Adverse Tracheal Intubation Events in Critically Ill Underweight and Obese Children: Retrospective Study of the National Emergency Airway for Children Registry (2013-2020).

OBJECTIVES: Extremes of patient body mass index are associated with difficult intubation and increased morbidity in adults. We aimed to determine the association between being underweight or obese with adverse airway outcomes, including adverse tracheal intubation (TI)-associated events (TIAEs) and/or severe peri-intubation hypoxemia (pulse oximetry oxygen saturation < 80%) in critically ill children. DESIGN/SETTING: Retrospective cohort using the National Emergency Airway for Children registry dataset of 2013-2020. PATIENTS: Critically ill children, 0 to 17 years old, undergoing TI in PICUs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Registry data from 24,342 patients who underwent TI between 2013 and 2020 were analyzed. Patients were categorized using the Centers for Disease Control and Prevention weight-for-age chart: normal weight (5th-84th percentile) 57.1%, underweight (< 5th percentile) 27.5%, overweight (85th to < 95th percentile) 7.2%, and obese (≥ 95th percentile) 8.2%. Underweight was most common in infants (34%); obesity was most common in children older than 8 years old (15.1%). Underweight patients more often had oxygenation and ventilation failure (34.0%, 36.2%, respectively) as the indication for TI and a history of difficult airway (16.7%). Apneic oxygenation was used more often in overweight and obese patients (19.1%, 19.6%) than in underweight or normal weight patients (14.1%, 17.1%; p < 0.001). TIAEs and/or hypoxemia occurred more often in underweight (27.1%) and obese (24.3%) patients ( p < 0.001). TI in underweight children was associated with greater odds of adverse airway outcome compared with normal weight children after adjusting for potential confounders (underweight: adjusted odds ratio [aOR], 1.09; 95% CI, 1.01-1.18; p = 0.016). Both underweight and obesity were associated with hypoxemia after adjusting for covariates and site clustering (underweight: aOR, 1.11; 95% CI, 1.02-1.21; p = 0.01 and obesity: aOR, 1.22; 95% CI, 1.07-1.39; p = 0.002). CONCLUSIONS: In underweight and obese children compared with normal weight children, procedures around the timing of TI are associated with greater odds of adverse airway events.

Microfluidic Digital Quantitative PCR to Measure Internal Cargo of Individual Liposomes.

Lipid nanoparticles serve as drug delivery vehicles for biopharmaceutical products. The lipid membrane shields internal nucleic-acid drug cargo from enzymatic degradation and facilitates cellular uptake of the drug. However, existing methods to assess drug loading within liposomes are limited to averaged bulk measurements, which obscures heterogeneity of the biopharmaceutical formulation. This report describes the development of a single-liposome analysis method to measure copy numbers of DNA within liposomes and assess population heterogeneity. This novel measurement was achieved by integrating two orthogonal polymerase chain reaction (PCR) techniques─digital PCR (dPCR) and quantitative PCR (qPCR)─within a single microfluidic assay. The dPCR dimension quantified liposomes to validate their capture in the single-liposome analysis regime. The qPCR dimension quantified DNA copy numbers packaged within each liposome. The ability of digital quantitative PCR (dqPCR) to analyze large numbers of individual liposomes in parallel revealed significant population heterogeneity, which could not be obtained from standard bulk analysis methods. Our innovative measurement of internal DNA cargo from single liposomes has the potential to inform liposome synthesis procedures and create more uniform liposomal biopharmaceutical formulations to enhance drug safety and efficacy.

Influence of microfabrication on digital PCR performance in bead-based microwell array assays.

Digital PCR (dPCR) is a highly sensitive analytical technique used to quantify DNA targets. Detection sensitivity can be further enhanced by capturing target sequences onto beads for preconcentration and sample cleanup prior to analysis in microfluidic microwell arrays. However, robust digital analysis requires individual beads to be interrogated within individual wells. Fabricating microwells with dimensions ≤ 3 µm is challenging, and the high surface area-to-volume ratio of the wells leaves PCR susceptible to inhibition stemming from materials used during device processing. This report describes the development of a microfabrication procedure to create ultralow-volume wells (100 fL) for bead-based dPCR and characterize the effects of microprocessing materials on assay performance. Standard microfabrication protocols used for creating microelectronics resulted in devices with nanoscopic debris originating from photoresists used during processing. A model dPCR assay was developed to characterize the effects of this debris, which revealed variable PCR inhibition. Debris within microwells attenuated digital and analog assay signals to a greater extent than debris on the device surface. Spatial heterogeneity of debris across devices was quantified to characterize regional PCR inhibition and intra- and inter-device variability. Ultimately, a fabrication procedure was developed to create pristine microfluidic arrays using dual processes to remove positive resist and forgoing use of negative resist entirely, which enabled robust amplification with digital signals matching theoretical predictions. Results from this work catalog the unique performance artifacts from device microfabrication and provide a guide for future studies seeking to conduct robust, high-sensitivity bead-based dPCR assays. Graphical abstract.

