Divergent responses of human intestinal organoid monolayers using commercial in vitro cytotoxicity assays.

In vitro models, such as primary cells and continuous cell lines routinely used for evaluating drug candidates, have limitations in their translational relevance to human diseases. Organotypic cultures are increasingly being used to assess therapeutics for various cancers and infectious diseases. Monitoring drug cytotoxicity in cell cultures is crucial in drug development, and several commercially available kits for cytotoxicity assessment offer distinct advantages and limitations. Given the complexity of organoid cultures, including donor-driven variability, we investigated drug-treated, tissue stem cell-derived human intestinal organoid responses with commonly used cell cytotoxicity assay kits. Using seven different compounds, we compared the cytotoxicity assay performance of two different leaky membrane-based and two metabolism-based assays. Significant variability was seen in reported viability outcomes across assays and organoid lines. High baseline activity of lactate dehydrogenase (LDH) in four human intestinal organoid lines required modification of the standard LDH assay protocol. Additionally, the LDH assay reported unique resilience to damage in a genetically-modified line contrasting results compared to other assays. This study highlights factors that can impact the measurement of cell cytotoxicity in intestinal organoid models, which are emerging as valuable new tools for research and pre-clinical drug testing and suggest the need for using multiple assay types to ensure reliable cytotoxicity assessment.

Pediatric Out-of-Hospital Cardiac Arrest: The Role of the Telecommunicator in Recognition of Cardiac Arrest and Delivery of Bystander Cardiopulmonary Resuscitation.

BACKGROUND: Telecommunicator CPR (T-CPR), whereby emergency dispatch facilitates cardiac arrest recognition and coaches CPR over the telephone, is an important strategy to increase early recognition and bystander CPR in adult out-of-hospital cardiac arrest (OHCA). Little is known about this treatment strategy in the pediatric population. We investigated the role of T-CPR and related performance among pediatric OHCA. METHODS AND RESULTS: This study was a retrospective cohort investigation of OHCA among individuals <18 years in King County, Washington, from April 1, 2013, to December 31, 2019. We reviewed the 911 audio recordings to determine if and how bystander CPR was delivered (unassisted or T-CPR), key time intervals in recognition of arrest, and key components of T-CPR delivery. Of the 185 eligible pediatric OHCAs, 23% (n=43) had bystander CPR initiated unassisted, 59% (n=109) required T-CPR, and 18% (n=33) did not receive CPR before emergency medical services arrival. Among all cases, cardiac arrest was recognized by the telecommunicator in 89% (n=165). Among those receiving T-CPR, the median (interquartile range) interval from start of call to OHCA recognition was 59 seconds (38-87) and first CPR intervention was 115 seconds (94-162). When stratified by age (≤8 versus >8), the older age group was less likely to receive CPR before emergency medical services arrival (88% versus 69%, P=0.002). For those receiving T-CPR, bystanders spent a median of 207 seconds (133-270) performing CPR. The median compression rate was 93 per minute (82-107) among those receiving T-CPR. CONCLUSIONS: T-CPR is an important strategy to increase early recognition and early CPR among pediatric OHCA.

Standardization of an antiviral pipeline for human norovirus in human intestinal enteroids demonstrates nitazoxanide has no to weak antiviral activity.

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis. In immunocompetent hosts, symptoms usually resolve within 3 days; however, in immunocompromised persons, HuNoV infection can become persistent, debilitating, and sometimes life-threatening. There are no licensed therapeutics for HuNoV due to a near half-century delay in its cultivation. Treatment for chronic HuNoV infection in immunosuppressed patients anecdotally includes nitazoxanide, a broad-spectrum antimicrobial licensed for treatment of parasite-induced gastroenteritis. Despite its off-label use for chronic HuNoV infection, nitazoxanide has not been clearly demonstrated to be an effective treatment. In this study, we standardized a pipeline for antiviral testing using multiple human small intestinal enteroid lines representing different intestinal segments and evaluated whether nitazoxanide inhibits replication of five HuNoV strains in vitro. Nitazoxanide did not exhibit high selective antiviral activity against any HuNoV strain tested, indicating it is not an effective antiviral for HuNoV infection. Human intestinal enteroids are further demonstrated as a model to serve as a preclinical platform to test antivirals against HuNoVs to treat gastrointestinal disease. Abstr.

A Standardized Antiviral Pipeline for Human Norovirus in Human Intestinal Enteroids Demonstrates No Antiviral Activity of Nitazoxanide.

