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The behaviour and natural history of Megaloastia mainae, a longlegged salticid spider that appears to be endemic to northwest Australia, was investigated under natural conditions in the Kimberley Region of Western Australia. Megaloastia mainae is commonly found on rock escarpments, where it spends most of its time on downward-facing horizontal surfaces of large rocks. The diet of M. mainae comprises a variety of insects and spiders, including other salticids and web-building spiders. Megaloastia mainae builds large nests on the surface of the rock escarpment, which are generally occupied for at least several weeks. Contrary to previous reports, we found no evidence that M. mainae builds a prey-capture web or uses its nest as a predatory device. Megaloastia mainae is active during the day, and usually remains within 2 m of its nest. Megaloastia mainae orients to face prey from up to 1.2 m, and can move very quickly across the rock escarpment. Hunting is generally by slow stalking approaches, followed by rapid attacks. The elongated legs of M. mainae may be the result of selection for rapid locomotion in inverted environments. We also found that M. mainae that built nests close to conspecifics were more likely to be missing legs, suggesting that agonistic interactions might be an important source of injury for these spiders. [ABSTRACT FROM AUTHOR]

This paper examines employers' views on the importance of health benefits and their perspective on policies aimed at expanding health coverage, reducing administrative expenses, and improving the quality of care. Employers of all sizes hold a positive view of the value of health benefits in attracting and retaining workers and in improving workers' health and productivity. Small employers support strategies that would make coverage more affordable; large employers support policies that reduce administrative costs and improve quality. Both support policies that would require additional administrative action as opposed to greater financial commitment on the part of firms in expanding coverage. [ABSTRACT FROM AUTHOR]

With health insurance moving toward greater patient cost sharing, this study finds a sharp increase in the number of underinsured people. Based on indicators of cost exposure relative to income, as of 2007 an estimated twenty-five million insured people ages 19-64 were underinsured -- a 60 percent increase since 2003. The rate of increase was steepest among those with incomes above 200 percent of poverty, where underinsurance rates nearly tripled. In total, 42 percent of U.S. adults were underinsured or uninsured. The underinsured report high levels of access problems and financial stress. The findings underscore the need for policy attention to benefit design, to assure care and affordability. [ABSTRACT FROM AUTHOR]

