Machine Learning Algorithm Detection of Confluent B-Lines.

OBJECTIVE: B-lines are a ring-down artifact of lung ultrasound that arise with increased alveolar water in conditions such as pulmonary edema and infectious pneumonitis. Confluent B-line presence may signify a different level of pathology compared with single B-lines. Existing algorithms aimed at B-line counting do not distinguish between single and confluent B-lines. The objective of this study was to test a machine learning algorithm for confluent B-line identification. METHODS: This study used a subset of 416 clips from 157 subjects, previously acquired in a prospective study enrolling adults with shortness of breath at two academic medical centers, using a hand-held tablet and a 14-zone protocol. After exclusions, random sampling generated a total of 416 clips (146 curvilinear, 150 sector and 120 linear) for review. A group of five experts in point-of-care ultrasound blindly evaluated the clips for presence/absence of confluent B-lines. Ground truth was defined as majority agreement among the experts and used for comparison with the algorithm. RESULTS: Confluent B-lines were present in 206 of 416 clips (49.5%). Sensitivity and specificity of confluent B-line detection by algorithm compared with expert determination were 83% (95% confidence interval [CI]: 0.77-0.88) and 92% (95% CI: 0.88-0.96). Sensitivity and specificity did not statistically differ between transducers. Agreement between algorithm and expert for confluent B-lines measured by unweighted κ was 0.75 (95% CI: 0.69-0.81) for the overall set. CONCLUSION: The confluent B-line detection algorithm had high sensitivity and specificity for detection of confluent B-lines in lung ultrasound point-of-care clips, compared with expert determination.

Interrater Reliability of Point-of-Care Cardiopulmonary Ultrasound in Patients With Septic Shock: An Analysis of Agreement Between Treating Clinician and Expert Reviewers.

BACKGROUND: Cardiopulmonary ultrasound (CPUS) is commonly used to assess cardiac function and preload status in patients with septic shock. However, the reliability of CPUS findings at the point of care is unknown. OBJECTIVE: To assess interrater reliability (IRR) of CPUS in patients with suspected septic shock between treating emergency physicians (EPs) vs emergency ultrasound (EUS) experts. METHODS: Single-center, prospective, observational cohort enrolling patients (n = 51) with hypotension and suspected infection. Treating EPs performed and interpreted CPUS for cardiac function parameters (left ventricular [LV] function and right ventricular [RV] function and size) and preload volume parameters (inferior vena cava [IVC] diameter and pulmonary B-lines). The primary outcome was IRR (assessed by Kappa values [κ] and intraclass correlation coefficient [ICC]) between EP and EUS-expert consensus. Secondary analyses examined the effects on IRR of operator experience, respiratory rate, and known difficult views on a Cardiology-performed echocardiogram. RESULTS: IRR was fair for LV function, κ = 0.37, 95% confidence interval (CI) 0.1-0.64; poor for RV function, κ = -0.05, 95% CI -0.6-0.5; moderate for RV size, κ = 0.47, 95% CI 0.07-0.88; and substantial for B-lines, κ = 0.73, 95% CI 0.51-0.95 and IVC size, ICC = 0.87, 95% CI 0.2-0.99. Involvement of ultrasound-trained faculty was associated with improved IRR of RV size (p = 0.002), but not other CPUS domains. CONCLUSIONS: Our study demonstrated high IRR for preload volume parameters (IVC size and presence of B-lines), but not for cardiac parameters (LV function and RV function and size) in patients presenting with concern for septic shock. Future research must focus on determining sonographer and patient-specific factors affecting CPUS interpretation in real-time.

A Multicenter, Prospective Study Comparing Subxiphoid and Parasternal Views During Brief Echocardiography: Effect on Image Quality, Acquisition Time, and Visualized Anatomy.