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis. In immunocompetent hosts, symptoms usually resolve within three days; however, in immunocompromised persons, HuNoV infection can become persistent, debilitating, and sometimes life-threatening. There are no licensed therapeutics for HuNoV due to a near half-century delay in its cultivation. Treatment for chronic HuNoV infection in immunosuppressed patients anecdotally includes nitazoxanide, a broad-spectrum antimicrobial licensed for treatment of parasite-induced gastroenteritis. Despite its off-label use for chronic HuNoV infection, nitazoxanide has not been clearly demonstrated to be an effective treatment. In this study, we established a standardized pipeline for antiviral testing using multiple human small intestinal enteroid (HIE) lines representing different intestinal segments and evaluated whether nitazoxanide inhibits replication of 5 HuNoV strains in vitro . Nitazoxanide did not exhibit high selective antiviral activity against any HuNoV strains tested, indicating it is not an effective antiviral for norovirus infection. HIEs are further demonstrated as a model to serve as a pre-clinical platform to test antivirals against human noroviruses to treat gastrointestinal disease.

Oral health needs of U.S. children with developmental disorders: a population-based study.

BACKGROUND: Children with Special Health Care Needs (CSHCN) have higher rates of oral diseases and tooth decay compared with the general population. Children with developmental disorders/ disabilities (DD) are a subset of CSHCN whose oral health has not been specifically addressed. Therefore, this study had two objectives: to describe the oral health needs (OHN) of children with DD compared with children without DD; and to assess barriers to access to care, utilization of dental services, and their association with oral health needs for children with DD. METHODS: This cross-sectional study utilized a sample of 30,530 noninstitutionalized children from the 2018 National Survey of Children's Health (NSCH). Analysis was conducted using descriptive and inferential statistics. RESULTS: The analysis identified 6501 children with DD and 24,029 children without DD. Children with DD had significantly higher prevalence of OHN (20.3% vs. 12.2%, respectively), unmet dental needs (3.5% vs 1.2%), and utilization of any dental visits (86.1% vs 76.1%), (P-value < . 001). The adjusted logistic model identified four factors that contributed to the higher odds of OHN among children with DD: poverty (< 100% of the Federal Poverty Level (AOR = 2.27, CI: 1.46-3.51), being uninsured (AOR = 2.12, 95% CI: 1.14-3.95), a high level of disability (AOR = 1.89, CI: 1.23-2.78), and living in the western United States (AOR = 1.61, CI: 1.09-2.37. CONCLUSION: Despite higher utilization of dental services, children with DD had poorer oral health and more unmet dental needs than children without DD. Advocacy efforts and policy changes are needed to develop affordable access that assesses, as early as possible, children with DD whose conditions impact their ability a great deal so that their potential OHN may be alleviated more effectively.

"Of Course, Data Can Never Fully Represent Reality": Assessing the Relationship between "Indigenous Data" and "Indigenous Knowledge," "Traditional Ecological Knowledge," and "Traditional Knowledge".

Multiple terms describe Indigenous peoples' creative expressions, including "Indigenous knowledge" (IK), "traditional ecological knowledge" (TEK), "traditional knowledge" (TK), and increasingly, "Indigenous data" (ID). Variation in terms contributes to disciplinary divides, challenges in organizing and finding prior studies about Indigenous peoples' creative expressions, and intellectually divergent chains of reference. The authors applied a decolonial, digital, feminist, ethics-of-care approach to citation analysis of records about Indigenous peoples knowledge and data, including network analyses of author-generated keywords and research areas, and content analysis of peer-reviewed studies about ID. Results reveal ambiguous uses of the term "Indigenous data"; the influence of ecology and environmental studies in research areas and topics associated with IK, TEK, and TK; and the influence of public administration and governance studies in research areas and topics associated with ID studies. Researchers of ID would benefit from applying a more nuanced and robust vocabulary, one informed by studies of IK, TEK, and TK. Researchers of TEK and TK would benefit from the more people-centered approaches of IK. Researchers and systems designers who work with data sets can practice relational accountability by centering the Indigenous peoples from whom observations are sourced, combining narrative methodologies with computational methods to sustain the holism favored by Indigenous science and the relationality of Indigenous peoples.

Genetic Manipulation of Human Intestinal Enteroids Demonstrates the Necessity of a Functional Fucosyltransferase 2 Gene for Secretor-Dependent Human Norovirus Infection.

