Rapid adaptation to CDK2 inhibition exposes intrinsic cell-cycle plasticity.

CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.

O-GlcNAcylation regulates neurofilament-light assembly and function and is perturbed by Charcot-Marie-Tooth disease mutations.

The neurofilament (NF) cytoskeleton is critical for neuronal morphology and function. In particular, the neurofilament-light (NF-L) subunit is required for NF assembly in vivo and is mutated in subtypes of Charcot-Marie-Tooth (CMT) disease. NFs are highly dynamic, and the regulation of NF assembly state is incompletely understood. Here, we demonstrate that human NF-L is modified in a nutrient-sensitive manner by O-linked-ß-N-acetylglucosamine (O-GlcNAc), a ubiquitous form of intracellular glycosylation. We identify five NF-L O-GlcNAc sites and show that they regulate NF assembly state. Interestingly, NF-L engages in O-GlcNAc-mediated protein-protein interactions with itself and with the NF component α-internexin, implying that O-GlcNAc is a general regulator of NF architecture. We further show that NF-L O-GlcNAcylation is required for normal organelle trafficking in primary neurons, underlining its functional significance. Finally, several CMT-causative NF-L mutants exhibit perturbed O-GlcNAc levels and resist the effects of O-GlcNAcylation on NF assembly state, indicating a potential link between dysregulated O-GlcNAcylation and pathological NF aggregation. Our results demonstrate that site-specific glycosylation regulates NF-L assembly and function, and aberrant NF O-GlcNAcylation may contribute to CMT and other neurodegenerative disorders.

Successful Explantation of Berlin Heart Excor in Two Young Children.

Berlin Heart EXCOR ventricular assist device (VAD) implantation in children is widely used as bridge-to-heart transplantation. Berlin left ventricular assist device (LVAD) support as a bridge to recovery is rare. There is a scarcity of literature describing systematic evaluation in pediatric VAD explantation. Patient 1. A 3 month old boy presented with acute heart failure secondary to myocarditis. An echocardiogram demonstrated severely depressed left ventricular function. He required ECMO cannulation and was decannulated 11 days later. He continued to be hemodynamically unstable and required Berlin LVAD implantation with the intent to bridge to transplantation. Patient 2. A 3 month old boy presented initially with a heart rate of 250 beats/min and an electrocardiogram showed multifocal atrial tachycardia. An echocardiogram showed severely decreased left ventricular function. He was placed on ECMO due to unstable hemodynamics. He did not tolerate ECMO decannulation due to persistent chaotic atrial rhythm and underwent Berlin LVAD implantation with the intent to bridge to transplantation. After both patients showed evidence of myocardial recovery, they underwent a weaning protocol that includes: screening, trial-off with echocardiography, and trial-off in the catheterization suite. Our patients met the criteria and underwent successful explantation. Explantation of VAD can be successfully performed even in young children with appropriate candidate selection and a multidisciplinary and systematic approach.

Fluorescent pulse-chase labeling to monitor long-term mitochondrial degradation in primary hippocampal neurons.

The accumulation of dysfunctional mitochondria is a hallmark of neurodegenerative diseases, yet the dynamics of mitochondrial turnover in neurons are unclear. Here, we describe a protocol to monitor the degradation of spectrally distinct, "aged" mitochondrial populations. We describe the preparation and transfection of primary rat hippocampal neuron cultures. We detail a mitochondrial-damaging assay, a SNAP pulse-chase labeling paradigm, and live imaging to visualize the mitochondrial network. Finally, we provide steps to quantify mitochondrial turnover via lysosomal fusion. For complete details on the use and execution of this protocol, please refer to Evans and Holzbaur (2020a).

Extracorporeal membrane oxygenation for multisystem inflammatory syndrome in children.

Early reports suggested that pediatric COVID-19 cases were less severe in children. Most children requiring intensive care admission in these reports had underlying medical conditions. Shortly after the surge of adult COVID-19 cases in Detroit, Michigan, previously healthy children began to present with shock with multiorgan dysfunction, elevated inflammatory markers, and physical exam findings with features of Kawasaki disease. This disease process was later called multisystem inflammatory syndrome in children (MIS-C.) In this case series, we describe three previously healthy children who presented with severe manifestations of MIS-C, including cardiogenic shock and profound systemic inflammation. These children developed severely depressed myocardial function with end-organ injury and were cannulated to veno-arterial extracorporeal membrane oxygenation (VA-ECMO) due to cardiogenic shock with arrhythmia. All three children improved with VA-ECMO support and anti-inflammatory treatment. All had complete recovery of myocardial function at discharge and 6-month follow-up with no significant morbidity.

