Dachsous1b cadherin regulates actin and microtubule cytoskeleton during early zebrafish embryogenesis.

Dachsous (Dchs), an atypical cadherin, is an evolutionarily conserved regulator of planar cell polarity, tissue size and cell adhesion. In humans, DCHS1 mutations cause pleiotropic Van Maldergem syndrome. Here, we report that mutations in zebrafish dchs1b and dchs2 disrupt several aspects of embryogenesis, including gastrulation. Unexpectedly, maternal zygotic (MZ) dchs1b mutants show defects in the earliest developmental stage, egg activation, including abnormal cortical granule exocytosis (CGE), cytoplasmic segregation, cleavages and maternal mRNA translocation, in transcriptionally quiescent embryos. Later, MZdchs1b mutants exhibit altered dorsal organizer and mesendodermal gene expression, due to impaired dorsal determinant transport and Nodal signaling. Mechanistically, MZdchs1b phenotypes can be explained in part by defective actin or microtubule networks, which appear bundled in mutants. Accordingly, disruption of actin cytoskeleton in wild-type embryos phenocopied MZdchs1b mutant defects in cytoplasmic segregation and CGE, whereas interfering with microtubules in wild-type embryos impaired dorsal organizer and mesodermal gene expression without perceptible earlier phenotypes. Moreover, the bundled microtubule phenotype was partially rescued by expressing either full-length Dchs1b or its intracellular domain, suggesting that Dchs1b affects microtubules and some developmental processes independent of its known ligand Fat. Our results indicate novel roles for vertebrate Dchs in actin and microtubule cytoskeleton regulation in the unanticipated context of the single-celled embryo.

Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival.

Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy.

Rates of Body Mass Index Increase in Children During the COVID-19 Pandemic.

Pediatric obesity is a significant public health concern, and the COVID-19 pandemic altered many of its risk factors. Understanding this impact can help pediatricians and public health officials prioritize initiatives and identify high-risk subgroups. We performed a retrospective longitudinal cohort study of 596 children and adolescents in a primary care clinic to determine changes in weight gain during the pandemic. A significant rise in normalized BMI was found during the prepandemic period across all age groups and initial BMI classification groups. This rate of change increased during the pandemic for those with pre-existing overweight or obesity status who were ≥10 years of age. Children with regular clinic visits in the prepandemic study period, but without a clinic visit during the pandemic, had significantly higher baseline normalized BMI and were older. These changes in the rate of weight gain during the COVID-19 pandemic period raise the possibility that pandemic influences may have exacerbated the rate of weight gain in children and adolescents with pre-existing obese or overweight range BMI. The difference in composition of the cohort without a pandemic time-period visit highlights the need for future studies in this area, especially those focused on adolescents.

Crawling toward obsolescence: The extended lifespan of amylase for pancreatitis.

The correlation between hyperamylasemia and acute pancreatitis was discovered in 1929, yet another test, lipase, was shown to provide better diagnostic performance in the late 1980s and early 1990s. Subsequent studies demonstrated co-ordering amylase with lipase did not provide additional benefit, only added cost. We sought to investigate the impact of studies advocating for the obsolescence of amylase on its clinical demand. We reviewed 1.3 million reportable results for amylase over 14 years (2009-2022). The trend in utilization of amylase over this period declined by 66% along a linear trajectory (R2 = 0.97). Despite demand for amylase decreasing by an average of 17,003 tests per year, the last year of the study (2022) recorded over 100,000 results for amylase. By interpolating the decline of amylase until the utilization reached zero, we calculated amylase orders will continue for 6 more years until 2028. Tests for creatinine and lipase changed <3% over the same period. Despite a multitude of studies advocating for the obsolescence of amylase, robust demand continues. Many important clinical guidelines, a source many practicing physicians rely on, have yet to acknowledge the preference for lipase over amylase. They frequently treat the two tests as equivalent, neglecting their head-to-head comparison studies and subsequent studies advocating against co-ordering both tests simultaneously. To expedite the obsolescence of amylase, which we anticipate lasting 46 years in our case study from its initial call for obsolescence to the last orders placed, metrics created specifically to monitor the utilization of unnecessary tests are also needed.

Predicting physician departure with machine learning on EHR use patterns: A longitudinal cohort from a large multi-specialty ambulatory practice.