Human noroviruses (HuNoVs) are the leading cause of nonbacterial gastroenteritis worldwide. Histo-blood group antigen (HBGA) expression is an important susceptibility factor for HuNoV infection based on controlled human infection models and epidemiologic studies that show an association of secretor status with infection caused by several genotypes. The fucosyltransferase 2 gene (FUT2) affects HBGA expression in intestinal epithelial cells; secretors express a functional FUT2 enzyme, while nonsecretors lack this enzyme and are highly resistant to infection and gastroenteritis caused by many HuNoV strains. These epidemiologic associations are confirmed by infections in stem cell-derived human intestinal enteroid (HIE) cultures. GII.4 HuNoV does not replicate in HIE cultures derived from nonsecretor individuals, while HIEs from secretors are permissive to infection. However, whether FUT2 expression alone is critical for infection remains unproven, since routinely used secretor-positive transformed cell lines are resistant to HuNoV replication. To evaluate the role of FUT2 in HuNoV replication, we used CRISPR or overexpression to genetically manipulate FUT2 gene function to produce isogenic HIE lines with or without FUT2 expression. We show that FUT2 expression alone affects both HuNoV binding to the HIE cell surface and susceptibility to HuNoV infection. These findings indicate that initial binding to a molecule(s) glycosylated by FUT2 is critical for HuNoV infection and that the HuNoV receptor is present in nonsecretor HIEs. In addition to HuNoV studies, these isogenic HIE lines will be useful tools to study other enteric microbes where infection and/or disease outcome is associated with secretor status.IMPORTANCE Several studies have demonstrated that secretor status is associated with susceptibility to human norovirus (HuNoV) infection; however, previous reports found that FUT2 expression is not sufficient to allow infection with HuNoV in a variety of continuous laboratory cell lines. Which cellular factor(s) regulates susceptibility to HuNoV infection remains unknown. We used genetic manipulation of HIE cultures to show that secretor status determined by FUT2 gene expression is necessary and sufficient to support HuNoV replication based on analyses of isogenic lines that lack or express FUT2. Fucosylation of HBGAs is critical for initial binding and for modification of another putative receptor(s) in HIEs needed for virus uptake or uncoating and necessary for successful infection by GI.1 and several GII HuNoV strains.

Determining witnessed status for out-of-hospital cardiac arrest.

OBJECTIVE: Witnessed status is considered a core variable in reporting cardiac arrest data and can be ascertained from either the emergency dispatch recording or the pre-hospital record. The purpose of this study is to compare and assess the quality and consistency of these information sources. METHODS: This retrospective analysis included 1896 cases of out-of-hospital cardiac arrest occurring between September 1, 2012 and December 31, 2014. RESULTS: We found that there was minimal (kappa=0.30, 95% CI 0.27-0.33) to moderate (kappa=0.64, 95% CI 0.59-0.69) agreement between the pre-hospital record and the emergency dispatch recording when these sources of information are used to determine witnessed status. Witnessed status could not be determined from the emergency dispatch recording in 36.2% (n=684) of eligible cases. Survival was similar regardless of the method used to determine witnessed status. Using a combination of the pre-hospital record and the emergency dispatch recording yielded the highest number of witnessed cases. CONCLUSION: The determination of witnessed status in out-of-hospital cardiac arrest may be challenging, as evidenced by the discrepancies in witnessed status when comparing different sources of information. The large number of cases where the witnessed status could not be determined from the emergency dispatch recording precludes its use as the sole source of information. It is reasonable to use the patient care record alone, however it should be recognized that there is misclassification of witnessed status regardless of the method used and this may affect the strength of association between witnessed status and survival.

Extracellular Matrix Deposition in Engineered Micromass Cartilage Pellet Cultures: Measurements and Modelling.

This article explores possible mechanisms governing extracellular matrix deposition in engineered cartilaginous cell pellets. A theoretical investigation is carried out alongside an experimental study measuring proteoglycan and collagen volume fractions within murine chondrogenic (ATDC-5) cell pellets. The simple mathematical model, which adopts a nutrient-dependent proteoglycan production rate, successfully reproduces the periphery-dominated proteoglycan deposition, characteristic of the growth pattern observed experimentally within pellets after 21 days of culture. The results suggest that this inhomogeneous proteoglycan production is due to nutrient deficiencies at the pellet centre. Our model analysis further indicates that a spatially uniform distribution of proteoglycan matrix could be maintained by initiating the culture process with a smaller-sized pellet. Finally, possible extensions are put forward with an aim to improve the model predictions for the early behaviour, where different mechanisms appear to dominate the matrix production within the pellets.

Cytokine Spatzle binds to the Drosophila immunoreceptor Toll with a neurotrophin-like specificity and couples receptor activation.