Insulin signaling and osmotic stress response regulate arousal and developmental progression of C. elegans at hatching.

The progression of animal development from embryonic to juvenile life depends on the coordination of organism-wide responses with environmental conditions. We found that two transcription factors that function in interneuron differentiation in Caenorhabditis elegans, fax-1, and unc-42, are required for arousal and progression from embryogenesis to larval life by potentiating insulin signaling. The combination of mutations in either transcription factor and a mutation in daf-2 insulin receptor results in a novel perihatching arrest phenotype; embryos are fully developed but inactive, often remaining trapped within the eggshell, and fail to initiate pharyngeal pumping. This pathway is opposed by an osmotic sensory response pathway that promotes developmental arrest and a sleep state at the end of embryogenesis in response to elevated salt concentration. The quiescent state induced by loss of insulin signaling or by osmotic stress can be reversed by mutations in genes that are required for sleep. Therefore, countervailing signals regulate late embryonic arousal and developmental progression to larval life, mechanistically linking the two responses. Our findings demonstrate a role for insulin signaling in an arousal circuit, consistent with evidence that insulin-related regulation may function in control of sleep states in many animals. The opposing quiescent arrest state may serve as an adaptive response to the osmotic threat from high salinity environments.

Image Processing for Public Health Surveillance of Tobacco Point-of-Sale Advertising: Machine Learning-Based Methodology.

BACKGROUND: With a rapidly evolving tobacco retail environment, it is increasingly necessary to understand the point-of-sale (POS) advertising environment as part of tobacco surveillance and control. Advances in machine learning and image processing suggest the ability for more efficient and nuanced data capture than previously available. OBJECTIVE: The study aims to use machine learning algorithms to discover the presence of tobacco advertising in photographs of tobacco POS advertising and their location in the photograph. METHODS: We first collected images of the interiors of tobacco retailers in West Virginia and the District of Columbia during 2016 and 2018. The clearest photographs were selected and used to create a training and test data set. We then used a pretrained image classification network model, Inception V3, to discover the presence of tobacco logos and a unified object detection system, You Only Look Once V3, to identify logo locations. RESULTS: Our model was successful in identifying the presence of advertising within images, with a classification accuracy of over 75% for 8 of the 42 brands. Discovering the location of logos within a given photograph was more challenging because of the relatively small training data set, resulting in a mean average precision score of 0.72 and an intersection over union score of 0.62. CONCLUSIONS: Our research provides preliminary evidence for a novel methodological approach that tobacco researchers and other public health practitioners can apply in the collection and processing of data for tobacco or other POS surveillance efforts. The resulting surveillance information can inform policy adoption, implementation, and enforcement. Limitations notwithstanding, our analysis shows the promise of using machine learning as part of a suite of tools to understand the tobacco retail environment, make policy recommendations, and design public health interventions at the municipal or other jurisdictional scale.

Duration of significant patent ductus arteriosus and bronchopulmonary dysplasia in extremely preterm infants.

OBJECTIVE: To demonstrate the association between the duration of significant patent ductus arteriosus (PDA) and bronchopulmonary dysplasia (BPD) in extremely preterm infants. METHODS: All extremely preterm infants (<29 weeks) treated in our Neonatal Intensive Care Unit from January 2013 to March 2016 were included if their PDA status was confirmed at <7 days of life. Infants with genetic syndromes, complex congenital anomalies and insignificant PDAs were excluded. Total duration of significant PDA was estimated by reviewing serial echocardiograms. Significant PDA was diagnosed using our scoring system that was based upon echocardiographic parameters and clinical status of the infants. Study cohort was divided into four groups based on the duration of significant PDA. Group A-No PDA, Group B-PDA <1-week, Group C- PDA 1-2 weeks, and Group D-PDA >2 weeks. ANOVA and multivariate analysis were performed to compare the groups. RESULTS: There were 147 infants with no PDA (Group A), 50, 35, and 41 infants were enrolled in Groups B, C, and D, respectively. There were no differences in maternal and neonatal variables among groups except for the following: maternal smoking, chorioamnionitis, antenatal indomethacin, gestation, birth weight, mode of delivery and incidence of death or BPD. Logistic regression analysis showed that longer duration of significant PDA was associated with higher risk for death or BPD (adjusted OR 1.37, 95% CI 1.03-1.82). CONCLUSION: Longer duration of significant PDA is associated with the higher risk for BPD/death in extremely preterm infants.