Physician turnover places a heavy burden on the healthcare industry, patients, physicians, and their families. Having a mechanism in place to identify physicians at risk for departure could help target appropriate interventions that prevent departure. We have collected physician characteristics, electronic health record (EHR) use patterns, and clinical productivity data from a large ambulatory based practice of non-teaching physicians to build a predictive model. We use several techniques to identify possible intervenable variables. Specifically, we used gradient boosted trees to predict the probability of a physician departing within an interval of 6 months. Several variables significantly contributed to predicting physician departure including tenure (time since hiring date), panel complexity, physician demand, physician age, inbox, and documentation time. These variables were identified by training, validating, and testing the model followed by computing SHAP (SHapley Additive exPlanation) values to investigate which variables influence the model's prediction the most. We found these top variables to have large interactions with other variables indicating their importance. Since these variables may be predictive of physician departure, they could prove useful to identify at risk physicians such who would benefit from targeted interventions.

Adoption of Emergency Department-Initiated Buprenorphine for Patients With Opioid Use Disorder: Secondary Analysis of a Cluster Randomized Trial.

Importance: Emergency department (ED) initiation of buprenorphine is safe and effective but underutilized in practice. Understanding the factors affecting adoption of this practice could inform more effective interventions. Objective: To quantify the factors, including social contagion, associated with the adoption of the practice of ED initiation of buprenorphine for patients with opioid use disorder. Design, Setting, and Participants: This is a secondary analysis of the EMBED (Emergency Department-Initiated Buprenorphine For Opioid Use Disorder) trial, a multicentered, cluster randomized trial of a clinical decision support intervention targeting ED initiation of buprenorphine. The trial occurred from November 2019 to May 2021. The study was conducted at ED clusters across health care systems from the northeast, southeast, and western regions of the US and included attending physicians, resident physicians, and advanced practice practitioners. Data analysis was performed from August 2022 to June 2023. Exposures: This analysis included both the intervention and nonintervention groups of the EMBED trial. Graph methods were used to construct the network of clinicians who shared in the care of patients for whom buprenorphine was initiated during the trial before initiating the practice themselves, termed exposure. Main Outcomes and Measures: Cox proportional hazard modeling with time-dependent covariates was performed to assess the association of the number of these exposures with self-adoption of the practice of ED initiation of buprenorphine while adjusting for clinician role, health care system, and intervention site status. Results: A total of 1026 unique clinicians in 18 ED clusters across 5 health care systems were included. Analysis showed associations of the cumulative number of exposures to others initiating buprenorphine with the self-practice of buprenorphine initiation. This increased in a dose-dependent manner (1 exposure: hazard ratio [HR], 1.31; 95% CI, 1.16-1.48; 5 exposures: HR, 2.85; 95% CI, 1.66-4.89; 10 exposures: HR, 3.55; 95% CI, 1.47-8.58). Intervention site status was associated with practice adoption (HR, 1.50; 95% CI, 1.04-2.18). Health care system and clinician role were also associated with practice adoption. Conclusions and Relevance: In this secondary analysis of a multicenter, cluster randomized trial of a clinical decision support tool for buprenorphine initiation, the number of exposures to ED initiation of buprenorphine and the trial intervention were associated with uptake of ED initiation of buprenorphine. Although systems-level approaches are necessary to increase the rate of buprenorphine initiation, individual clinicians may change practice of those around them. Trial Registration: ClinicalTrials.gov Identifier: NCT03658642.

PROSER: A Web-Based Peripheral Blood Smear Interpretation Support Tool Utilizing Electronic Health Record Data.

OBJECTIVES: Peripheral blood smear (PBS) interpretation represents a cornerstone of pathology practice and resident training but has remained largely static for decades. Here, we describe a novel PBS interpretation support tool. METHODS: In a mixed-methods quality improvement study, a web-based clinical decision support (CDS) tool to assist pathologists in PBS interpretation, PROSER, was deployed in an academic hospital over a 2-month period in 2022. PROSER interfaced with the hospital system's electronic health record and data warehouse to obtain and display relevant demographic, laboratory, and medication information for patients with pending PBS consults. PROSER used these data along with morphologic findings entered by the pathologist to draft a PBS interpretation using rule-based logic. We evaluated users' perceptions of PROSER with a Likert-type survey. RESULTS: PROSER displayed 46 laboratory values with corresponding reference ranges and abnormal flags, allowed for entry of 14 microscopy findings, and computed 2 calculations based on laboratory values; it composed automated PBS reports using a library of 92 prewritten phrases. Overall, PROSER was well received by residents. CONCLUSIONS: In this quality improvement study, we successfully deployed a web-based CDS tool for PBS interpretation. Future work is needed to quantitatively evaluate this intervention's effects on clinical outcomes and resident training.

