Performance characteristics of a polymerase chain reaction-based assay for the detection of EGFR mutations in plasma cell-free DNA from patients with non-small cell lung cancer using cell-free DNA collection tubes.

Survival rates in non-small cell lung cancer (NSCLC) are low. Detection of circulating tumor DNA in liquid biopsy (plasma) is increasingly used to identify targeted therapies for clinically actionable mutations, including EGFR mutations in NSCLC. The cobas® EGFR Mutation Test v2 (cobas EGFR test) is FDA-approved for EGFR mutation detection in tissue or liquid biopsy from NSCLC. Standard K2EDTA tubes require plasma separation from blood within 4 to 8 hours; however, Roche Cell-Free DNA (cfDNA) Collection Tubes (Roche cfDNA tube) enable whole blood stability for up to 7 days prior to plasma separation. This analysis assessed performance of Roche cfDNA tubes with the cobas EGFR test for the detection of EGFR mutations in plasma from healthy donors or patients with NSCLC. Overall, test performance was equally robust with either blood collection tube, eg, regarding limit of detection, linearity, and reproducibility, making Roche cfDNA tubes suitable for routine clinical laboratory use in this setting. Importantly, the Roche cfDNA tubes provided more flexibility for specimen handling versus K2EDTA tubes, eg, in terms of tube mixing, plasma separation, and sample stability, and do not require processing of blood within 8 hours thereby increasing the reach of plasma biopsies in NSCLC.

Biallelic variants in FLII cause pediatric cardiomyopathy by disrupting cardiomyocyte cell adhesion and myofibril organization.

Pediatric cardiomyopathy (CM) represents a group of rare, severe disorders that affect the myocardium. To date, the etiology and mechanisms underlying pediatric CM are incompletely understood, hampering accurate diagnosis and individualized therapy development. Here, we identified biallelic variants in the highly conserved flightless-I (FLII) gene in 3 families with idiopathic, early-onset dilated CM. We demonstrated that patient-specific FLII variants, when brought into the zebrafish genome using CRISPR/Cas9 genome editing, resulted in the manifestation of key aspects of morphological and functional abnormalities of the heart, as observed in our patients. Importantly, using these genetic animal models, complemented with in-depth loss-of-function studies, we provided insights into the function of Flii during ventricular chamber morphogenesis in vivo, including myofibril organization and cardiomyocyte cell adhesion, as well as trabeculation. In addition, we identified Flii function to be important for the regulation of Notch and Hippo signaling, crucial pathways associated with cardiac morphogenesis and function. Taken together, our data provide experimental evidence for a role for FLII in the pathogenesis of pediatric CM and report biallelic variants as a genetic cause of pediatric CM.

Core outcomes for orofacial clefts: reconciling traditional and ICHOM minimum datasets.

OBJECTIVE/DESIGN/SETTING: This retrospective study sought voluntary participation from leading cleft centres from Europe and Brazil regarding core outcome measures. The results of this study would inform the debate on core outcome consensus pertaining to the European Reference Network for rare diseases (ERN CRANIO) and achieve a core outcome set for cleft care providers worldwide. INTERVENTION/METHOD: Five orofacial cleft (OFC) disciplines were identified, within which all of the International Consortium of Health Outcomes Measurement (ICHOM) outcomes fall. One questionnaire was designed for each discipline and comprised 1. the relevant ICHOM's outcomes within that discipline, and 2. a series of questions targeted to clinicians. What core outcomes are currently measured and when, did these align with the ICHOM minimum, if not how did they differ, and would they recommend modified or additional outcomes?. RESULTS: For some disciplines participants agreed with the ICHOM minimums but urged for earlier and more frequent intervention. Some clinicians felt that some of the ICHOM standards were compatible but that different ages were preferred and for others the ICHOM standards were acceptable but developmental stages should be preferred to absolute time points. CONCLUSION/IMPLICATIONS: Core outcomes for OFC were supported in principle but there are differences between the ICHOM recommendations and the 2002 WHO global consensus. The latter are established in many centres with historical archives of OFC outcome data, and it was concluded that with some modifications ICHOM could be moulded into useful core outcomes data for inter-centre comparisons worldwide.