BACKGROUND: Recent literature has suggested echocardiography (echo) may prolong pauses in chest compressions during cardiac arrest. OBJECTVES: We sought to determine the impact of the sonographic approach (subxiphoid [SX] vs. parasternal long [PSL]) on time to image completion, image quality, and visualization of cardiac anatomy during echo, as performed during Advanced Cardiac Life Support. METHODS: This was a multicenter, randomized controlled trial conducted at 29 emergency departments (EDs) assessing the time to image acquisition and image quality between SX and PSL views for echo. Patients were enrolled in the ED and imaged in a simulated cardiac arrest scenario. Clinicians experienced in echo performed both SX and PSL views, first view in random order. Image quality and time to image acquisition were recorded. Echos were evaluated for identification of cardiac landmarks. Data are presented as percentages or medians with interquartile ranges (IQRs). RESULTS: We obtained 6247 echo images, comprising 3124 SX views and 3123 PSL. Overall time to image acquisition was 9.0 s (IQR 6.7-14.1 s). Image acquisition was shorter using PSL (8.8 s, IQR 6.5-13.5 s) compared with SX (9.3 s, IQR 6.7-15.0 s). The image quality was better with the PSL view (3.86 vs. 3.54; p < 0.0001), twice as many SX images scoring in the worst quality category compared with PSL (8.6% vs. 3.7%). Imaging of the pericardium, cardiac chambers, and other anatomic landmarks was superior with PSL imaging. CONCLUSIONS: Echo was performed in < 10 s in > 50% of patients using either imaging technique. Imaging using PSL demonstrated improved image quality and improved identification of cardiac landmarks.

Assessment of dynamic changes in cardiac function during resuscitation of patients with suspected septic shock: A prospective, observational, cohort study.

STUDY OBJECTIVE: To describe changes in cardiac function throughout the course of resuscitation of patients with suspected septic shock. METHODS: Prospective observational cohort study of Point-of-Care Transthoracic Echocardiograms (TTE) obtained in Emergency Department (ED) patients with a presumed infectious cause of hypotension within one hour of initiating IV fluid resuscitation. Findings of this pre-resuscitation TTE were compared to mid-resuscitation TTE (obtained upon disposition from the ED), and post-resuscitation TTE (obtained after admission to hospital). RESULTS: 22 enrolled patients had a second TTE available for comparison to the initial, pre-resuscitation TTE. 12 patients had a mid-resuscitation TTE and 16 patients had a post-resuscitation TTE. We observed a high incidence of changes on TTE during the clinical course of resuscitation (14/22 [64%]). Patients who developed LV or RV dysfunction during resuscitation were more likely to require vasopressor infusion and ICU admission (Spearman's coefficients [95% CI] of 0.68 [0.36-0.86] and 0.47 [0.04;0.75] respectively). Development of RV dysfunction alone was associated with increased use of positive pressure ventilation and vasopressor infusion (Spearman's coefficients [95% CI] of 0.43 [0;0.72] and 0.47 [0.05,0.75] respectively). CONCLUSIONS: Cardiac function changes assessed by TTE are common during the resuscitation of patients with septic shock. These changes likely reflect the underlying physiology of patients with septic shock and correlate with need for interventions and higher level of care. Further work is required to characterize these changes and to elucidate how to use these physiologic data to guide management.

Identifying and Overcoming Barriers to Resident Use of Point-of-Care Ultrasound.

INTRODUCTION: Emergency medicine residency programs have rigorous point-of-care ultrasound (POCUS) curricula. However, this training does not always readily translate to routine use in clinical decision-making. This study sought to identify and overcome barriers that could prevent resident physicians from performing POCUS during clinical shifts. METHODS: This was a two-step process improvement study. First, a survey was deployed to all residents of a three-year academic residency program to identify barriers to clinical use of POCUS. This survey identified the perceived lack of a uniform documenting protocol as the most important barrier to performing POCUS on shift. Second, as an intervention to overcome this barrier, a streamlined documentation protocol was developed and presented to residents. The primary outcome was the number of patients who had POCUS used in medical decision-making one year before and after intervention. Secondary outcomes were the level of training of residents performing exams and whether faculty overseeing exams were trained through an ultrasound fellowship program. RESULTS: POCUS use by residents increased from 82 to 223 patients before and after the intervention, respectively. Per resident, this translates to an absolute increase from 2.2 (95% confidence intervall [CI], 1.4, 3) to 5.8 (95% CI, 4, 7.6) or 3.6 (95% CI, 1.8, 5.4) exams/resident over the study period. We observed no significant difference in the proportions of scans attributable to the resident level of training (χ2 = 0.5, p = 0.47). The proportion of exams by non-ultrasound fellowship trained faculty increased significantly more compared to fellowship trained faculty (χ2 = 19, p<0.0001); however, both ultrasound fellowship trained and non-ultrasound fellowship trained faculty increased the absolute number of exams performed. CONCLUSION: A key perceived barrier to resident-performed POCUS is unfamiliarity with documenting ultrasounds for medical decision-making. Educating residents in person about a POCUS documentation protocol may help overcome this barrier. Incorporating resident input and motivation into POCUS incentivization may increase utilization. Future studies in optimizing POCUS on shift will need to focus on streamlining documentation, addressing time constraints, and faculty support for resident-performed POCUS.