Evaluation of Pharmacist Intervention on Discharge Medication Reconciliation.

BACKGROUND: Discharge medication reconciliation (Discharge MedRec) was implemented on one unit at a large urban teaching hospital, and was to be expanded across the rest of the hospital and the health authority's various sites by the end of 2018. Clinical pharmacists on the Acute Care for the Elderly unit carried out discharge planning and led Discharge MedRec during a pilot period, to inform the future implementation. OBJECTIVES: The primary objective was to examine the number and type of medication discrepancies before and after implementation of Discharge MedRec. The secondary objectives were to compare documented medication changes, pharmacist recommendations, discharge counselling, communication with community pharmacists, polypharmacy, and 30-day readmission rates. METHODS: Patients seen in December 2015 constituted the control (pre-implementation) group, who received usual care. Patients seen from January to April 2016 constituted the intervention group, for whom pharmacists performed Discharge MedRec and other discharge activities as per the hospital-to-home checklist of the Institute for Safe Medication Practices Canada. RESULTS: There were 66 patients in the control group and 306 in the intervention group. Median discrepancies per patient decreased from 6.5 to 3 (p = 0.007), median number of documented changes without rationale increased from 2 to 3 (p = 0.01), and median number of documented changes with rationale increased from 1 to 2 (p < 0.001). Pharmacists made a per-patient median of 1 progress note recommendation in the control group and 2 progress note recommendations in the intervention group (p = 0.007), and a per-patient median of 2 orders in both the control and intervention groups (p = 0.62). Median recommendation acceptance was 100% for both groups, but twice as many recommendations were made per patient for the intervention group. Discharge counselling increased from 22.7% to 65%. Communication with community pharmacists increased from 10.6% to 60.8%. CONCLUSIONS: Clinical pharmacist involvement improved Discharge MedRec planning and documentation. Decreases in medication discrepancies, combined with an increase in discharge counselling, should improve continuity of care across the health care team and increase patient adherence with medication therapy. This study further demonstrates the leadership role that pharmacists play in the assessment and clear documentation of medication changes at all transitions of care.

CONTEXTE: Le processus de bilan comparatif des médicaments au moment du congé a été mis en place dans une unité d'un important hôpital universitaire en milieu urbain et devait être mis en place dans le reste de l'hôpital et dans les différents sites de la régie de santé avant la fin de 2018. Des pharmaciens cliniciens de l'Unité de soins gériatriques de courte durée ont réalisé la planification des congés et ont dirigé le processus de bilan comparatif des médicaments au moment du congé, au cours d'une période d'essai, afin de contribuer à une future mise en place d'un tel processus. OBJECTIFS: L'objectif principal consistait en l'étude du nombre et du type de divergences relatives aux médicaments avant et après la mise en place du processus de bilan comparatif des médicaments au moment du congé. Les objectifs secondaires portaient sur la comparaison des éléments suivants : les changements apportés à la pharmacothérapie, les recommandations des pharmaciens, l'offre de conseils au moment du congé, les échanges avec les pharmaciens communautaires, la polypharmacie et les taux de réadmissions dans les 30 jours suivant le congé. MÉTHODES: Les patients rencontrés en décembre 2015 constituaient le groupe témoin (avant la mise en place du processus) ayant reçu les soins habituels. Les patients rencontrés entre janvier et avril 2016 formaient le groupe expérimental pour lequel les pharmaciens avaient réalisé un processus de bilan comparatif des médicaments au moment du congé et d'autres activités en lien avec le congé, en fonction de la liste de vérification du transfert de l'hôpital à la maison de l'Institut pour la sécurité des médicaments aux patients du Canada. RÉSULTATS: Il y avait 66 patients dans le groupe témoin et 306 dans le groupe expérimental. Le nombre médian de divergences par patient a diminué et est passé de 6,5 à 3 (p = 0,007), le nombre médian de changements consignés, apportés sans raison apparente a augmenté et est passé de 2 à 3 (p = 0,01) et le nombre médian de changements consignés, dont la raison apparaissait aux dossiers a augmenté et est passé de 1 à 2 (p < 0,001). Le nombre médian de recommandations par patient dans les notes d'évolution réalisées par les pharmaciens était de un dans le groupe témoin et de deux dans le groupe expérimental (p = 0,007) et le nombre médian d'ordonnances par patient réalisées par des pharmaciens était de deux, tant dans le groupe témoin que dans le groupe expérimental (p = 0,62). Les taux médians d'acceptation des recommandations étaient de 100 % dans les deux groupes, mais il y a eu deux fois plus de recommandations par patient réalisées dans le groupe expérimental. L'offre de conseils au moment du congé a augmenté et est passée de 22,7 % à 65 %. Les échanges avec les pharmaciens communautaires ont augmenté et sont passés de 10,6 % à 60,8 %. CONCLUSIONS: La participation des pharmaciens cliniciens a amélioré la planification et l'enregistrement du bilan comparatif des médicaments au moment du congé. Une réduction des divergences concernant les médicaments, associée à une augmentation de l'offre de conseils au moment du congé, devrait améliorer la continuité des soins au sein de l'équipe de soins de santé et accroître l'observance thérapeutique du patient. La présente étude est un nouvel exemple du rôle de leader que les pharmaciens jouent dans l'évaluation et la description claire des changements apportés à la pharmacothérapie à chaque transfert des soins.