Prehospital Ultrasound Use to Guide Resuscitative Thoracotomy in Blunt Traumatic Cardiac Arrest.

Traumatic cardiac arrest is frequently encountered in the air medical transport environment, and resuscitative thoracotomy is a procedure that is sometimes performed in an attempt to salvage these critically injured patients. Focused assessment with sonography for trauma (FAST) is a point-of-care ultrasound protocol commonly used in trauma patients to detect the presence of free fluid in the intraperitoneal and pericardial spaces. The authors present a case of an adult female victim of a motor vehicle collision whose prehospital FAST scan revealed significant hemoperitoneum without hemopericardium. When she developed cardiac arrest, these ultrasound findings aided in the decision to perform resuscitative thoracotomy and helped guide the sequence of maneuvers with prioritization given to cross-clamping the aorta. This case highlights the utility of prehospital ultrasound in yielding timely, actionable diagnostic information that can inform the performance of a high-acuity low-occurrence procedure in the air medical transport environment.

Optogenetic control of YAP cellular localisation and function.

YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attach a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression and cell proliferation. Similarly, optofYap can be used in zebrafish embryos to modulate target genes. We demonstrate that optoYAP can override a cell's response to substrate stiffness to generate anchorage-independent growth. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.

Initiation of Inhaled Nitric Oxide by an Air Transport Team in Adult Coronavirus Disease 2019 Respiratory Failure.

The coronavirus disease 2019 (COVID-19) pandemic has caused a significant increase in the volume of critical care flight transports between outlying referral hospitals and tertiary care facilities. Because of the tropism of severe acute respiratory syndrome coronavirus 2, flight crews are often asked to transport mechanically ventilated patients in refractory hypoxemic respiratory failure. The authors present a case series of 5 patients with COVID-19 acute respiratory distress syndrome (ARDS) who were initiated on inhaled nitric oxide (iNO) by the transport team before rotor wing transport and survived the journey in stable or improved condition upon arrival. Previously, no case reports have described adults with COVID-19 ARDS transported after iNO initiation by the transport team. This case series shows the feasibility of iNO initiation by trained air medical transport teams and suggests a short-term stabilizing effect of iNO in patients with ARDS from COVID-19.

DNA-damage induced cell death in yap1;wwtr1 mutant epidermal basal cells.

In a previous study, it was reported that Yap1 and Wwtr1 in zebrafish regulates the morphogenesis of the posterior body and epidermal fin fold (Kimelman et al., 2017). We report here that DNA damage induces apoptosis of epidermal basal cells (EBCs) in zebrafish yap1-/-;wwtr1-/- embryos. Specifically, these mutant EBCs exhibit active Caspase-3, Caspase-8, and γH2AX, consistent with DNA damage serving as a stimulus of the extrinsic apoptotic pathway in epidermal cells. Live imaging of zebrafish epidermal cells reveals a steady growth of basal cell size in the developing embryo, but this growth is inhibited in mutant basal cells followed by apoptosis, leading to the hypothesis that factors underscoring cell size play a role in this DNA damage-induced apoptosis phenotype. We tested two of these factors using cell stretching and substrate stiffness assays, and found that HaCaT cells cultured on stiff substrates exhibit more numerous γH2AX foci compared to ones cultured on soft substrates. Thus, our experiments suggest that substrate rigidity may modulate genomic stress in epidermal cells, and that Yap1 and Wwtr1 promotes their survival.

Curriculum to Develop Documentation Proficiency Among Medical Students in an Emergency Medicine Clerkship.

