Cincinnati from October 2020 through March 2022. Semistructured interviews were conducted with TIMELESS residents (n = 9) approximately every 6 months to capture experiences over time. The authors used inductive thematic analysis to develop themes and compared their findings with existing theories of learner motivation. RESULTS: The authors developed 2 themes: TIMELESS had variable effects on residents' motivation for learning and TIMELESS increased resident engagement with and awareness of the program of assessment. Participants reported increased motivation to learn and seek assessment, though some felt a tension between performance (e.g., advancement through the residency program) and growth (e.g., improvement as a physician). Participants became more aware of the quality of assessments they received, in part due to TIMELESS increasing the perceived stakes of assessment, and reported being more deliberate when assessing other residents. CONCLUSIONS: Resident motivation for learning, assessment, and feedback was impacted in ways that the authors contextualize using current theories of learner motivation (i.e., goal orientation theory and attribution theory). Future research should investigate how interventions, such as coaching, guided learner reflection, or various CBTVT implementation strategies, can help keep learners oriented toward mastery learning rather than toward performance.

Transcriptomic Analysis of Diffuse Intrinsic Pontine Glioma (DIPG) Identifies a Targetable ALDH-Positive Subset of Highly Tumorigenic Cancer Stem-like Cells.

Understanding the cancer stem cell (CSC) landscape in diffuse intrinsic pontine glioma (DIPG) is desperately needed to address treatment resistance and identify novel therapeutic approaches. Patient-derived DIPG cells demonstrated heterogeneous expression of aldehyde dehydrogenase (ALDH) and CD133 by flow cytometry. Transcriptome-level characterization identified elevated mRNA levels of MYC, E2F, DNA damage repair (DDR) genes, glycolytic metabolism, and mTOR signaling in ALDH+ compared with ALDH-, supporting a stem-like phenotype and indicating a druggable target. ALDH+ cells demonstrated increased proliferation, neurosphere formation, and initiated tumors that resulted in decreased survival when orthotopically implanted. Pharmacologic MAPK/PI3K/mTOR targeting downregulated MYC, E2F, and DDR mRNAs and reduced glycolytic metabolism. In vivo PI3K/mTOR targeting inhibited tumor growth in both flank and an ALDH+ orthotopic tumor model likely by reducing cancer stemness. In summary, we describe existence of ALDH+ DIPGs with proliferative properties due to increased metabolism, which may be regulated by the microenvironment and likely contributing to drug resistance and tumor recurrence. IMPLICATIONS: Characterization of ALDH+ DIPGs coupled with targeting MAPK/PI3K/mTOR signaling provides an impetus for molecularly targeted therapy aimed at addressing the CSC phenotype in DIPG.

Increasing Physician Reporting of Diagnostic Learning Opportunities.

BACKGROUND: An estimated 10% of Americans experience a diagnostic error annually, yet little is known about pediatric diagnostic errors. Physician reporting is a promising method for identifying diagnostic errors. However, our pediatric hospital medicine (PHM) division had only 1 diagnostic-related safety report in the preceding 4 years. We aimed to improve attending physician reporting of suspected diagnostic errors from 0 to 2 per 100 PHM patient admissions within 6 months. METHODS: Our improvement team used the Model for Improvement, targeting the PHM service. To promote a safe reporting culture, we used the term diagnostic learning opportunity (DLO) rather than diagnostic error, defined as a "potential opportunity to make a better or more timely diagnosis." We developed an electronic reporting form and encouraged its use through reminders, scheduled reflection time, and monthly progress reports. The outcome measure, the number of DLO reports per 100 patient admissions, was tracked on an annotated control chart to assess the effect of our interventions over time. We evaluated DLOs using a formal 2-reviewer process. RESULTS: Over the course of 13 weeks, there was an increase in the number of reports filed from 0 to 1.6 per 100 patient admissions, which met special cause variation, and was subsequently sustained. Most events (66%) were true diagnostic errors and were found to be multifactorial after formal review. CONCLUSIONS: We used quality improvement methodology, focusing on psychological safety, to increase physician reporting of DLOs. This growing data set has generated nuanced learnings that will guide future improvement work.

Comparing Two Proximal Measures of Unrecognized Clinical Deterioration in Children.

Critical deterioration events (CDEs) and emergency transfers (ETs) are two proximal measures to cardiopulmonary arrest, and both aim to evaluate how systems recognize and respond to clinical deterioration in children. This retrospective observational study sought to (1) characterize CDEs and ETs by timing, overlap, and intervention category, and (2) evaluate the performance of the watcher identification system and the pediatric early warning score (PEWS) to identify patients who experience these events. A total of 359 CDEs and 88 ETs occurred during the study period. Respiratory events were most common and accounted for 80.5% of CDEs and 47.7% of ETs. A narrow majority of patients were identified as watchers (55.4% of CDEs and 51.1% of ETs). In total, 85.5% of CDEs and 87.5% of ETs were identified as watchers, elevated PEWS, or both. Opportunities exist for improved escalation plans for high-risk patients to prevent the need for emergent intervention.

Association Between Statewide School Closure and COVID-19 Incidence and Mortality in the US.