The Effect of COVID-19 Pandemic Restrictions on Lead Screening in a Primary Care Clinic.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) has disrupted outpatient pediatrics, postponing well-child care to address immediate patient safety concerns. Screening for lead toxicity is a critical component of this care. Children may be at increased risk for lead exposure at home because of social restrictions. We present data on how COVID-19 restrictions have impacted lead screening in a primary care practice. METHOD: Lead testing data on 658 children in a primary care practice were analyzed to determine the effect of COVID-19 restrictions on lead screening rates, levels, and deficiencies. RESULTS: Lead screening significantly decreased during peak restrictions, leading to increased screening deficiencies. Despite this decrease, screening lead levels increased during peak restrictions. DISCUSSION: These data show how COVID-19 restrictions have disrupted routine care and highlight the importance of continued lead screening in at-risk populations. The electronic medical record can be leveraged to identify deficiencies to be targeted by quality improvement initiatives.

Epithelial vertex models with active biochemical regulation of contractility can explain organized collective cell motility.

Collective motions of groups of cells are observed in many biological settings such as embryo development, tissue formation, and cancer metastasis. To effectively model collective cell movement, it is important to incorporate cell specific features such as cell size, cell shape, and cell mechanics, as well as active behavior of cells such as protrusion and force generation, contractile forces, and active biochemical signaling mechanisms that regulate cell behavior. In this paper, we develop a comprehensive model of collective cell migration in confluent epithelia based on the vertex modeling approach. We develop a method to compute cell-cell viscous friction based on the vertex model and incorporate RhoGTPase regulation of cortical myosin contraction. Global features of collective cell migration are examined by computing the spatial velocity correlation function. As active cell force parameters are varied, we found rich dynamical behavior. Furthermore, we find that cells exhibit nonlinear phenomena such as contractile waves and vortex formation. Together our work highlights the importance of active behavior of cells in generating collective cell movement. The vertex modeling approach is an efficient and versatile approach to rigorously examine cell motion in the epithelium.

TWIST1 induces expression of discoidin domain receptor 2 to promote ovarian cancer metastasis.

The mesenchymal gene program has been shown to promote the metastatic progression of ovarian cancer; however, specific proteins induced by this program that lead to these metastatic behaviors have not been identified. Using patient derived tumor cells and established human ovarian tumor cell lines, we find that the Epithelial-to-Mesenchymal Transition inducing factor TWIST1 drives expression of discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase (RTK) that recognizes fibrillar collagen as ligand. The expression and action of DDR2 was critical for mesothelial cell clearance, invasion and migration in ovarian tumor cells. It does so, in part, by upregulating expression and activity of matrix remodeling enzymes that lead to increased cleavage of fibronectin and spreading of tumor cells. Additionally, DDR2 stabilizes SNAIL1, allowing for sustained mesenchymal phenotype. In patient derived ovarian cancer specimens, DDR2 expression correlated with enhanced invasiveness. DDR2 expression was associated with advanced stage ovarian tumors and metastases. In vivo studies demonstrated that the presence of DDR2 is critical for ovarian cancer metastasis. These findings indicate that the collagen receptor DDR2 is critical for multiple steps of ovarian cancer progression to metastasis, and thus, identifies DDR2 as a potential new target for the treatment of metastatic ovarian cancer.

Past matrix stiffness primes epithelial cells and regulates their future collective migration through a mechanical memory.

During morphogenesis and cancer metastasis, grouped cells migrate through tissues of dissimilar stiffness. Although the influence of matrix stiffness on cellular mechanosensitivity and motility are well-recognized, it remains unknown whether these matrix-dependent cellular features persist after cells move to a new microenvironment. Here, we interrogate whether priming of epithelial cells by a given matrix stiffness influences their future collective migration on a different matrix - a property we refer to as the 'mechanical memory' of migratory cells. To prime cells on a defined matrix and track their collective migration onto an adjoining secondary matrix of dissimilar stiffness, we develop a modular polyacrylamide substrate through step-by-step polymerization of different PA compositions. We report that epithelial cells primed on a stiff matrix migrate faster, display higher actomyosin expression, form larger focal adhesions, and retain nuclear YAP even after arriving onto a soft secondary matrix, as compared to their control behavior on a homogeneously soft matrix. Priming on a soft ECM causes a reverse effect. The depletion of YAP dramatically reduces this memory-dependent migration. Our results present a previously unidentified regulation of mechanosensitive collective cell migration by past matrix stiffness, in which mechanical memory depends on YAP activity.