INTRODUCTION: Documenting a clinical encounter is a core skill for entering residency, but medical students often receive scant dedicated documentation training, leading to a high rate of inadequate information. Utilizing adult experiential learning theory, we created and implemented an educational resource to train medical students on how to proficiently document an emergency department (ED) patient encounter. METHODS: One hundred and five third- and fourth-year medical students participating in an emergency medicine clerkship took part in a brief orientation day documentation curriculum that included a group didactic, a review of reference materials, a standardized patient activity, a sample patient note writing assignment with individualized feedback, and supervising faculty physician feedback on real patient notes. Students were subsequently entrusted with primary documentation responsibility for all ED patients whose care they participated in. RESULTS: After completing this curriculum, students' self-rated comfort with writing a high-quality note increased from 4.1 to 5.9 (p < .001) and knowledge about billing and coding increased from 2.9 to 5.5 (p < .001) on a 7-point scale. Among faculty physicians, 93% found student notes to always, usually, or frequently be clinically useful, and 86% reported that student notes always, usually, or frequently contained enough information for billing and coding. DISCUSSION: This curriculum was effective at training medical students on proficient patient care documentation in emergency medicine. The relatively short amount of synchronous learning time required could aid in implementation, and the allowance of medical student notes to count for billing purposes could facilitate student and faculty buy-in.

Effects of extended pharmacological disruption of zebrafish embryonic heart biomechanical environment on cardiac function, morphology, and gene expression.

BACKGROUND: Biomechanical stimuli are known to be important to cardiac development, but the mechanisms are not fully understood. Here, we pharmacologically disrupted the biomechanical environment of wild-type zebrafish embryonic hearts for an extended duration and investigated the consequent effects on cardiac function, morphological development, and gene expression. RESULTS: Myocardial contractility was significantly diminished or abolished in zebrafish embryonic hearts treated for 72 hours from 2 dpf with 2,3-butanedione monoxime (BDM). Image-based flow simulations showed that flow wall shear stresses were abolished or significantly reduced with high oscillatory shear indices. At 5 dpf, after removal of BDM, treated embryonic hearts were maldeveloped, having disrupted cardiac looping, smaller ventricles, and poor cardiac function (lower ejected flow, bulboventricular regurgitation, lower contractility, and slower heart rate). RNA sequencing of cardiomyocytes of treated hearts revealed 922 significantly up-regulated genes and 1,698 significantly down-regulated genes. RNA analysis and subsequent qPCR and histology validation suggested that biomechanical disruption led to an up-regulation of inflammatory and apoptotic genes and down-regulation of ECM remodeling and ECM-receptor interaction genes. Biomechanics disruption also prevented the formation of ventricular trabeculation along with notch1 and erbb4a down-regulation. CONCLUSIONS: Extended disruption of biomechanical stimuli caused maldevelopment, and potential genes responsible for this are identified.

Periphery signals generated by Piezo-mediated stomach stretch and Neuromedin-mediated glucose load regulate the Drosophila brain nutrient sensor.

Nutrient sensors allow animals to identify foods rich in specific nutrients. The Drosophila nutrient sensor, diuretic hormone 44 (DH44) neurons, helps the fly to detect nutritive sugar. This sensor becomes operational during starvation; however, the mechanisms by which DH44 neurons or other nutrient sensors are regulated remain unclear. Here, we identified two satiety signals that inhibit DH44 neurons: (1) Piezo-mediated stomach/crop stretch after food ingestion and (2) Neuromedin/Hugin neurosecretory neurons in the ventral nerve cord (VNC) activated by an increase in the internal glucose level. A subset of Piezo+ neurons that express DH44 neuropeptide project to the crop. We found that DH44 neuronal activity and food intake were stimulated following a knockdown of piezo in DH44 neurons or silencing of Hugin neurons in the VNC, even in fed flies. Together, we propose that these two qualitatively distinct peripheral signals work in concert to regulate the DH44 nutrient sensor during the fed state.

Use of graded responsibility and common entrustment considerations among United States emergency medicine residency programs.

PURPOSE: The Accreditation Council for Graduate Medical Education (ACGME) requires all residency programs to provide increasing autonomy as residents progress through training, known as graded responsibility. However, there is little guidance on how to implement graded responsibility in practice and a paucity of literature on how it is currently implemented in emergency medicine (EM). We sought to determine how emergency medicine (EM) residency programs apply graded responsibility across a variety of activities and to identify which considerations are important in affording additional responsibilities to trainees. METHODS: We conducted a cross-sectional study of EM residency programs using a 23-question survey that was distributed by email to 162 ACGME-accredited EM program directors. Seven different domains of practice were queried. RESULTS: We received 91 responses (56.2% response rate) to the survey. Among all domains of practice except for managing critically ill medical patients, the use of graded responsibility exceeded 50% of surveyed programs. When graded responsibility was applied, post-graduate year (PGY) level was ranked an "extremely important" or "very important" consideration between 80.9% and 100.0% of the time. CONCLUSION: The majority of EM residency programs are implementing graded responsibility within most domains of practice. When decisions are made surrounding graded responsibility, programs still rely heavily on the time-based model of PGY level to determine advancement.