Background: Limited data exist to guide oxygen administration during one-lung ventilation for thoracic surgery. We hypothesised that high intraoperative inspired oxygen fraction during lung resection surgery requiring one-lung ventilation is independently associated with postoperative pulmonary complications (PPCs)., Methods: We performed this retrospective multicentre study using two integrated perioperative databases (Multicenter Perioperative Outcomes Group and Society of Thoracic Surgeons General Thoracic Surgery Database) to study adult thoracic surgical procedures using one-lung ventilation. The primary outcome was a composite of PPCs (atelectasis, acute respiratory distress syndrome, pneumonia, respiratory failure, reintubation, and prolonged ventilation >48 h). The exposure of interest was high inspired oxygen fraction (FiO 2 ), defined by area under the curve of a FiO 2 threshold > 80%. Univariate analysis and logistic regression modelling assessed the association between intraoperative FiO 2 and PPCs., Results: Across four US medical centres, 141/2733 (5.2%) procedures conducted in 2716 patients (55% female; mean age 66 yr) resulted in PPCs. FiO 2 was univariately associated with PPCs (adjusted OR [aOR]: 1.17, 95% confidence interval [CI]: 1.04-1.33, P=0.012). Logistic regression modelling showed that duration of one-lung ventilation (aOR: 1.20, 95% CI: 1.03-1.41, P=0.022), but not the time-weighted average FiO 2 (aOR: 1.01, 95% CI: 1.00-1.02, P=0.165), was associated with PPCs., Conclusions: Our results do not support limiting the inspired oxygen fraction for the purpose of reducing postoperative pulmonary complications in thoracic surgery involving one-lung ventilation., (Copyright © 2024 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Study Objective: Hindsight bias is the tendency to overestimate the predictability of an event after it has already occurred. We aimed to evaluate whether hindsight bias influences the retrospective interpretation of clinical scenarios in the field of anesthesiology, which relies on clinicians making rapid decisions in the setting of perioperative adverse events., Design: Two clinical scenarios were developed (intraoperative hypotension and intraoperative hypoxia) with 3 potential diagnoses for each. Participants completed a crossover study reviewing one case without being informed of the supposed ultimate diagnosis (i.e., no 'anchor' diagnosis), referred to as their foresight case, and the other as a hindsight case wherein they were informed in the leading sentence of the scenario that 1 of the 3 conditions provided was the ultimate diagnosis (i.e., the diagnosis the participant might 'anchor' to if given this information at the start). Participants were randomly assigned to (1) which scenario (hypotension or hypoxia) was presented as the initial foresight case and (2) which of the 3 potential diagnoses for the second case (the hindsight case, which defaulted to whichever case the participant was not assigned for the first case) was presented as the ultimate diagnosis in the leading sentence in a 2 (scenario order) x 3 (hindsight case anchor) between-subjects factorial design (6 possible randomization assignments)., Setting: Two academic medical centers., Participants: Faculty, fellow, and resident anesthesiologists and certified nurse anesthetists (CRNAs)., Interventions: None., Measurements: After reading each clinical scenario, participants were asked to rate the probability (%) of each of three potential diagnoses to have caused the hypotension or hypoxia. Compositional data analysis (CoDA) was used to compare whether diagnosis probabilities differ between the hindsight and the foresight case., Main Results: 113 participants completed the study. 59 participants (52%) were resident anesthesiologists. Participants randomized to the hypotension scenario as a hindsight case were 2.82 times more likely to assign higher probability to the pulmonary embolus diagnosis if provided as an anchor (95% CI, 1.35-5.90; P = 0.006) and twice as likely to assign higher probability to the myocardial infarction diagnosis if provided as an anchor (95% CI, 1.12-3.58; P = 0.020). Participants randomized to the hypoxia scenario as a hindsight case were 1.78 times more likely to assign higher probability to the mainstem bronchus intubation diagnosis if provided in the anchor statement (95% CI, 1.00-3.14; P = 0.048) and 3.72 times more likely to assign higher probability to the pulmonary edema diagnosis if provided as an anchor (95% CI, 1.88-7.35; P < 0.001)., Conclusions: Hindsight bias influences the clinical diagnosis probabilities assigned by anesthesia providers. Clinicians should be educated on hindsight bias in perioperative medicine and be cognizant of the effect of hindsight bias when interpreting clinical outcomes., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Research on neutrophil biology has been limited by the short life span and limited genetic manipulability of these cells, driving the need for representative and efficient model cell lines. The promyelocytic cell line HL-60 and its subline PLB-985 can be differentiated into neutrophil-like cells (NLCs) and have been used to study neutrophil functions including chemotaxis, phagocytosis, endocytosis, and degranulation. Compared to neutrophils derived from hematopoietic stem cells, NLCs serve as a cost-effective neutrophil model. NLCs derived from both HL-60 and PLB-985 cells have been shown to perform degranulation, an important neutrophil function. However, no study has directly compared the two lines as models for degranulation including their release of different types of mobilizable organelles. Furthermore, Nutridoma, a commercially available supplement, has recently been shown to improve the chemotaxis, phagocytosis, and oxidative burst abilities of NLCs derived from promyelocytic cells, however it is unknown whether this reagent also improves the degranulation ability of NLCs. Here, we show that NLCs derived from both HL-60 and PLB-985 cells are capable of degranulating, with each showing markers for the release of multiple types of secretory organelles, including primary granules. We also show that differentiating HL-60 cells using Nutridoma does not enhance their degranulation activity over NLCs differentiated using Dimethyl Sulfoxide (DMSO) plus Granulocyte-colony stimulating factor (G-CSF). Finally, we show that promyelocytic cells can be genetically engineered and differentiated using these methods, to yield NLCs with a defect in degranulation. Our results indicate that both cell lines serve as effective models for investigating the mechanisms of neutrophil degranulation, which can advance our understanding of the roles of neutrophils in inflammation and immunity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. There has been renewed interest in phage research in the last decade, in part due to the host-specific killing capabilities of lytic phages, which offer a promising tool to counter the increasing threat of antimicrobial resistant bacteria. Furthermore, recent studies demonstrating that phages adhere to intestinal mucus suggest they may have a protective role in preventing bacterial invasion into the underlying epithelium. Importantly, like bacterial microbiomes, disrupted phageomes have been associated with worsened outcomes in diseases such as inflammatory bowel disease. Previous studies have demonstrated that phages can modulate the microbiome of animals and humans through fecal filtrate transplants, benefiting the host's health. With this recent wave of research comes the necessity to establish and standardize protocols for studying phages in the context of the gut microbiome. This protocol provides a set of procedures to study isolated T4 phages and their bacterial host, Escherichia coli, in the context of the murine gastrointestinal tract. The methods described here outline how to start from a phage lysate, administer it to mice and assess effects on bacterial host and phage levels. This protocol can be modified and applied to other phage-bacterial pairs and provides a starting point for studying host-phage dynamics in vivo.

Human neutrophils respond to multiple chemoattractants to guide their migration from the vasculature to sites of infection and injury, where they clear pathogens and amplify inflammation. To properly focus their responses during this complex navigation, neutrophils prioritize pathogen- and injury-derived signals over long-range inflammatory signals, such as the leukotriene LTB4, secreted by host cells. Different chemoattractants can also drive qualitatively different modes of migration even though their receptors couple to the same Gα i family of G proteins. Here, we used live-cell imaging to demonstrate that the responses differed in their signaling dynamics. Low-priority chemoattractants caused transient responses, whereas responses to high-priority chemoattractants were sustained. We observed this difference in both primary neutrophils and differentiated HL-60 cells, for downstream signaling mediated by Ca 2+ , a major regulator of secretion, and Cdc42, a primary regulator of polarity and cell steering. The rapid attenuation of Cdc42 activation in response to LTB4 depended on the phosphorylation sites Thr 308 and Ser 310 in the carboxyl-terminal tail of its receptor LTB4R in a manner independent of endocytosis. Mutation of these residues to alanine impaired chemoattractant prioritization, although it did not affect chemoattractant-dependent differences in migration persistence. Our results indicate that distinct temporal regulation of shared signaling pathways distinguishes between receptors and contributes to chemoattractant prioritization.