Disruption of the Cdc42/Par6/aPKC or Dlg/Scrib/Lgl Polarity Complex Promotes Epithelial Proliferation via Overlapping Mechanisms.

The establishment and maintenance of apical-basal polarity is a defining characteristic and essential feature of functioning epithelia. Apical-basal polarity (ABP) proteins are also tumor suppressors that are targeted for disruption by oncogenic viruses and are commonly mutated in human carcinomas. Disruption of these ABP proteins is an early event in cancer development that results in increased proliferation and epithelial disorganization through means not fully characterized. Using the proliferating Drosophila melanogaster wing disc epithelium, we demonstrate that disruption of the junctional vs. basal polarity complexes results in increased epithelial proliferation via distinct downstream signaling pathways. Disruption of the basal polarity complex results in JNK-dependent proliferation, while disruption of the junctional complex primarily results in p38-dependent proliferation. Surprisingly, the Rho-Rok-Myosin contractility apparatus appears to play opposite roles in the regulation of the proliferative phenotype based on which polarity complex is disrupted. In contrast, non-autonomous Tumor Necrosis Factor (TNF) signaling appears to suppress the proliferation that results from apical-basal polarity disruption, regardless of which complex is disrupted. Finally we demonstrate that disruption of the junctional polarity complex activates JNK via the Rho-Rok-Myosin contractility apparatus independent of the cortical actin regulator, Moesin.

AJUBA LIM Proteins Limit Hippo Activity in Proliferating Cells by Sequestering the Hippo Core Kinase Complex in the Cytosol.

The Hippo pathway controls organ growth and is implicated in cancer development. Whether and how Hippo pathway activity is limited to sustain or initiate cell growth when needed is not understood. The members of the AJUBA family of LIM proteins are negative regulators of the Hippo pathway. In mammalian epithelial cells, we found that AJUBA LIM proteins limit Hippo regulation of YAP, in proliferating cells only, by sequestering a cytosolic Hippo kinase complex in which LATS kinase is inhibited. At the plasma membranes of growth-arrested cells, AJUBA LIM proteins do not inhibit or associate with the Hippo kinase complex. The ability of AJUBA LIM proteins to inhibit YAP regulation by Hippo and to associate with the kinase complex directly correlate with their capacity to limit Hippo signaling during Drosophila wing development. AJUBA LIM proteins did not influence YAP activity in response to cell-extrinsic or cell-intrinsic mechanical signals. Thus, AJUBA LIM proteins limit Hippo pathway activity in contexts where cell proliferation is needed.

Cell density and actomyosin contractility control the organization of migrating collectives within an epithelium.

The mechanisms underlying collective migration are important for understanding development, wound healing, and tumor invasion. Here we focus on cell density to determine its role in collective migration. Our findings show that increasing cell density, as might be seen in cancer, transforms groups from broad collectives to small, narrow streams. Conversely, diminishing cell density, as might occur at a wound front, leads to large, broad collectives with a distinct leader-follower structure. Simulations identify force-sensitive contractility as a mediator of how density affects collectives, and guided by this prediction, we find that the baseline state of contractility can enhance or reduce organization. Finally, we test predictions from these data in an in vivo epithelium by using genetic manipulations to drive collective motion between predicted migratory phases. This work demonstrates how commonly altered cellular properties can prime groups of cells to adopt migration patterns that may be harnessed in health or exploited in disease.

The Action of Discoidin Domain Receptor 2 in Basal Tumor Cells and Stromal Cancer-Associated Fibroblasts Is Critical for Breast Cancer Metastasis.

High levels of collagen deposition in human and mouse breast tumors are associated with poor outcome due to increased local invasion and distant metastases. Using a genetic approach, we show that, in mice, the action of the fibrillar collagen receptor discoidin domain receptor 2 (DDR2) in both tumor and tumor-stromal cells is critical for breast cancer metastasis yet does not affect primary tumor growth. In tumor cells, DDR2 in basal epithelial cells regulates the collective invasion of tumor organoids. In stromal cancer-associated fibroblasts (CAFs), DDR2 is critical for extracellular matrix production and the organization of collagen fibers. The action of DDR2 in CAFs also enhances tumor cell collective invasion through a pathway distinct from the tumor-cell-intrinsic function of DDR2. This work identifies DDR2 as a potential therapeutic target that controls breast cancer metastases through its action in both tumor cells and tumor-stromal cells at the primary tumor site.

