Lineage dynamics of murine pancreatic development at single-cell resolution.

Organogenesis requires the complex interactions of multiple cell lineages that coordinate their expansion, differentiation, and maturation over time. Here, we profile the cell types within the epithelial and mesenchymal compartments of the murine pancreas across developmental time using a combination of single-cell RNA sequencing, immunofluorescence, in situ hybridization, and genetic lineage tracing. We identify previously underappreciated cellular heterogeneity of the developing mesenchyme and reconstruct potential lineage relationships among the pancreatic mesothelium and mesenchymal cell types. Within the epithelium, we find a previously undescribed endocrine progenitor population, as well as an analogous population in both human fetal tissue and human embryonic stem cells differentiating toward a pancreatic beta cell fate. Further, we identify candidate transcriptional regulators along the differentiation trajectory of this population toward the alpha or beta cell lineages. This work establishes a roadmap of pancreatic development and demonstrates the broad utility of this approach for understanding lineage dynamics in developing organs.

Tricellular junctions regulate intestinal stem cell behaviour to maintain homeostasis.

Ageing results in loss of tissue homeostasis across taxa. In the intestine of Drosophila melanogaster, ageing is correlated with an increase in intestinal stem cell (ISC) proliferation, a block in terminal differentiation of progenitor cells, activation of inflammatory pathways, and increased intestinal permeability. However, causal relationships between these phenotypes remain unclear. Here, we demonstrate that ageing results in altered localization and expression of septate junction proteins in the posterior midgut, which is quite pronounced in differentiated enterocytes (ECs) at tricellular junctions (TCJs). Acute loss of the TCJ protein Gliotactin (Gli) in ECs results in increased ISC proliferation and a block in differentiation in intestines from young flies, demonstrating that compromised TCJ function is sufficient to alter ISC behaviour in a non-autonomous manner. Blocking the Jun N-terminal kinase signalling pathway is sufficient to suppress changes in ISC behaviour, but has no effect on loss of intestinal barrier function, as a consequence of Gli depletion. Our work demonstrates a pivotal link between TCJs, stem cell behaviour, and intestinal homeostasis and provides insights into causes of age-onset and gastrointestinal diseases.

Determining the Learning Curve for Acquiring Core Sonographic Skills for Ultrasound-Guided Axillary Brachial Plexus Block.

BACKGROUND AND OBJECTIVES: The objectives of this study were to determine the learning curve for capturing sonograms and identifying anatomical structures relevant to ultrasound-guided axillary brachial plexus block and to determine if massed was superior to distributed practice for this core sonographic skill. METHODS: Ten University of Melbourne, third- or fourth-year Doctor of Medicine students were randomized to massed or distributed practice. Participants performed 15 supervised learning sessions comprising scanning followed by feedback. A "sonographic proficiency score" was calculated by summing parameters in acquiring and interpreting the sonogram, and identifying relevant anatomical structures. RESULTS: Between the 1st and 10th sessions, the proficiency scores increased (P = 0.043). Except for one, all participants had relatively rapid increases in their "sonographic proficiency scores." There was no difference in proficiency scores between the 15th and 10th sessions (P > 0.05). There was no difference in scores between groups for the first session, (P = 0.40), 15th session (P = 0.10), or at any time. There was no difference in the slope of the increase in "sonographic proficiency score" over the first 10 scanning sessions between groups [massed, 1.1 (0.32); distributed, 0.90 (0.15); P = 0.22) presented as mean (SD)]. The 95% confidence interval for the difference in slopes between massed and distributed groups was -0.15 to 0.56. CONCLUSIONS: The proficiency of participants in capturing sonograms and identifying anatomical structures improved significantly over 8 to 10 learning sessions. Because of sample size issues, we cannot make a firm conclusion regarding massed versus distributed practice for this core sonographic skill.

A Randomized Controlled Trial of Ultrasound Versus Nerve Stimulator Guidance for Axillary Brachial Plexus Block.

BACKGROUND: Ultrasound-guided techniques improve outcomes in regional anesthesia when compared with traditional techniques; however, this assertion has not been studied with novices. The primary objective of this study was to compare sensory and motor block after axillary brachial plexus block when performed by novice trainees allocated to an ultrasound- or nerve-stimulator-guided group. A secondary objective was to compare the rates of skill acquisition between the 2 groups. METHODS: This study was a prospective, randomized, observer-blinded, 2-arm controlled trial. Anesthesia trainees participating in this trial were novices to axillary brachial plexus block and sonography. All trainee participants underwent a standardized training program. The primary outcome was combined sensory and motor block in the relevant territories 30 minutes after completion of block. A global rating scale was used to assess trainee block performance. RESULTS: The study was ceased after 12 trainees completed 153 blocks. There was no difference between groups in combined motor/sensory score (P = 0.28) or as a function of block number (P = 0.38). There was no difference in onset between groups (P = 0.38). In both groups, there was an increase in the global rating scale score (P < 0.0001) and reduced preblock survey and block performance times (P = 0.001) with experience. CONCLUSIONS: We were unable to demonstrate a difference in the efficacy of axillary brachial plexus block performed by novices when ultrasound guidance was compared with a nerve stimulator technique. There was evidence of similarly improved clinical performance of novices in both groups.

Insulin- and warts-dependent regulation of tracheal plasticity modulates systemic larval growth during hypoxia in Drosophila melanogaster.