Neutrophils are the most abundant leukocyte in humans and provide a critical early line of defense as part of our innate immune system. We perform a comprehensive, genome-wide assessment of the molecular factors critical to proliferation, differentiation, and cell migration in a neutrophil-like cell line. Through the development of multiple migration screen strategies, we specifically probe directed (chemotaxis), undirected (chemokinesis), and 3D amoeboid cell migration in these fast-moving cells. We identify a role for mTORC1 signaling in cell differentiation, which influences neutrophil abundance, survival, and migratory behavior. Across our individual migration screens, we identify genes involved in adhesion-dependent and adhesion-independent cell migration, protein trafficking, and regulation of the actomyosin cytoskeleton. This genome-wide screening strategy, therefore, provides an invaluable approach to the study of neutrophils and provides a resource that will inform future studies of cell migration in these and other rapidly migrating cells., (© 2023. Springer Nature Limited.)

Changes to gut environmental factors such as pH and osmolality due to disease or drugs correlate with major shifts in microbiome composition; however, we currently cannot predict which species can tolerate such changes or how the community will be affected. Here, we assessed the growth of 92 representative human gut bacterial strains spanning 28 families across multiple pH values and osmolalities in vitro . The ability to grow in extreme pH or osmolality conditions correlated with the availability of known stress response genes in many cases, but not all, indicating that novel pathways may participate in protecting against acid or osmotic stresses. Machine learning analysis uncovered genes or subsystems that are predictive of differential tolerance in either acid or osmotic stress. For osmotic stress, we corroborated the increased abundance of these genes in vivo during osmotic perturbation. The growth of specific taxa in limiting conditions in isolation in vitro correlated with survival in complex communities in vitro and in an in vivo mouse model of diet-induced intestinal acidification. Our data show that in vitro stress tolerance results are generalizable and that physical parameters may supersede interspecies interactions in determining the relative abundance of community members. This study provides insight into the ability of the microbiota to respond to common perturbations that may be encountered in the gut and provides a list of genes that correlate with increased ability to survive in these conditions. IMPORTANCE To achieve greater predictability in microbiota studies, it is crucial to consider physical environmental factors such as pH and particle concentration, as they play a pivotal role in influencing bacterial function and survival. For example, pH is significantly altered in various diseases, including cancers, inflammatory bowel disease, as well in the case of over-the-counter drug use. Additionally, conditions like malabsorption can affect particle concentration. In our study, we investigate how changes in environmental pH and osmolality can serve as predictive indicators of bacterial growth and abundance. Our research provides a comprehensive resource for anticipating shifts in microbial composition and gene abundance during complex perturbations. Moreover, our findings underscore the significance of the physical environment as a major driver of bacterial composition. Finally, this work emphasizes the necessity of incorporating physical measurements into animal and clinical studies to better understand the factors influencing shifts in microbiota abundance., Competing Interests: The authors declare no conflict of interest.

Targeting common oncogenic drivers of glioblastoma multiforme (GBM) in patients has remained largely ineffective, raising the possibility that alternative pathways may contribute to tumor aggressiveness. Here we demonstrate that Vangl1 and Fzd7, components of the non-canonical Wnt planar cell polarity (Wnt/PCP) signaling pathway, promote GBM malignancy by driving cellular proliferation, migration, and invasiveness, and engage Rho GTPases to promote cytoskeletal rearrangements and actin dynamics in migrating GBM cells. Mechanistically, we uncover the existence of a novel Vangl1/Fzd7 complex at the leading edge of migrating GBM cells and propose that this complex is critical for the recruitment of downstream effectors to promote tumor progression. Moreover, we observe that depletion of FZD7 results in a striking suppression of tumor growth and latency and extends overall survival in an intracranial mouse xenograft model. Our observations support a novel mechanism by which Wnt/PCP components Vangl1 and Fzd7 form a complex at the leading edge of migratory GBM cells to engage downstream effectors that promote actin cytoskeletal rearrangements dynamics. Our findings suggest that interference with Wnt/PCP pathway function may offer a novel therapeutic strategy for patients diagnosed with GBM., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: In light of the growing appreciation for the role of collective cell motility in metastasis, a deeper understanding of the underlying signaling pathways will be critical to translating these observations to the treatment of advanced cancers. Here, we examine the contribution of Wnt/planar cell polarity (Wnt/PCP), one of the non-canonical Wnt signaling pathways and defined by the involvement of the tetraspanin-like proteins Vangl1 and Vangl2, to breast tumor cell motility, collective cell invasiveness and mammary tumor metastasis., Methods: Vangl1 and Vangl2 knockdown and overexpression and Wnt5a stimulation were employed to manipulate Wnt/PCP signaling in a battery of breast cancer cell lines representing all breast cancer subtypes, and in tumor organoids from MMTV-PyMT mice. Cell migration was assessed by scratch and organoid invasion assays, Vangl protein subcellular localization was assessed by confocal fluorescence microscopy, and RhoA activation was assessed in real time by fluorescence imaging with an advanced FRET biosensor. The impact of Wnt/PCP suppression on mammary tumor growth and metastasis was assessed by determining the effect of conditional Vangl2 knockout on the MMTV-NDL mouse mammary tumor model., Results: We observed that Vangl2 knockdown suppresses the motility of all breast cancer cell lines examined, and overexpression drives the invasiveness of collectively migrating MMTV-PyMT organoids. Vangl2-dependent RhoA activity is localized in real time to a subpopulation of motile leader cells displaying a hyper-protrusive leading edge, Vangl protein is localized to leader cell protrusions within leader cells, and actin cytoskeletal regulator RhoA is preferentially activated in the leader cells of a migrating collective. Mammary gland-specific knockout of Vangl2 results in a striking decrease in lung metastases in MMTV-NDL mice, but does not impact primary tumor growth characteristics., Conclusions: We conclude that Vangl-dependent Wnt/PCP signaling promotes breast cancer collective cell migration independent of breast tumor subtype and facilitates distant metastasis in a genetically engineered mouse model of breast cancer. Our observations are consistent with a model whereby Vangl proteins localized at the leading edge of leader cells in a migrating collective act through RhoA to mediate the cytoskeletal rearrangements required for pro-migratory protrusion formation., (© 2023. The Author(s).)