Drosophila Toll functions in embryonic development and innate immunity and is activated by an endogenous ligand, Spätzle (Spz). The related Toll-like receptors in vertebrates also function in immunity but are activated directly by pathogen-associated molecules such as bacterial endotoxin. Here, we present the crystal structure at 2.35-Å resolution of dimeric Spz bound to a Toll ectodomain encompassing the first 13 leucine-rich repeats. The cystine knot of Spz binds the concave face of the Toll leucine-rich repeat solenoid in an area delineated by N-linked glycans and induces a conformational change. Mutagenesis studies confirm that the interface observed in the crystal structure is relevant for signaling. The asymmetric binding mode of Spz to Toll is similar to that of nerve growth factor (NGF) in complex with the p75 neurotrophin receptor but is distinct from that of microbial ligands bound to the Toll-like receptors. Overall, this study indicates an allosteric signaling mechanism for Toll in which ligand binding to the N terminus induces a conformational change that couples to homodimerization of juxtamembrane structures in the Toll ectodomain C terminus.

Dispatcher-assisted cardiopulmonary resuscitation: time to identify cardiac arrest and deliver chest compression instructions.

BACKGROUND: Dispatcher-assisted cardiopulmonary resuscitation (DA-CPR), in which 9-1-1 dispatchers provide CPR instructions over the telephone, has been shown to nearly double the rate of bystander CPR. We sought to identify factors that hampered the identification of cardiac arrest by 9-1-1 dispatchers and prevented or delayed the provision of dispatcher-assisted CPR chest compressions. METHODS AND RESULTS: We reviewed dispatch recordings for 476 out-of-hospital cardiac arrests occurring between January 1, 2011, and December 31, 2011. We found that the dispatcher correctly identified cardiac arrest in 80% of reviewed cases and 92% of cases in which they were able to assess patient consciousness and breathing. The median time to recognition of the arrest was 75 seconds. Chest compressions following dispatcher-assisted CPR instructions occurred in 62% of cases when the dispatcher had the opportunity to asses for consciousness and breathing and bystander CPR was not already started. The median time to first dispatcher-assisted CPR chest compression was 176 seconds. CONCLUSIONS: Dispatchers are able to accurately diagnose cardiac arrest over the telephone, but recognition is likely not possible in all circumstances. In some cases, recognition of cardiac arrest may be improved through training in the detection of agonal respirations. Delays in the delivery of dispatcher-assisted CPR chest compressions are common and are attributable to a mixture of dispatcher behavior and factors beyond the control of the dispatcher. Performance standards for the successful and quick recognition of cardiac arrest and delivery of first chest compressions should be adopted as metrics against which emergency medical services systems can measure their performance.

Functional insights from the crystal structure of the N-terminal domain of the prototypical toll receptor.

Drosophila melanogaster Toll is the founding member of an important family of pathogen-recognition receptors in humans, the Toll-like receptor (TLR) family. In contrast, the prototypical receptor is a cytokine-like receptor for Spätzle (Spz) protein and plays a dual role in both development and immunity. Here, we present the crystal structure of the N-terminal domain of the receptor that encompasses the first 201 amino acids at 2.4 Å resolution. To our knowledge, the cysteine-rich cap adopts a novel fold unique to Toll-1 orthologs in insects and that is not critical for ligand binding. However, we observed that an antibody directed against the first ten LRRs blocks Spz signaling in a Drosophila cell-based assay. Supplemented by point mutagenesis and deletion analysis, our data suggests that the region up to LRR 14 is involved in Spz binding. Comparison with mammalian TLRs reconciles previous contradictory findings about the mechanism of Toll activation.

Heterogeneous proliferation within engineered cartilaginous tissue: the role of oxygen tension.

This article investigates heterogeneous proliferation within a seeded three-dimensional scaffold structure with the purpose of improving protocols for engineered tissue growth. A simple mathematical model is developed to examine the very strong interaction between evolving oxygen profiles and cell distributions within cartilaginous constructs. A comparison between predictions based on the model and experimental evidence is given for both spatial and temporal evolution of the oxygen tension and cell number density, showing that behaviour for the first 14 days can be explained well by the mathematical model. The dependency of the cellular proliferation rate on the oxygen tension is examined and shown to be similar in size to previous work but linear in form. The results show that cell-scaffold constructs that rely solely on diffusion for their supply of nutrients will inevitably produce proliferation-dominated regions near the outer edge of the scaffold in situations when the cell number density and oxygen consumption rate exceed a critical level. Possible strategies for reducing such non-uniform proliferation, including the conventional methods of enhancing oxygen transport, are outlined based on the model predictions.