Air pollution particulate SRM 1648 causes oxidative stress in RAW 264.7 macrophages leading to production of prostaglandin E2, a potential Th2 mediator.

Particulates in air pollution have been strongly associated with asthma symptoms. These particulates are a conglomeration of many components, including metals, polyaromatic hydrocarbons, and lipopolysaccharide, that may cause oxidative stress upon uptake by alveolar macrophages. The objective of this study was to assess whether uptake of a model air particulate (SRM 1648) causes oxidative stress in macrophages resulting in the production of the eicosanoid mediator prostaglandin E(2) (PGE(2)) that might exacerbate asthma. SRM 1648 suspended in phosphate-buffered saline (PBS) was introduced into wells with plated RAW 264.7 monocyte/macrophages. Following incubation of SRM 1648 with RAW 264.7 macrophages, prostaglandin E(2) was measured by enzyme immunosorbent assay (EIA), and oxidative stress was assessed by the levels of intracellular reduced glutathione (GSH) as well as by the oxidation of dihydrodichlorofluorescein (H(2)DCFDA) to the fluorescent dichlorofluoresecein (DCF). The results indicated that SRM 1648 caused oxidative stress in RAW 264.7 macrophages, as shown by a compensatory increase in GSH levels in comparison to the controls of titanium dioxide and media alone. Prostaglandin E(2) levels significantly increased at the 3-, 6-, and 12-h time points. Introduction of GSH ester to buffer against oxidative stress was able to block the elevation of PGE(2). The data show that SRM 1648 causes oxidative stress in RAW 264.7 macrophages resulting in formation of the potential Th2 mediator prostaglandin E(2).

Environmental oxygen tension affects phenotype in cultured bone marrow-derived macrophages.

This study tested the hypothesis that the unique phenotype of alveolar macrophages (AM) is maintained through adaptation to the relatively high oxygen partial pressure (P(O2)) of the lung, through modification of redox-sensitive transcription factors. BALB/c mouse bone marrow-derived macrophages (BMC) were differentiated under different P(O2) and compared functionally to AM and peritoneal macrophages (PM). BMC differentiated in normoxia (P(O2) 140 Torr, BMC(high)) were similar to AM in having low phagocytic and antigen presenting cell (APC) activities. However, BMC grown in low oxygen tension as found in other tissues (<40 Torr, BMC(low)) were better phagocytes and APCs, similar to PM. BMC(high) were more oxidative intracellularly than BMC(low), based on oxidation of dichlorofluorescein and higher glutathione disulfide/glutathione (GSH) ratios, despite having more GSH. Finally, lipopolysaccharide-induced nuclear factor-kappaB translocation, measured by laser scanning cytometry, was reduced in BMC(high) and AM, compared with BMC(low) and PM, respectively. These data suggest that regulation of the AM phenotype may occur, at least in part, via inhibition of NF-kappaB by the unique redox environment.