Integration of Lung Point-of-care Ultrasound into Clinical Decision Making for Medical Students in Simulated Cases.

BACKGROUND: Point-of-care ultrasound (POCUS) has an emerging presence in medical student education; however, there is limited evidence that this translates into appropriate clinical care. We aimed to evaluate the ability of medical students to integrate newly obtained POCUS knowledge into simulated clinical cases. METHODS: We conducted an observational study of medical students participating in a mandatory rotation during their clinical years. Students in small groups underwent formalized lung POCUS lectures and hands-on training. Students participated in simulated "dyspnea" cases focused on either congestive heart failure (CHF) or chronic obstructive pulmonary disease (COPD). They were observed for critical actions including elements related to medical decision-making and ultrasound use and interpretation. Ultrasound-specific written knowledge was gauged with a short assessment after the first lecture and at week 4. RESULTS: A total of 62 students participated and were observed during simulations. All groups correctly identified and treated CHF in the simulated case. Most groups (7 out of 9) attempted to use ultrasound in the CHF case; five groups correctly recognized B-lines; and four groups correctly interpreted B-lines as pulmonary edema. No groups used ultrasound in the COPD case. CONCLUSION: Most students attempted to use ultrasound during simulated CHF cases after a brief didactic intervention; however, many students struggled with clinical application. Interestingly, no students recognized the need to apply ultrasound for diagnosis and management of COPD. Future studies are needed to better understand how to optimize teaching for medical students to improve translation into POCUS skills and improved clinical practice.

An autism-linked missense mutation in SHANK3 reveals the modularity of Shank3 function.

Genome sequencing has revealed an increasing number of genetic variations that are associated with neuropsychiatric disorders. Frequently, studies limit their focus to likely gene-disrupting mutations because they are relatively easy to interpret. Missense variants, instead, have often been undervalued. However, some missense variants can be informative for developing a more profound understanding of disease pathogenesis and ultimately targeted therapies. Here we present an example of this by studying a missense variant in a well-known autism spectrum disorder (ASD) causing gene SHANK3. We analyzed Shank3's in vivo phosphorylation profile and identified S685 as one phosphorylation site where one ASD-linked variant has been reported. Detailed analysis of this variant revealed a novel function of Shank3 in recruiting Abelson interactor 1 (ABI1) and the WAVE complex to the post-synaptic density (PSD), which is critical for synapse and dendritic spine development. This function was found to be independent of Shank3's other functions such as binding to GKAP and Homer. Introduction of this human ASD mutation into mice resulted in a small subset of phenotypes seen previously in constitutive Shank3 knockout mice, including increased allogrooming, increased social dominance, and reduced pup USV. Together, these findings demonstrate the modularity of Shank3 function in vivo. This modularity further indicates that there is more than one independent pathogenic pathway downstream of Shank3 and correcting a single downstream pathway is unlikely to be sufficient for clear clinical improvement. In addition, this study illustrates the value of deep biological analysis of select missense mutations in elucidating the pathogenesis of neuropsychiatric phenotypes.

A glucose-sensing neuron pair regulates insulin and glucagon in Drosophila.