Maintaining persistent migration in complex environments is critical for neutrophils to reach infection sites. Neutrophils avoid getting trapped, even when obstacles split their front into multiple leading edges. How they re-establish polarity to move productively while incorporating receptor inputs under such conditions remains unclear. Here, we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels to probe cell-intrinsic processes during the resolution of competing fronts. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the process, rather than amplifying structural asymmetries or early fluctuations. Using optogenetic tools, we show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, a retracting edge commits to retraction, with ROCK limiting sensitivity to receptor inputs until the retraction completes. Collectively, our results suggest that cell edges locally adopt highly stable protrusion/retraction programs that are modulated by mechanical feedback., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

In the mammalian intestine, flagellar motility can provide microbes competitive advantage, but also threatens the spatial segregation established by the host at the epithelial surface. Unlike microbicidal defensins, previous studies indicated that the protective activities of human α-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, resides in its remarkable ability to bind microbial surface proteins and self-assemble into protective fibers and nets. Given its ability to bind flagellin, we proposed that HD6 might be an effective inhibitor of bacterial motility. Here, we utilized advanced automated live cell fluorescence imaging to assess the effects of HD6 on actively swimming Salmonella enterica in real time. We found that HD6 was able to effectively restrict flagellar motility of individual bacteria. Flagellin-specific antibody, a classic inhibitor of flagellar motility that utilizes a mechanism of agglutination, lost its activity at low bacterial densities, whereas HD6 activity was not diminished. A single amino acid variant of HD6 that was able to bind flagellin, but not self-assemble, lost ability to inhibit flagellar motility. Together, these results suggest a specialized role of HD6 self-assembly into polymers in targeting and restricting flagellar motility., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Akahoshi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Compartmentalized protein recruitment is a fundamental feature of signal transduction. Accordingly, the cell cortex is a primary site of signaling supported by the recruitment of protein regulators to the plasma membrane. Recent emergence of optogenetic strategies designed to control localized protein recruitment has offered valuable toolsets for investigating spatiotemporal dynamics of associated signaling mechanisms. However, determining proper recruitment parameters is important for optimizing synthetic control. In this chapter, we describe a stepwise process for building linear differential equation models that characterize the kinetics and spatial distribution of optogenetic protein recruitment to the plasma membrane. Specifically, we outline how to construct (1) ordinary differential equations that capture the kinetics, efficiency, and magnitude of recruitment and (2) partial differential equations that model spatial recruitment dynamics and diffusion. Additionally, we explore how these models can be used to evaluate the overall system performance and determine how component parameters can be tuned to optimize synthetic recruitment., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Clinical reasoning encompasses the process of data collection, synthesis, and interpretation to generate a working diagnosis and make management decisions. Situated cognition theory suggests that knowledge is relative to contextual factors, and clinical reasoning in urgent situations is framed by pressure of consequential, time-sensitive decision-making for diagnosis and management. These unique aspects of urgent clinical care may limit the effectiveness of traditional tools to assess, teach, and remediate clinical reasoning., Methods: Using two validated frameworks, a multidisciplinary group of clinicians trained to remediate clinical reasoning and with experience in urgent clinical care encounters designed the novel Rapid Evaluation Assessment of Clinical Reasoning Tool (REACT). REACT is a behaviorally anchored assessment tool scoring five domains used to provide formative feedback to learners evaluating patients during urgent clinical situations. A pilot study was performed to assess fourth-year medical students during simulated urgent clinical scenarios. Learners were scored using REACT by a separate, multidisciplinary group of clinician educators with no additional training in the clinical reasoning process. REACT scores were analyzed for internal consistency across raters and observations., Results: Overall internal consistency for the 41 patient simulations as measured by Cronbach's alpha was 0.86. A weighted kappa statistic was used to assess the overall score inter-rater reliability. Moderate reliability was observed at 0.56., Discussion: To our knowledge, REACT is the first tool designed specifically for formative assessment of a learner's clinical reasoning performance during simulated urgent clinical situations. With evidence of reliability and content validity, this tool guides feedback to learners during high-risk urgent clinical scenarios, with the goal of reducing diagnostic and management errors to limit patient harm., (© 2022. The Author(s) under exclusive licence to Society of General Internal Medicine.)

Changes in the microbiota composition are associated with many human diseases, but factors that govern strain abundance remain poorly defined. We show that a commensal Escherichia coli strain and a pathogenic Salmonella enterica serovar Typhimurium isolate both utilize nitrate for intestinal growth, but each accesses this resource in a distinct biogeographical niche. Commensal E. coli utilizes epithelial-derived nitrate, whereas nitrate in the niche occupied by S. Typhimurium is derived from phagocytic infiltrates. Surprisingly, avirulent S. Typhimurium was shown to be unable to utilize epithelial-derived nitrate because its chemotaxis receptors McpB and McpC exclude the pathogen from the niche occupied by E. coli. In contrast, E. coli invades the niche constructed by S. Typhimurium virulence factors and confers colonization resistance by competing for nitrate. Thus, nutrient niches are not defined solely by critical resources, but they can be further subdivided biogeographically within the host into distinct microhabitats, thereby generating new niche opportunities for distinct bacterial species., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