Tracing the Equilibrium Phase of Cancer Immunoediting in Epidermal Neoplasms via Longitudinal Intravital Imaging.

Recognition of transformed cells by the immune system can sometimes generate a rate-limiting equilibrium phase, wherein tumor outgrowth is prevented without complete neoplasm elimination. Targeting premalignancies during this immune-controlled bottleneck is a promising strategy for rational cancer prevention. Thus far, immune equilibrium has been difficult to model in a traceable way, and most immunoediting systems have been limited to mesenchymal tumor types. Here, we introduce a mouse model for fluorescent tracing of somatic epithelial transformation. We demonstrate that transplantation can be used to prevent a confounding artificial tolerance that affects autochthonous inducible models. Using this system, we observe the expected dichotomy of outcomes: immune-deficient contexts permit rapid tumorigenesis, whereas initiated clones in immunocompetent mice undergo elimination or equilibrium. The equilibrium phase correlates with localization within hair follicles, which have been characterized previously as relatively immune-privileged sites. Given this, we posit that valleys in the immune surveillance landscape of a normal tissue can provide a cell-extrinsic alternative to the canonical cell-intrinsic adaptations believed to establish the equilibrium phase. Our model is a prototype for tracing immunoediting in vivo and could serve as a novel screening platform for therapies targeted against immune-controlled premalignancies.

No evidence of a role of the ß4 subunit of the nicotinic acetylcholine receptor in alcohol-related behaviors.

BACKGROUND: Nicotinic acetylcholine receptors have gained attention in the last several years as mediators of alcohol-related behaviors. The genes that code for the α5, α3, and ß4 subunits (Chrna5, Chrna3, and Chrnb4, respectively) map adjacent to each other on human chromosome 15/mouse chromosome 9. Genetic variants in this region have been associated with alcohol phenotypes and mice that overexpress these three subunits have reduced ethanol intake. In the present experiments, we examined the role of the Chrnb4 gene in three ethanol behaviors: consumption, ataxia, and sedation. Wildtype, heterozygous, and knockout mice were tested for ethanol consumption with a 2-bottle choice procedure and the drinking-in-the-dark paradigm. Ethanol-induced ataxia was measured with the balance beam and dowel test. Finally, the sedative effects of ethanol were measured with the loss of righting reflex paradigm. RESULTS: We observed no significant genotypic effects on any of the ethanol behaviors examined, suggesting that the ß4 subunit is not involved in mediating these responses. CONCLUSIONS: While we found no evidence for the involvement of the ß4 subunit in ethanol responses, it is possible that this subunit modulates other behaviors not tested and further work should address this before completely ruling out its involvement.

Mesolimbic transcriptional response to hedonic substitution of voluntary exercise and voluntary ethanol consumption.

The mesolimbic dopaminergic pathway has been implicated in many rewarding behaviors, including the consumption of ethanol and voluntary exercise. It has become apparent that different rewarding stimuli activate this pathway, and therefore it is possible for these behaviors to influence each other, i.e. hedonic substitution. Using adult female C57BL/6J mice, we demonstrate that voluntary access to a running wheel substantially reduces the consumption and preference of ethanol. Furthermore, we examined gene expression of several genes involved in regulating the mesolimbic dopaminergic pathway, which we hypothesized to be the main pathway involved in hedonic substitution. In the striatum, we observed a reduction in mRNA expression of Drd1a due to exercise. Hippocampal Bdnf mRNA increased in response to exercise and decreased in response to ethanol. Furthermore, there was an interaction effect of exercise and ethanol on the expression of Slc18a2 in the midbrain. These data suggest an important role for this pathway, and especially for Bdnf and Slc18a2 in regulating hedonic substitution.