Although glucose-sensing neurons were identified more than 50 years ago, the physiological role of glucose sensing in metazoans remains unclear. Here we identify a pair of glucose-sensing neurons with bifurcated axons in the brain of Drosophila. One axon branch projects to insulin-producing cells to trigger the release of Drosophila insulin-like peptide 2 (dilp2) and the other extends to adipokinetic hormone (AKH)-producing cells to inhibit secretion of AKH, the fly analogue of glucagon. These axonal branches undergo synaptic remodelling in response to changes in their internal energy status. Silencing of these glucose-sensing neurons largely disabled the response of insulin-producing cells to glucose and dilp2 secretion, disinhibited AKH secretion in corpora cardiaca and caused hyperglycaemia, a hallmark feature of diabetes mellitus. We propose that these glucose-sensing neurons maintain glucose homeostasis by promoting the secretion of dilp2 and suppressing the release of AKH when haemolymph glucose levels are high.

Induction of interferon-stimulated genes and cellular stress pathways by morpholinos in zebrafish.

The phenotypes caused by morpholino-mediated interference of gene function in zebrafish are often not observed in the corresponding mutant(s). We took advantage of the availability of a relatively large collection of transcriptomic datasets to identify common signatures that characterize morpholino-injected animals (morphants). In addition to the previously reported activation of tp53 expression, we observed increased expression of the interferon-stimulated genes (ISGs), isg15 and isg20, the cell death pathway gene casp8, and other cellular stress response genes including phlda3, mdm2 and gadd45aa. Studies involving segmentation stage embryos were more likely to show upregulation of these genes. We also found that the expression of these genes could be upregulated by increasing doses of an egfl7 morpholino, or even high doses of the standard control morpholino. Thus, these data show that morpholinos can induce the expression of ISGs in zebrafish embryos and further our understanding of morpholino effects.

Loss of pyruvate kinase M2 limits growth and triggers innate immune signaling in endothelial cells.

Despite their inherent proximity to circulating oxygen and nutrients, endothelial cells (ECs) oxidize only a minor fraction of glucose in mitochondria, a metabolic specialization that is poorly understood. Here we show that the glycolytic enzyme pyruvate kinase M2 (PKM2) limits glucose oxidation, and maintains the growth and epigenetic state of ECs. We find that loss of PKM2 alters mitochondrial substrate utilization and impairs EC proliferation and migration in vivo. Mechanistically, we show that the NF-κB transcription factor RELB is responsive to PKM2 loss, limiting EC growth through the regulation of P53. Furthermore, S-adenosylmethionine synthesis is impaired in the absence of PKM2, resulting in DNA hypomethylation, de-repression of endogenous retroviral elements (ERVs) and activation of antiviral innate immune signalling. This work reveals the metabolic and functional consequences of glucose oxidation in the endothelium, highlights the importance of PKM2 for endothelial growth and links metabolic dysfunction with autoimmune activation in ECs.

Zebrafish mutants and TEAD reporters reveal essential functions for Yap and Taz in posterior cardinal vein development.

As effectors of the Hippo signaling cascade, YAP1 and TAZ are transcriptional regulators playing important roles in development, tissue homeostasis and cancer. A number of different cues, including mechanotransduction of extracellular stimuli, adhesion molecules, oncogenic signaling and metabolism modulate YAP1/TAZ nucleo-cytoplasmic shuttling. In the nucleus, YAP1/TAZ tether with the DNA binding proteins TEADs, to activate the expression of target genes that regulate proliferation, migration, cell plasticity, and cell fate. Based on responsive elements present in the human and zebrafish promoters of the YAP1/TAZ target gene CTGF, we established zebrafish fluorescent transgenic reporter lines of Yap1/Taz activity. These reporter lines provide an in vivo view of Yap1/Taz activity during development and adulthood at the whole organism level. Transgene expression was detected in many larval tissues including the otic vesicles, heart, pharyngeal arches, muscles and brain and is prominent in endothelial cells. Analysis of vascular development in yap1/taz zebrafish mutants revealed specific defects in posterior cardinal vein (PCV) formation, with altered expression of arterial/venous markers. The overactivation of Yap1/Taz in endothelial cells was sufficient to promote an aberrant vessel sprouting phenotype. Our findings confirm and extend the emerging role of Yap1/Taz in vascular development including angiogenesis.

Effect of Mutations on the Global and Site-Specific Stability and Folding of an Elementary Protein Structural Motif.