To control their movement, cells need to coordinate actin assembly with the geometric features of their substrate. Here, we uncover a role for the actin regulator WASP in the 3D migration of neutrophils. We show that WASP responds to substrate topology by enriching to sites of inward, substrate-induced membrane deformation. Superresolution imaging reveals that WASP preferentially enriches to the necks of these substrate-induced invaginations, a distribution that could support substrate pinching. WASP facilitates recruitment of the Arp2/3 complex to these sites, stimulating local actin assembly that couples substrate features with the cytoskeleton. Surprisingly, WASP only enriches to membrane deformations in the front half of the cell, within a permissive zone set by WASP's front-biased regulator Cdc42. While WASP KO cells exhibit relatively normal migration on flat substrates, they are defective at topology-directed migration. Our data suggest that WASP integrates substrate topology with cell polarity by selectively polymerizing actin around substrate-induced membrane deformations in the front half of the cell., (© 2021 Brunetti et al.)

To migrate efficiently to target locations, cells must integrate receptor inputs while maintaining polarity: a distinct front that leads and a rear that follows. Here we investigate what is necessary to overwrite pre-existing front-rear polarity in neutrophil-like HL60 cells migrating inside straight microfluidic channels. Using subcellular optogenetic receptor activation, we show that receptor inputs can reorient weakly polarized cells, but the rear of strongly polarized cells is refractory to new inputs. Transient stimulation reveals a multi-step repolarization process, confirming that cell rear sensitivity to receptor input is the primary determinant of large-scale directional reversal. We demonstrate that the RhoA/ROCK/myosin II pathway limits the ability of receptor inputs to signal to Cdc42 and reorient migrating neutrophils. We discover that by tuning the phosphorylation of myosin regulatory light chain we can modulate the activity and localization of myosin II and thus the amenability of the cell rear to 'listen' to receptor inputs and respond to directional reprogramming., (© 2021. The Author(s).)

During chemotaxis, neutrophils use cell surface G Protein Coupled Receptors to detect chemoattractant gradients. The downstream signaling system is wired with multiple feedback loops that amplify weak inputs and promote spatial separation of cell front and rear activities. Positive feedback could promote rapid signal spreading, yet information from the receptors is transmitted with high spatial fidelity, enabling detection of small differences in chemoattractant concentration across the cell. How the signal transduction network achieves signal amplification while preserving spatial information remains unclear. The GTPase Cdc42 is a cell-front polarity coordinator that is predictive of cell turning, suggesting an important role in spatial processing. Here we directly measure information flow from receptors to Cdc42 by pairing zebrafish parapinopsina, an optogenetic G Protein Coupled Receptor with reversible ON/OFF control, with a spectrally compatible red/far red Cdc42 Fluorescence Resonance Energy Transfer biosensor. Using this toolkit, we show that positive and negative signals downstream of G proteins shape a rapid, dose-dependent Cdc42 response. Furthermore, F-actin and Cdc42 itself provide two distinct negative signals that limit the duration and spatial spread of Cdc42 activation, maintaining output signals local to the originating receptors., (© 2021. The Author(s).)

Optogenetic protein dimerization systems are powerful tools to investigate the biochemical networks that cells use to make decisions and coordinate their activities. These tools, including the improved Light-Inducible Dimer (iLID) system, offer the ability to selectively recruit components to subcellular locations, such as micron-scale regions of the plasma membrane. In this way, the role of individual proteins within signaling networks can be examined with high spatiotemporal resolution. Currently, consistent recruitment is limited by heterogeneous optogenetic component expression, and spatial precision is diminished by protein diffusion, especially over long time scales. Here, we address these challenges within the iLID system with alternative membrane anchoring domains and fusion configurations. Using live cell imaging and mathematical modeling, we demonstrate that the anchoring strategy affects both component expression and diffusion, which in turn impact recruitment strength, kinetics, and spatial dynamics. Compared to the commonly used C-terminal iLID fusion, fusion proteins with large N-terminal anchors show stronger local recruitment, slower diffusion of recruited components, efficient recruitment over wider gene expression ranges, and improved spatial control over signaling outputs. We also define guidelines for component expression regimes for optimal recruitment for both cell-wide and subcellular recruitment strategies. Our findings highlight key sources of imprecision within light-inducible dimer systems and provide tools that allow greater control of subcellular protein localization across diverse cell biological applications.

Protein modification by SUMO helps orchestrate the elaborate events of meiosis to faithfully produce haploid gametes. To date, only a handful of meiotic SUMO targets have been identified. Here, we delineate a multidimensional SUMO-modified meiotic proteome in budding yeast, identifying 2747 conjugation sites in 775 targets, and defining their relative levels and dynamics. Modified sites cluster in disordered regions and only a minority match consensus motifs. Target identities and modification dynamics imply that SUMOylation regulates all levels of chromosome organization and each step of meiotic prophase I. Execution-point analysis confirms these inferences, revealing functions for SUMO in S-phase, the initiation of recombination, chromosome synapsis and crossing over. K15-linked SUMO chains become prominent as chromosomes synapse and recombine, consistent with roles in these processes. SUMO also modifies ubiquitin, forming hybrid oligomers with potential to modulate ubiquitin signaling. We conclude that SUMO plays diverse and unanticipated roles in regulating meiotic chromosome metabolism., Competing Interests: NB, SO, MI, JB, PP, AD, SK, MM, OD, SC, JJ, NK, NH No competing interests declared, (© 2021, Bhagwat et al.)