Importance: In the US, states enacted nonpharmaceutical interventions, including school closure, to reduce the spread of coronavirus disease 2019 (COVID-19). All 50 states closed schools in March 2020 despite uncertainty if school closure would be effective. Objective: To determine if school closure and its timing were associated with decreased COVID-19 incidence and mortality. Design, Setting, and Participants: US population-based observational study conducted between March 9, 2020, and May 7, 2020, using interrupted time series analyses incorporating a lag period to allow for potential policy-associated changes to occur. To isolate the association of school closure with outcomes, state-level nonpharmaceutical interventions and attributes were included in negative binomial regression models. States were examined in quartiles based on state-level COVID-19 cumulative incidence per 100 000 residents at the time of school closure. Models were used to derive the estimated absolute differences between schools that closed and schools that remained open as well as the number of cases and deaths if states had closed schools when the cumulative incidence of COVID-19 was in the lowest quartile compared with the highest quartile. Exposures: Closure of primary and secondary schools. Main Outcomes and Measures: COVID-19 daily incidence and mortality per 100 000 residents. Results: COVID-19 cumulative incidence in states at the time of school closure ranged from 0 to 14.75 cases per 100 000 population. School closure was associated with a significant decline in the incidence of COVID-19 (adjusted relative change per week, -62% [95% CI, -71% to -49%]) and mortality (adjusted relative change per week, -58% [95% CI, -68% to -46%]). Both of these associations were largest in states with low cumulative incidence of COVID-19 at the time of school closure. For example, states with the lowest incidence of COVID-19 had a -72% (95% CI, -79% to -62%) relative change in incidence compared with -49% (95% CI, -62% to -33%) for those states with the highest cumulative incidence. In a model derived from this analysis, it was estimated that closing schools when the cumulative incidence of COVID-19 was in the lowest quartile compared with the highest quartile was associated with 128.7 fewer cases per 100 000 population over 26 days and with 1.5 fewer deaths per 100 000 population over 16 days. Conclusions and Relevance: Between March 9, 2020, and May 7, 2020, school closure in the US was temporally associated with decreased COVID-19 incidence and mortality; states that closed schools earlier, when cumulative incidence of COVID-19 was low, had the largest relative reduction in incidence and mortality. However, it remains possible that some of the reduction may have been related to other concurrent nonpharmaceutical interventions.

Improving laboratory workflow through automated pre-processing of SurePath specimens for human papillomavirus testing with the Abbott RealTime assay.

BACKGROUND: Cervical specimens collected in liquid-based cytology (LBC) are used for cervical screening in the UK. The Abbott RealTime high-risk human papillomavirus (hrHPV) assay for the detection of 14 hrHPV types is approved by the English NHS Cervical Screening Programme (NHSCSP). As per manufacturer's instructions, pre-analytic processing of LBC involves a manual vortex and liquid transfer into a secondary tube. In high-throughput settings, hands-on time for pre-processing is considerable and poses the potential for human error. Implementation of hrHPV primary screening planned for 2019 accompanied by centralisation of services is a major change for the NHSCSP, increasing the demand for availability of automated pre-analytics. This study evaluated a custom-configured work-table setup of the Tecan Freedom EVO designed to automate pre-processing of BD SurePath LBC prior to HPV testing on the Abbott m2000. METHODS: Automatically and manual pre-processed specimen results were compared (primary screening population n = 307; triage population n = 169). RESULTS: Excellent agreement of overall hrHPV results (98.1%; k: 0.95) was observed. On average, it takes approximately 1.5 minutes hands-on-time per sample to process manually compared to 45 minutes to aliquot 48 samples using the Freedom EVO 150. CONCLUSION: The Tecan worktable configuration designed to automate and control pre-analytics required for preparing SurePath LBC samples for HPV testing using Abbott RealTime resulted in assay performance comparable to that following the manufacturer validated manual process. It significantly reduces hands-on-time and allows for complete specimen identification tracking and documentation of process control.

Hsp27 associates with actin and limits injury in energy depleted renal epithelia.

The purpose of the study was to determine whether Hsp27 interacts with actin and could protect against selected manifestations of injury from energy depletion in renal epithelia. LLC-PK1 cells were stably transfected to overexpress human Hsp27 tagged with green fluorescence protein (GFP). Transfected expression of the labeled Hsp27 did not reduce endogenous Hsp25 levels in the cells compared with either nontransfected cells or cells transfected with GFP alone used as the transfectant control (G). By fluorescence energy transfer (FRET) between GFP-tagged Hsp27 and rhodamine phalloidin-decorated actin, minimal interaction was found in uninjured control cells. In ATP-depleted cells, Hsp27 was associated closely with F-actin at lateral cell boundaries and with aggregated actin within the cell body. Less Hsp27 interaction with actin was found during recovery; but when adjusted for total phalloidin fluorescence, FRET between Hsp27 and F-actin did not change between 2-h ATP depletion and 4-h recovery. Where Hsp27 association with actin persisted during recovery, it was principally with the residual aggregates of actin in the cell body. Detachment of Na,K-ATPase from the cytoskeleton at 2-h ATP depletion was significantly less in Hsp27 cells compared with transfectant control G cells but not at 4-h ATP depletion. Detachment of ezrin from the cytoskeleton during ATP depletion was nearly complete and was not prevented in the Hsp27 cells. Protection of the Hsp27 cells was not attributable to preservation of cellular ATP levels. Hsp27 appears to have specific actions in renal epithelia subjected to energy depletion, including interacting with actin to preserve architecture in specific intracellular domains.