Adaptation to dynamic environmental cues during organismal development requires coordination of tissue growth with available resources. More specifically, the effects of oxygen availability on body size have been well-documented, but the mechanisms through which hypoxia restricts systemic growth have not been fully elucidated. Here, we characterize the larval growth and metabolic defects in Drosophila that result from hypoxia. Hypoxic conditions reduced fat body opacity and increased lipid droplet accumulation in this tissue, without eliciting lipid aggregation in hepatocyte-like cells called oenocytes. Additionally, hypoxia increased the retention of Dilp2 in the insulin-producing cells of the larval brain, associated with a reduction of insulin signaling in peripheral tissues. Overexpression of the wildtype form of the insulin receptor ubiquitously and in the larval trachea rendered larvae resistant to hypoxia-induced growth restriction. Furthermore, Warts downregulation in the trachea was similar to increased insulin receptor signaling during oxygen deprivation, which both rescued hypoxia-induced growth restriction, inhibition of tracheal molting, and developmental delay. Insulin signaling and loss of Warts function increased tracheal growth and augmented tracheal plasticity under hypoxic conditions, enhancing oxygen delivery during periods of oxygen deprivation. Our findings demonstrate a mechanism that coordinates oxygen availability with systemic growth in which hypoxia-induced reduction of insulin receptor signaling decreases plasticity of the larval trachea that is required for the maintenance of systemic growth during times of limiting oxygen availability.

Evaluation of a task-specific checklist and global rating scale for ultrasound-guided regional anesthesia.

UNLABELLED: Checklists and global rating scales (GRSs) are used for assessment of anesthesia procedural skills. The purpose of this study was to evaluate the reliability and validity of a recently proposed assessment tool comprising a checklist and GRS specific for ultrasound-guided regional anesthesia. METHODS: In this prospective, fully crossed study, we videotaped 30 single-target nerve block procedures performed by anesthesia trainees. Following pilot assessment and observer training, videos were assessed in random order by 6 blinded, expert observers. Interrater reliability was evaluated with intraclass correlation coefficients (ICCs) based on a 2-way random-effects model that took into account both agreement and correlation between observer results. Construct validity and feasibility were also evaluated. RESULTS: The ICC between assessors' total scores was 0.44 (95% confidence interval, 0.27-0.62). All 6 observers scored "experienced trainees" higher than "inexperienced trainees" (median total score 76.7 vs 54.2, P = 0.01), supporting the test's construct validity. The median time to assess the videos was 4 minutes 29 seconds. CONCLUSIONS: This is the first study to evaluate the reliability and validity of a combined checklist and GRS for ultrasound-guided regional anesthesia using multiple observers and taking into account both absolute agreement and correlation in determining the ICC of 0.44 for interrater reliability. There was evidence to support construct validity.

Ultrasound-guided regional anesthesia: how much practice do novices require before achieving competency in ultrasound needle visualization using a cadaver model.

BACKGROUND AND OBJECTIVES: Ultrasound needle visualization is a fundamental skill required for competency in ultrasound-guided regional anesthesia. The primary objective of this study using a cadaver model was to quantify the number of procedures that novices need to perform before competency, using a predefined dynamic scoring system was achieved in ultrasound needle visualization skills. METHODS: Fifteen trainees, novices to ultrasound-guided regional anesthesia, performed 30 simulated sciatic nerve blocks in cadavers. After each procedure, a supervisor provided feedback regarding quality-compromising behaviors. Learning curves were constructed for each individual trainee by calculating cusum statistics. Trainees were categorized into those who were proficient, not proficient, and undetermined. A mathematical model predicted the number of procedures required before an acceptable success rate would be attained. Logistic regression was used to identify factors associated with success. RESULTS: There was wide variability in individual cusum curves. The mean number of trials required to achieve competency in this cohort was 28. Trainees were categorized as proficient (n = 6), not proficient (n = 5), and undetermined (n = 4). With each subsequent procedure, there was a significant increase in the likelihood of success for trainees categorized as not proficient (P = 0.023) or undetermined (P = 0.024) but not for trainees categorized as proficient (P = 0.076). Participants recruited later in the study had an increased likelihood of success (P < 0.001). CONCLUSIONS: Trainees became competent in ultrasound needle visualization at a variable rate. This study estimates that novices would require approximately 28 supervised trials with feedback before competency in ultrasound needle visualization is achieved.

Sonographic location of the radial nerve confirmed by nerve stimulation during axillary brachial plexus blockade.

BACKGROUND AND OBJECTIVES: : Visualization of the radial nerve can be a challenge during ultrasound-guided axillary blockade. The objective of this study was to plot the location and examine the sonographic appearance of the radial nerve in the axilla using both ultrasound visualization and nerve stimulator verification in patients undergoing axillary blockade. METHODS: : Fifty-one patients requiring an axillary block for surgery were enrolled. Sonograms of the radial nerve at the point of best nerve stimulator response were recorded. The needle tip and center of the radial nerve were both plotted on a 2-dimensional scattergram using coordinates relative to the center of the axillary artery. A video tracing the path of the radial nerve was also recorded. RESULTS: : The radial nerve location was confirmed with both ultrasound imaging and nerve stimulation in 46 patients (90%). Five nerves (11%) were immediately deep to the axillary artery (ie, at a 6-o'clock position), 6 (13%) were cephalad, and the remaining 35 (76%) were caudad to the axillary artery. The 2-dimensional (2D) scattergrams reveal the variability in radial nerve location and clarity, of which 14 (30%) of the 46 visible nerves were difficult to trace precisely. A fascial plane between the medial and long heads of the triceps containing the radial nerve could be imaged clearly in 41 (91%) of the videos. CONCLUSIONS: : The radial nerve uncommonly lies immediately deep to the axillary artery. It is frequently located on the caudad side of the artery.