Canonical Notch Signaling Directs the Fate of Differentiating Neurocompetent Progenitors in the Mammalian Olfactory Epithelium.

The adult olfactory epithelium (OE) has the remarkable capacity to regenerate fully both neurosensory and non-neuronal cell types after severe epithelial injury. Lifelong persistence of two stem cell populations supports OE regeneration when damaged: the horizontal basal cells (HBCs), dormant and held in reserve; and globose basal cells, a heterogeneous population most of which are actively dividing. Both populations regenerate all cell types of the OE after injury, but the mechanisms underlying neuronal versus non-neuronal lineage commitment after recruitment of the stem cell pools remains unknown. We used both retroviral transduction and mouse lines that permit conditional cell-specific genetic manipulation as well as the tracing of progeny to study the role of canonical Notch signaling in the determination of neuronal versus non-neuronal lineages in the regenerating adult OE. Excision of either Notch1 or Notch2 genes alone in HBCs did not alter progenitor fate during recovery from epithelial injury, whereas conditional knock-out of both Notch1 and Notch2 together, retroviral transduction of progenitors with a dominant-negative form of MAML (mastermind-like), or excision of the downstream cofactor RBPJ caused progeny to adopt a neuronal fate exclusively. Conversely, we show that overexpressing the Notch1-intracellular domain (N1ICD) either genetically or by transduction blocks neuronal differentiation completely. However, N1ICD overexpression requires both alleles of the canonical cofactor RBPJ to specify downstream lineage. Together, our results suggest that canonical RBPJ-dependent Notch signaling through redundant Notch1 and Notch2 receptors is both necessary and sufficient for determining neuronal versus non-neuronal differentiation in the regenerating adult OE.SIGNIFICANCE STATEMENT Despite the substantial reconstitution of the olfactory epithelium and its population of sensory neurons after injury, disruption and exhaustion of neurogenesis is a consequence of aging and a cause of olfactory dysfunction. Understanding the mechanisms underlying the generation of replacement neurons and non-neuronal cells is critical to any therapeutic strategy aimed at rebuilding a functional neuroepithelium. The results shown here demonstrate that canonical Notch signaling determines the balance between neurons and non-neuronal cells during restoration of the epithelium after injury. Moreover, the complexities of the multiple Notch pathways impinging on that decision are dissected in detail. Finally, RBPJ, the canonical Notch transcriptional cofactor, exhibits a heretofore unreported haploinsufficiency in setting the balance among the regenerating populations.

Notch1 maintains dormancy of olfactory horizontal basal cells, a reserve neural stem cell.

The remarkable capacity of the adult olfactory epithelium (OE) to regenerate fully both neurosensory and nonneuronal cell types after severe epithelial injury depends on life-long persistence of two stem cell populations: the horizontal basal cells (HBCs), which are quiescent and held in reserve, and mitotically active globose basal cells. It has recently been demonstrated that down-regulation of the ΔN form of the transcription factor p63 is both necessary and sufficient to release HBCs from dormancy. However, the mechanisms by which p63 is down-regulated after acute OE injury remain unknown. To identify the cellular source of potential signaling mechanisms, we assessed HBC activation after neuron-only and sustentacular cell death. We found that ablation of sustentacular cells is sufficient for HBC activation to multipotency. By expression analysis, next-generation sequencing, and immunohistochemical examination, down-regulation of Notch pathway signaling is coincident with HBC activation. Therefore, using HBC-specific conditional knockout of Notch receptors and overexpression of N1ICD, we show that Notch signaling maintains p63 levels and HBC dormancy, in contrast to its suppression of p63 expression in other tissues. Additionally, Notch1, but not Notch2, is required to maintain HBC dormancy after selective neuronal degeneration. Taken together, our data indicate that the activation of HBCs observed after tissue injury or sustentacular cell ablation is caused by the reduction/elimination of Notch signaling on HBCs; elimination of Jagged1 expressed by sustentacular cells may be the ligand responsible.