Understanding the folding mechanism of proteins requires detailed knowledge of the roles of individual amino acid residues in stabilization of specific elements and local segments of the native structure. Recently, we have utilized the combination of circular dichroism (CD) and site-specific 13C isotopically edited infrared spectroscopy (IR) coupled with the Ising-like model for protein folding to map the thermal unfolding at the residue level of a de novo designed helix-turn-helix motif αtα. Here we use the same methodology to study how the sequence of local thermal unfolding is affected by selected mutations introduced into the most and least stable parts of the motif. Seven different mutants of αtα are screened to find substitutions with the most pronounced effects on the overall stability. Subsequently, thermal unfolding of two mutated αtα sequences is studied with site-specific resolution, using four distinct 13C isotopologues of each. The data are analyzed with the Ising-like model, which builds on a previous parametrization for the original αtα sequence and tests different ways of incorporating the amino acid substitution. We show that for both more and less stable mutants only the adjustment of all interaction parameters of the model can yield a satisfactory fit to the experimental data. The stabilizing and destabilizing mutations result, respectively, in a similar increase and decrease of the stability of all probed local segments, irrespective of their position with respect to the mutation site. Consequently, the relative order of their unfolding remains essentially unchanged. These results underline the importance of the interconnectivity of the stabilizing interaction network and cooperativity of the protein structure, which is evident even in a small motif with apparently noncooperative, heterogeneous unfolding. Overall, our findings are consistent with the native structure being the dominant factor in determining the folding mechanism, regardless of the details of its overall or local thermodynamic stabilization.

The Hippo pathway effector Wwtr1 regulates cardiac wall maturation in zebrafish.

Cardiac trabeculation is a highly regulated process that starts with the delamination of compact layer cardiomyocytes. The Hippo signaling pathway has been implicated in cardiac development but many questions remain. We have investigated the role of Wwtr1, a nuclear effector of the Hippo pathway, in zebrafish and find that its loss leads to reduced cardiac trabeculation. However, in mosaic animals, wwtr1-/- cardiomyocytes contribute more frequently than wwtr1+/- cardiomyocytes to the trabecular layer of wild-type hearts. To investigate this paradox, we examined the myocardial wall at early stages and found that compact layer cardiomyocytes in wwtr1-/- hearts exhibit disorganized cortical actin structure and abnormal cell-cell junctions. Accordingly, wild-type cardiomyocytes in mosaic mutant hearts contribute less frequently to the trabecular layer than when present in mosaic wild-type hearts, indicating that wwtr1-/- hearts are not able to support trabeculation. We also found that Nrg/Erbb2 signaling, which is required for trabeculation, could promote Wwtr1 nuclear export in cardiomyocytes. Altogether, these data suggest that Wwtr1 establishes the compact wall architecture necessary for trabeculation, and that Nrg/Erbb2 signaling negatively regulates its nuclear localization and therefore its activity.

A new parameter-rich structure-aware mechanistic model for amino acid substitution during evolution.

Improvements in the description of amino acid substitution are required to develop better pseudo-energy-based protein structure-aware models for use in phylogenetic studies. These models are used to characterize the probabilities of amino acid substitution and enable better simulation of protein sequences over a phylogeny. A better characterization of amino acid substitution probabilities in turn enables numerous downstream applications, like detecting positive selection, ancestral sequence reconstruction, and evolutionarily-motivated protein engineering. Many existing Markov models for amino acid substitution in molecular evolution disregard molecular structure and describe the amino acid substitution process over longer evolutionary periods poorly. Here, we present a new model upgraded with a site-specific parameterization of pseudo-energy terms in a coarse-grained force field, which describes local heterogeneity in physical constraints on amino acid substitution better than a previous pseudo-energy-based model with minimum cost in runtime. The importance of each weight term parameterization in characterizing underlying features of the site, including contact number, solvent accessibility, and secondary structural elements was evaluated, returning both expected and biologically reasonable relationships between model parameters. This results in the acceptance of proposed amino acid substitutions that more closely resemble those observed site-specific frequencies in gene family alignments. The modular site-specific pseudo-energy function is made available for download through the following website: https://liberles.cst.temple.edu/Software/CASS/index.html.