Stromal senescence establishes an immunosuppressive microenvironment that drives tumorigenesis.

Age is a significant risk factor for the development of cancer. However, the mechanisms that drive age-related increases in cancer remain poorly understood. To determine if senescent stromal cells influence tumorigenesis, we develop a mouse model that mimics the aged skin microenvironment. Using this model, here we find that senescent stromal cells are sufficient to drive localized increases in suppressive myeloid cells that contributed to tumour promotion. Further, we find that the stromal-derived senescence-associated secretory phenotype factor interleukin-6 orchestrates both increases in suppressive myeloid cells and their ability to inhibit anti-tumour T-cell responses. Significantly, in aged, cancer-free individuals, we find similar increases in immune cells that also localize near senescent stromal cells. This work provides evidence that the accumulation of senescent stromal cells is sufficient to establish a tumour-permissive, chronic inflammatory microenvironment that can shelter incipient tumour cells, thus allowing them to proliferate and progress unabated by the immune system.

Stromal-Initiated Changes in the Bone Promote Metastatic Niche Development.

More than 85% of advanced breast cancer patients suffer from metastatic bone lesions, yet the mechanisms that facilitate these metastases remain poorly understood. Recent studies suggest that tumor-derived factors initiate changes within the tumor microenvironment to facilitate metastasis. However, whether stromal-initiated changes are sufficient to drive increased metastasis in the bone remains an open question. Thus, we developed a model to induce reactive senescent osteoblasts and found that they increased breast cancer colonization of the bone. Analysis of senescent osteoblasts revealed that they failed to mineralize bone matrix and increased local osteoclastogenesis, the latter process being driven by the senescence-associated secretory phenotype factor, IL-6. Neutralization of IL-6 was sufficient to limit senescence-induced osteoclastogenesis and tumor cell localization to bone, thereby reducing tumor burden. Together, these data suggest that a reactive stromal compartment can condition the niche, in the absence of tumor-derived signals, to facilitate metastatic tumor growth in the bone.

Rho1 regulates adherens junction remodeling by promoting recycling endosome formation through activation of myosin II.

Once adherens junctions (AJs) are formed between polarized epithelial cells they must be maintained because AJs are constantly remodeled in dynamic epithelia. AJ maintenance involves endocytosis and subsequent recycling of E-cadherin to a precise location along the basolateral membrane. In the Drosophila pupal eye epithelium, Rho1 GTPase regulates AJ remodeling through Drosophila E-cadherin (DE-cadherin) endocytosis by limiting Cdc42/Par6/aPKC complex activity. We demonstrate that Rho1 also influences AJ remodeling by regulating the formation of DE-cadherin-containing, Rab11-positive recycling endosomes in Drosophila postmitotic pupal eye epithelia. This effect of Rho1 is mediated through Rok-dependent, but not MLCK-dependent, stimulation of myosin II activity yet independent of its effects upon actin remodeling. Both Rho1 and pMLC localize on endosomal vesicles, suggesting that Rho1 might regulate the formation of recycling endosomes through localized myosin II activation. This work identifies spatially distinct functions for Rho1 in the regulation of DE-cadherin-containing vesicular trafficking during AJ remodeling in live epithelia.

Redox-active tetrahydrosalen (salan) complexes of titanium.

A diarylamino-substituted N-methyl tetrahydrosalen (salan) ligand, (An2N)LH(2), is prepared in four steps and overall 53% yield from 5-bromosalicylaldehyde, with the key step a palladium-catalysed Hartwig-Buchwald amination of the tert-butyldimethylsilyl-protected 5-bromo-N-methylsalan ligand. Reaction of (An2N)LH(2) or its bromo analogue with Ti(O(i)Pr)(4) or TiF(4) results in metalation of the ligand. The isopropoxide groups are readily exchanged with α- or ß-hydroxyacids to form chelated complexes. X-ray crystallography and NMR spectroscopy indicate that the salan ligands are quite flexible, with (An2N)LTiF(2), for example, showing four stereoisomers in its (19)F NMR spectrum. The major stereoisomer of (salan)Ti(X)(Y) depends principally on the trans influence of the X and Y groups. Complexes of (An2N)L show two reversible, closely spaced redox couples at approximately + 0.1 V vs. ferrocene/ferrocenium, and a second set of two closely spaced redox couples at ~ + 0.8 V vs. Fc/Fc(+).