Background: Uncovering patients' biases toward characteristics of anesthesiologists may inform ways to improve the patient-anesthesiologist relationship. The authors previously demonstrated that patients prefer anesthesiologists displaying confident body language, but did not detect a sex bias. The effect of anesthesiologists' age on patient perceptions has not been studied. In this follow-up study, it was hypothesized that patients would prefer older-appearing anesthesiologists over younger-appearing anesthesiologists and male over female anesthesiologists., Methods: Three hundred adult, English-speaking patients were recruited in the Preanesthesia Evaluation and Testing Center. Patients were randomized (150 per group) to view a set of four videos in random order. Each 90-s video featured an older female, older male, younger female, or younger male anesthesiologist reciting the same script describing general anesthesia. Patients ranked each anesthesiologist on confidence, intelligence, and likelihood of choosing the anesthesiologist to care for their family member. Patients also chose the one anesthesiologist who seemed most like a leader., Results: Three hundred patients watched the videos and completed the questionnaire. Among patients younger than age 65 yr, the older anesthesiologists had greater odds of being ranked more confident (odds ratio, 1.92; 95% CI, 1.41 to 2.64; P < 0.001) and more intelligent (odds ratio, 2.24; 95% CI, 1.62 to 3.11; P < 0.001), and had greater odds of being considered a leader (odds ratio, 2.62; 95% CI, 1.72 to 4.00; P < 0.001) when compared with younger anesthesiologists. The preference for older anesthesiologists was not observed in patients age 65 and older. Female anesthesiologists had greater odds of being ranked more confident (odds ratio, 1.46; 95% CI, 1.13 to 1.87; P = 0.003) and more likely to be chosen to care for one's family member (odds ratio, 1.80; 95% CI, 1.40 to 2.31; P < 0.001) compared with male anesthesiologists. The ranking preference for female anesthesiologists on these two measures was observed among white patients and not among nonwhite patients., Conclusions: Patients preferred older anesthesiologists on the measures of confidence, intelligence, and leadership. Patients also preferred female anesthesiologists on the measures of confidence and likelihood of choosing the anesthesiologist to care for one's family member., (Copyright © 2020, the American Society of Anesthesiologists, Inc. All Rights Reserved.)

The Affordable Care Act was designed to provide financial protection to Americans in their use of the health care system. This required addressing two intertwined problems: cost barriers to accessing coverage and care, and barriers to comprehensive risk protection provided by insurance. We reviewed the evidence on whether the law was effective in achieving these goals. We found that the Affordable Care Act generated substantial, widespread improvements in protecting Americans against the financial risks of illness. The coverage expansions reduced uninsurance rates, especially relative to earlier forecasts; improved access to care; and lowered out-of-pocket spending. The insurance market reforms also made it easier for people to get and stay enrolled in coverage and ensured that those who were insured had true financial risk protection. But subsequent court decisions and congressional and executive branch actions have left millions uninsured and allowed the risk of inadequate insurance to resurface.

Background: Residency programs utilize night float systems to adhere to duty hour restrictions; however, the influence of night float on resident sleep has not been described. The study aim was to determine the influence of night float on resident sleep patterns and quality of sleep. We hypothesized that total sleep time decreases during night float, increases as residents acclimate to night shift work, and returns to baseline during recovery., Methods: This was a single-center observational study of 30 anesthesia residents scheduled to complete six consecutive night float shifts. Electroencephalography sleep patterns were recorded during baseline (three nights), night float (six nights), and recovery (three nights) using the ZMachine Insight monitor (General Sleep Corporation, USA). Total sleep time; light, deep, and rapid eye movement sleep; sleep efficiency; latency to persistent sleep; and wake after sleep onset were observed., Results: Mean total sleep time ± SD was 5.9 ± 1.9 h (3.0 ± 1.2.1 h light; 1.4 ± 0.6 h deep; 1.6 ± 0.7 h rapid eye movement) at baseline. During night float, mean total sleep time was 4.5 ± 1.8 h (1.4-h decrease, 95% CI: 0.9 to 1.9, Cohen's d = -1.1, P < 0.001) with decreases in light (2.2 ± 1.1 h, 0.7-h decrease, 95% CI: 0.4 to 1.1, d = -1.0, P < 0.001), deep (1.1 ± 0.7 h, 0.3-h decrease, 95% CI: 0.1 to 0.4, d = -0.5, P = 0.005), and rapid eye movement sleep (1.2 ± 0.6 h, 0.4-h decrease, 95% CI: 0.3 to 0.6, d = -0.9, P < 0.001). Mean total sleep time during recovery was 5.4 ± 2.2 h, which did not differ significantly from baseline; however, deep (1.0 ± 0.6 h, 0.4-h decrease, 95% CI: 0.2 to 0.6, d = -0.6, P = 0.001 *, P = 0.001) and rapid eye movement sleep (1.2 ± 0.8 h, 0.4-h decrease, 95% CI: 0.2 to 0.6, d = -0.9, P < 0.001 P < 0.001) were significantly decreased., Conclusions: Electroencephalography monitoring demonstrates that sleep quantity is decreased during six consecutive night float shifts. A 3-day period of recovery is insufficient for restorative sleep (rapid eye movement and deep sleep) levels to return to baseline.