Transcription factor p63 controls the reserve status but not the stemness of horizontal basal cells in the olfactory epithelium.

Adult tissue stem cells can serve two broad functions: to participate actively in the maintenance and regeneration of a tissue or to wait in reserve and participate only when activated from a dormant state. The adult olfactory epithelium, a site for ongoing, life-long, robust neurogenesis, contains both of these functional stem cell types. Globose basal cells (GBCs) act as the active stem cell population and can give rise to all the differentiated cells found in the normal tissue. Horizontal basal cells (HBCs) act as reserve stem cells and remain dormant unless activated by tissue injury. Here we show that HBC activation following injury by the olfactotoxic gas methyl bromide is coincident with the down-regulation of protein 63 (p63) but anticipates HBC proliferation. Gain- and loss-of-function studies show that this down-regulation of p63 is necessary and sufficient for HBC activation. Moreover, activated HBCs give rise to GBCs that persist for months and continue to act as bona fide stem cells by participating in tissue maintenance and regeneration over the long term. Our analysis provides mechanistic insight into the dynamics between tissue stem cell subtypes and demonstrates that p63 regulates the reserve state but not the stem cell status of HBCs.

Mechanisms of permanent loss of olfactory receptor neurons induced by the herbicide 2,6-dichlorobenzonitrile: effects on stem cells and noninvolvement of acute induction of the inflammatory cytokine IL-6.

We explored the mechanisms underlying the differential effects of two olfactory toxicants, the herbicide 2,6-dichlorobenzonitrile (DCBN) and the anti-thyroid drug methimazole (MMZ), on olfactory receptor neuron (ORN) regeneration in mouse olfactory epithelium (OE). DCBN, but not MMZ, induced inflammation-like pathological changes in OE, and DCBN increased interleukin IL-6 levels in nasal-wash fluid to much greater magnitude and duration than did MMZ. At 24h after DCBN injection, the population of horizontal basal cells (HBCs; reserve, normally quiescent OE stem cells) lining the DMM became severely depleted as some of them detached from the basal lamina, and sloughed into the nasal cavity along with the globose basal cells (GBCs; heterogeneous population of stem and progenitor cells), neurons, and sustentacular cells of the neuroepithelium. In contrast, the layer of HBCs remained intact in MMZ-treated mice, as only the mature elements of the neuroepithelium were shed. Despite the respiratory metaplasia accompanying the greater severity of the DCBN lesion, residual HBCs that survived intoxication were activated by the injury and contributed to the metaplastic respiratory epithelium, as shown by tracing their descendants in a K5CreEr(T2)::fl(stop)TdTomato strain of mice in which recombination causes HBCs to express TdTomato in advance of the lesion. But, contrary to published observations with MMZ, the HBCs failed to form ORNs. A role for IL-6 in suppressing ORN regeneration in DCBN-treated mice was rejected by the failure of the anti-inflammatory drug dexamethasone to prevent the subsequent respiratory metaplasia in the DMM, suggesting that other factors lead to HBC neuro-incompetence.

DeltaNp63 regulates stem cell dynamics in the mammalian olfactory epithelium.

The ability of the olfactory epithelium (OE) to regenerate after injury is mediated by at least two populations of presumed stem cells-globose basal cells (GBCs) and horizontal basal cells (HBCs). Of the two, GBCs are molecularly and phenotypically analogous to the olfactory progenitors of the embryonic placode (OPPs). In contrast, HBCs are a reserve stem cell population that appears later in development and requires activation by severe epithelial damage before contributing to epithelial reconstitution. Neither HBC emergence nor the mechanism of activation after injury is understood. Here we show that the transcription factor p63 (Trp63), which is expressed selectively by adult HBCs, is required for HBC differentiation. The first evidence of HBC differentiation is the expression of p63 by cells that closely resemble embryonic OPPs and adult GBCs by morphology and expression of the transcription factors Sox2, Ascl1, and Hes1. HBC formation is delayed in Ascl1 knock-out OE and is completely abrogated in p63-null mice. Strikingly, other cell types of the OE form normally in the p63 knock-out OE. The role of p63 in HBC differentiation appears to be conserved in the regenerating rat OE, where HBCs disappear and then reappear after tissue lesion. Finally, p63 protein is downregulated in HBCs activated by lesion to become multipotent progenitor cells. Together, our data identify a novel mechanism for the generation of a reserve stem cell population and suggest that a p63-dependent molecular switch is responsible for activating reserve stem cells when they are needed.