Efficient chemotaxis requires rapid coordination between different parts of the cell in response to changing directional cues. Here, we investigate the mechanism of front-rear coordination in chemotactic neutrophils. We find that changes in the protrusion rate at the cell front are instantaneously coupled to changes in retraction at the cell rear, while myosin II accumulation at the rear exhibits a reproducible 9-15-s lag. In turning cells, myosin II exhibits dynamic side-to-side relocalization at the cell rear in response to turning of the leading edge and facilitates efficient turning by rapidly re-orienting the rear. These manifestations of front-rear coupling can be explained by a simple quantitative model incorporating reversible actin-myosin interactions with a rearward-flowing actin network. Finally, the system can be tuned by the degree of myosin regulatory light chain (MRLC) phosphorylation, which appears to be set in an optimal range to balance persistence of movement and turning ability., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Ultrasound-guided regional anesthesia is increasingly used in the perioperative period but performance requires a mastery of regional ultrasound anatomy. We aimed to study whether the use of generative retrieval to learn ultrasound anatomy would improve long-term recall., Methods: Fourth-year medical students without prior training in ultrasound techniques were randomized into standard practice (SP) and generative retrieval (GR) groups. An initial pre-test consisted of 74 regional anesthesia ultrasound images testing common anatomic structures. During the study/learning session, GR participants were required to verbally identify an unlabeled anatomical structure within 10 seconds of the ultrasound image appearing on the screen. A labeled image of the structure was then shown to the GR participant for 5 seconds. SP participants viewed the same ultrasound images labeled with the correct anatomical structure for 15 seconds. Retention was tested at 1 week and 1 month following the study session. Participants completed a satisfaction survey after each session., Results: Forty-five medical students were enrolled with forty included in the analysis. There was no statistically significant difference in baseline scores (GR = 11.5 ± 4.9; SP = 11.2 ± 6.2; P = 0.84). There was no difference in scores at both the 1-week (SP = 54.5 ± 13.3; GR = 53.9 ± 10.5; P = 0.88) and 1-month (SP = 54.0 ± 14.5; GR = 50.7 ± 11.1; P = 0.42) time points. There was no statistically significant difference in learner satisfaction metrics between the groups., Conclusions: The use of generative retrieval practice to learn regional anesthesia ultrasound anatomy did not yield significant differences in learning and retention compared with standard learning., Competing Interests: Conflicts: The authors declare no conflicts of interest., (© 2019 Society for Education in Anesthesia.)

With increasing medical knowledge, procedural, and diagnostic skills to learn, it is vital for educators to make the limited amount of teaching time available to students effective and efficient. Generative retrieval is an effective and efficient learning tool, improving long-term retention through the practice of retrieval from memory. Forty medical students were randomized to learn normal cardiovascular anatomy using transthoracic echocardiography video clips in a generative retrieval (GR) or standard practice (SP) group. GR participants were required to verbally identify each unlabeled cardiovascular structure after viewing the video. After answering, participants viewed the correctly labeled video. SP participants viewed the same video clips labeled with the correct cardiovascular structure for the same amount of total time without verbally generating an answer. All participants were tested for intermediate (1-wk), late (1-mo), and long-term (6- to 9-mo) retention of cardiovascular anatomy. Additionally, a three-question survey was incorporated to assess perceptions of the learning method. There was no difference in pretest scores. The GR group demonstrated a trend toward improvement in recall at 1 wk [GR = 74.3 (SD 12.3); SP = 65.4 (SD 16.7); P = 0.10] and 1 mo [GR = 69.9 (SD15.6); SP = 64.3 (SD 15.4); P = 0.33]. At the 6- to 9-mo time point, there was a statistically significant difference in scores [GR = 74.3 (SD 9.9); SP = 65.0 (SD 14.1); P = 0.042]. At nearly every time point, learners had a statistically significantly higher perception of effectiveness, enjoyment, and satisfaction with GR. In addition to improved recall, GR is associated with increased perceptions of effectiveness, enjoyment, and satisfaction, which may lead to increased engagement, time spent studying, and improved retention.

Background: Patient perception of physician competence is important. The role of body language and physician sex on patient perceptions has not been investigated. The authors hypothesized that patients perceive anesthesiologists displaying confident body language as more competent and that patients would prefer male anesthesiologists., Methods: Two hundred adult patients presenting to the Preanesthesia Evaluation and Testing Center at the University of Virginia Health System were recruited to participate using consecutive sampling. Patients viewed four 90-s videos in random order. Each video featured a male or female actor displaying confident, high-power poses or unconfident, low-power poses. Each actor recited the same script describing general anesthesia. Patients were randomized (100 per group) to view one of two sets of videos to account for any actor preferences. Participants ranked each actor anesthesiologist on perceived confidence, intelligence, and likelihood of choosing that anesthesiologist to care for their family member. Participants also chose the one actor anesthesiologist who seemed most like a leader., Results: Two hundred patients watched the videos and completed the questionnaire. Actor anesthesiologists displaying confident, high-power body language had greater odds of being ranked as more confident (odds ratio, 2.27; 95% CI, 1.76 to 2.92; P < 0.0001), more intelligent (odds ratio, 1.69; 95% CI, 1.13 to 2.18; P < 0.0001), more likely chosen to care for one's family member (odds ratio, 2.34; 95% CI, 1.82 to 3.02; P < 0.0001), and more likely to be considered a leader (odds ratio, 2.60; 95% CI, 1.86 to 3.65; P < 0.0001). Actor anesthesiologist sex was not associated with ranking for any response measures., Conclusions: Patients perceive anesthesiologists displaying confident body language as more confident, more intelligent, more like a leader, and are more likely to choose that anesthesiologist to care for their family member. Differences in patient perceptions based on sex of the anesthesiologist were not detected.

Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-β aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

Issue: With encouragement from the Trump administration, 14 states have received approval for or are pursuing work requirements for nondisabled Medicaid beneficiaries. The requirements have sparked controversy, including two legal challenges., Goal: To predict the effect of work requirements on the insurance coverage of Medicaid enrollees over time., Methods: Analysis of the coverage patterns of a national cohort of nondisabled adults in the federal Medical Expenditure Panel Survey. Their experience is applied to a similar cohort of adults in Kentucky (which has received approval for work requirements, subject to a legal challenge) to project the potential effects of work requirements on their insurance coverage., Findings and Conclusions: Adding a new administrative hurdle in the form of work requirements in Kentucky would double the number of enrollees who disenroll from the program over a two-year period. We estimate that as many as 118,000 adults enrolled in Medicaid would either become uninsured for an extended period of time or experience a gap in insurance over a two-year period. These findings should be of concern to policymakers: research has found that adults who experience coverage gaps report problems getting health care or paying medical bills at rates nearly as high as those who are uninsured continuously.

Background: Human neutrophils are central players in innate immunity, a major component of inflammatory responses, and a leading model for cell motility and chemotaxis. However, primary neutrophils are short-lived, limiting their experimental usefulness in the laboratory. Thus, human myeloid cell lines have been characterized for their ability to undergo neutrophil-like differentiation in vitro. The HL-60 cell line and its PLB-985 sub-line are commonly used to model human neutrophil behavior, but how closely gene expression in differentiated cells resembles that of primary neutrophils has remained unclear., Results: In this study, we compared the effectiveness of differentiation protocols and used RNA sequencing (RNA-seq) to compare the transcriptomes of HL-60 and PLB-985 cells with published data for human and mouse primary neutrophils. Among commonly used differentiation protocols for neutrophil-like cell lines, addition of dimethyl sulfoxide (DMSO) gave the best combination of cell viability and expression of markers for differentiation. However, combining DMSO with the serum-free-supplement Nutridoma resulted in increased chemotactic response, phagocytic activity, oxidative burst and cell surface expression of the neutrophil markers FPR1 and CD11b without a cost in viability. RNA-seq analysis of HL-60 and PLB-985 cells before and after differentiation showed that differentiation broadly increases the similarity in gene expression between the cell lines and primary neutrophils. Furthermore, the gene expression pattern of the differentiated cell lines correlated slightly better with that of human neutrophils than the mouse neutrophil pattern did. Finally, we created a publicly available gene expression database that is searchable by gene name and protein domain content, where users can compare gene expression in HL-60, PLB-985 and primary human and mouse neutrophils., Conclusions: Our study verifies that a DMSO-based differentiation protocol for HL-60 and PLB-985 cell lines gives superior differentiation and cell viability relative to other common protocols, and indicates that addition of Nutridoma may be preferable for studies of chemotaxis, phagocytosis, or oxidative burst. Our neutrophil gene expression database will be a valuable tool to identify similarities and differences in gene expression between the cell lines and primary neutrophils, to compare expression levels for genes of interest, and to improve the design of tools for genetic perturbations.

The physicist Edward Purcell wrote in 1977 about mechanisms that cells could use to propel themselves in a low Reynolds number environment. Reporting in Developmental Cell, O'Neill et al. (2018) provide direct evidence for one of these mechanisms by optogenetically driving the migration of cells suspended in liquid through RhoA activation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

One-lung ventilation is routinely used to facilitate exposure for thoracic surgical procedures and can be achieved via several lung isolation techniques. The optimal method for lung isolation depends on a number of factors that include (1) the indication for lung isolation, (2) anatomic features of the upper and lower airway, (3) availability of equipment and devices, and (4) the anesthesiologist's proficiency and preferences. Though double-lumen endobronchial tubes (DLTs) are most commonly utilized to achieve lung isolation, the use of endobronchial blockers offer advantages in patients with challenging airway anatomy. Anesthesiologists should be familiar with existing alternatives to the DLT for lung isolation and alternative techniques for DLT placement in the patient with a difficult airway. Newer technologies such as videolaryngoscopy with or without adjunctive fiberoptic bronchoscopy may facilitate intubation and lung isolation in difficult airway management.

Neutrophils are key early responders in the innate immune response that use chemotaxis, the directed migration along chemical gradients, to reach sites of infection or inflammation. This process requires integrating inputs from cell surface receptors with the cell's polarity and motility signaling network, in which highly dynamic and interconnected signaling by Rho-family GTPases plays a central role. To understand this fundamentally important behavior, we describe a high-resolution, under-agarose chemotaxis assay for use with neutrophil-like cell lines (HL-60 or PLB-985) or with primary neutrophils. We also describe how to use optical uncaging of chemoattractants to stimulate cells in this assay. These techniques are compatible with epifluorescence, total internal reflection fluorescence (TIRF), and confocal microscopy. Additionally, we cover how to measure the activities of Rho-family GTPases in this context using Förster resonance energy transfer (FRET)-based biosensors. The specific experimental steps outlined in this chapter include how to (1) set up the under-agarose assay, (2) optically pattern chemoattractant gradients, (3) image cells, and (4) conduct basic image analysis for FRET biosensors.