Canonical Wnt signaling promotes the proliferation and neurogenesis of peripheral olfactory stem cells during postnatal development and adult regeneration.

The mammalian olfactory epithelium (OE) has a unique stem cell or progenitor niche, which is responsible for the constant peripheral neurogenesis throughout the lifespan of the animal. However, neither the signals that regulate the behavior of these cells nor the lineage properties of the OE stem cells are well understood. Multiple Wnt signaling components exhibit dynamic expression patterns in the developing OE. We generated Wnt signaling reporter TOPeGFP transgenic mice and found TOPeGFP activation predominantly in proliferating Sox2(+) OE basal cells during early postnatal development. FACS-isolated TOPeGFP(+) OE basal cells are required, but are not sufficient, for formation of spheres. Wnt3a significantly promotes the proliferation of the Sox2(+) OE sphere cells. Wnt-stimulated OE sphere cells maintain their multipotency and can differentiate into most types of neuronal and non-neuronal epithelial cells. Also, Wnt activators shift the production of differentiated cells toward olfactory sensory neurons. Moreover, TOPeGFP(+) cells are robustly increased in the adult OE after injury. In vivo administration of Wnt modulators significantly alters the regeneration potential. This study demonstrates the role of the canonical Wnt signaling pathway in the regulation of OE stem cells or progenitors during development and regeneration.

FBXW7 mutation in adult T-cell and B-cell acute lymphocytic leukemias.

The FBXW7 (also known as AGO, hCDC4, FBW7 and SEL-10) gene encodes a subunit of an ubiquitin protein ligase which regulates levels of cyclin E, NOTCH and other proteins. Engineered FBXW7 null cells display cell cycle and chromosome stability defects. Mutations of FBXW7 have been found in human colorectal, ovarian, endometrial tumors and T-cell acute lymphocytic leukemias. Prompted by these findings we have examined acute myeloid leukemia, non-Hodgkin's lymphoma, T-cell acute lymphocytic leukemia, B-cell acute lymphocytic leukemia and adult T-cell leukemia DNA for mutations of the FBXW7 gene. Mutations were detected by PCR-SSCP of all coding exons of the three isoforms of FBXW7, shifted bands were direct sequenced. As expected, mutations were found in T-cell acute lymphocytic leukemias. However mutations of FBXW7 were also found in four of 118 B-cell acute lymphocytic leukemias and one of 24 adult T-cell leukemia samples. The nucleotide changes consisted of an insertion, resulting in a frameshift mutation, and missense mutations of highly conserved residues. All mutations affected the FBXW7 target interacting domain. These observations suggest that disruption of FBXW7 has a role in several forms of lymphocytic leukemias and not exclusively T-cell acute lymphocytic leukemia.

Two ligands signal through the Drosophila PDGF/VEGF receptor to ensure proper salivary gland positioning.

The Drosophila embryonic salivary gland is a migrating tissue that undergoes a stereotypic pattern of migration into the embryo. We demonstrate that the migratory path of the salivary gland requires the PDGF/VEGF pathway. The PDGF/VEGF receptor, Pvr, is strongly expressed in the salivary glands, and Pvr mutations cause abnormal ventral curving of the glands, suggesting that Pvr is involved in gland migration. Although the Pvr ligands, Pvf1 and Pvf2, have distinct expression patterns in the Drosophila embryo, mutations for either one of the ligands result in salivary gland migration defects similar to those seen in embryos that lack Pvr. Rescue experiments indicate that the PDGF/VEGF pathway functions autonomously in the salivary gland. The results of this study demonstrate that the Drosophila PDGF/VEGF pathway is essential for proper positioning of the salivary glands.