A larval zebrafish model of cardiac physiological recovery following cardiac arrest and myocardial hypoxic damage.

Contemporary cardiac injury models in zebrafish larvae include cryoinjury, laser ablation, pharmacological treatment and cardiac dysfunction mutations. Although effective in damaging cardiomyocytes, these models lack the important element of myocardial hypoxia, which induces critical molecular cascades within cardiac muscle. We have developed a novel, tractable, high throughput in vivo model of hypoxia-induced cardiac damage that can subsequently be used in screening cardioactive drugs and testing recovery therapies. Our potentially more realistic model for studying cardiac arrest and recovery involves larval zebrafish (Danio rerio) acutely exposed to severe hypoxia (PO2=5-7 mmHg). Such exposure induces loss of mobility quickly followed by cardiac arrest occurring within 120 min in 5 days post fertilization (dpf) and within 40 min at 10 dpf. Approximately 90% of 5 dpf larvae survive acute hypoxic exposure, but survival fell to 30% by 10 dpf. Upon return to air-saturated water, only a subset of larvae resumed heartbeat, occurring within 4 min (5 dpf) and 6-8 min (8-10 dpf). Heart rate, stroke volume and cardiac output in control larvae before hypoxic exposure were 188±5 bpm, 0.20±0.001 nL and 35.5±2.2 nL/min (n=35), respectively. After briefly falling to zero upon severe hypoxic exposure, heart rate returned to control values by 24 h of recovery. However, reflecting the severe cardiac damage induced by the hypoxic episode, stroke volume and cardiac output remained depressed by ∼50% from control values at 24 h of recovery, and full restoration of cardiac function ultimately required 72 h post-cardiac arrest. Immunohistological staining showed co-localization of Troponin C (identifying cardiomyocytes) and Capase-3 (identifying cellular apoptosis). As an alternative to models employing mechanical or pharmacological damage to the developing myocardium, the highly reproducible cardiac effects of acute hypoxia-induced cardiac arrest in the larval zebrafish represent an alternative, potentially more realistic model that mimics the cellular and molecular consequences of an infarction for studying cardiac tissue hypoxia injury and recovery of function.

Application of intraoperative structured light scanning to enable post-operative evaluation of digital and 3D-printed penile models.

BACKGROUND: Penile phenotype in hypospadias is currently assessed visually or manually (e.g., ruler, goniometer) for clinical, education, and research applications. However, these methods lack precision and accuracy across raters and cannot be reevaluated retrospectively following a surgical repair. The project aim was to evaluate the precision and reliability of penile dimensions obtained from digital and three dimensional (3D) printed models created from intraoperative (OR) structured light scans (SLS) during primary pediatric penile procedures. METHODS: Boys ages 1 month to 6 years underwent first- or single-stage penile surgery at a single institution were enrolled in this prospective study (IRB #20-000143). For each patient, immediately following placement of a stay suture under consistent manual tension, intra-operative dimension measurements with a ruler were obtained. A digital 3D model was created prior to penile repositioning using an Artec Space Spider scanner and Artec Studio 13 software. Following the case, two different raters completed 10 digital measurements of each generated model in Autodesk Fusion 360. These digital models were subsequently 3D printed and two different raters completed 10 manual dimension measurements of each 3D printed model using a ruler. A one-way random effects intraclass correlation coefficient (ICC) evaluated measures of agreement between and within raters, respectively. Analyses were conducted in R version 4.2. RESULTS: Six scans were obtained (hypospadias: 4, circumcision: 2). Intra-rater assessments showed excellent precision across repeated digital measurements; manual measurements of 3D printed models had excellent reliability for glans width and penile length but poor to good reliability for glans height. Inter-rater reliability was good to excellent for glans width (0.77-0.95) and good for penile length (0.71-0.88). However, there was poor inter-rater reliability for glans height (0-0.14). Following training regarding glans height location, there was an improvement in precision and repeatability of manual and digital measurements. CONCLUSION: Digital measurement of OR-derived 3D models resulted in excellent repeatability for each rater and improved between-rater reliability over manual measurement of 3D printed models alone, ensuring that images can be compared by various surgeons both now and in the future. SLS is promising as a novel modality to digitally generate 3D models, thereby informing phenotypic analysis for research and education. Further development of digital measurement methods to ensure consistency between raters for quantitative assessment of additional parameters and assessment of the technology within the pre-operative environment for surgical planning is planned.

Metabolic immaturity and breastmilk bile acid metabolites are central determinants of heightened newborn vulnerability to norovirus diarrhea.

The pathogenic outcome of enteric virus infections is governed by a complex interplay between the virus, intestinal microbiota, and host immune factors, with metabolites serving as a key mediator. Noroviruses bind bile acid metabolites, which are produced by the host and then modified by commensal bacteria. Paradoxically, bile acids can have both proviral and antiviral roles during norovirus infections. Working in an infant mouse model of norovirus infection, we demonstrate that microbiota and their bile acid metabolites protect from norovirus diarrhea, whereas host bile acids promote disease. We also find that maternal bile acid metabolism determines the susceptibility of newborn mice to norovirus diarrhea during breastfeeding. Finally, targeting maternal and neonatal bile acid metabolism can protect newborn mice from norovirus disease. In summary, neonatal metabolic immaturity and breastmilk bile acids are central determinants of heightened newborn vulnerability to norovirus disease.

Metabolic immaturity of newborns and breast milk bile acid metabolites are the central determinants of heightened neonatal vulnerability to norovirus diarrhea.

Noroviruses are the leading global cause of acute gastroenteritis, responsible for 685 million annual cases. While all age groups are susceptible to noroviruses, children are vulnerable to more severe infections than adults, underscored by 200 million pediatric cases and up to 200,000 deaths in children annually. Understanding the basis for the increased vulnerability of young hosts is critical to developing effective treatments. The pathogenic outcome of any enteric virus infection is governed by a complex interplay between the virus, intestinal microbiota, and host immune factors. A central mediator in these complex relationships are host- and microbiota-derived metabolites. Noroviruses bind a specific class of metabolites, bile acids, which are produced by the host and then modified by commensal bacterial enzymes. Paradoxically, bile acids can have both proviral and antiviral roles during norovirus infections. Considering these opposing effects, the microbiota-regulated balance of the bile acid pool may be a key determinant of the pathogenic outcome of a norovirus infection. The bile acid pool in newborns is unique due to immaturity of host metabolic pathways and developing gut microbiota, which could underlie the vulnerability of these hosts to severe norovirus infections. Supporting this concept, we demonstrate herein that microbiota and their bile acid metabolites protect from severe norovirus diarrhea whereas host-derived bile acids promote disease. Remarkably, we also report that maternal bile acid metabolism determines neonatal susceptibility to norovirus diarrhea during breastfeeding by delivering proviral bile acids to the newborn. Finally, directed targeting of maternal and neonatal bile acid metabolism can protect the neonatal host from norovirus disease. Altogether, these data support the conclusion that metabolic immaturity in newborns and ingestion of proviral maternal metabolites in breast milk are the central determinants of heightened neonatal vulnerability to norovirus disease.

Microbiota regulates neonatal disease tolerance to virus-evoked necrotizing enterocolitis by shaping the STAT1-NLRC5 axis in the intestinal epithelium.

Microbiota and feeding modes influence the susceptibility of premature newborns to necrotizing enterocolitis (NEC) through mechanisms that remain unknown. Here, we show that microbiota colonization facilitated by breastmilk feeding promotes NOD-like receptor family CARD domain containing 5 (Nlrc5) gene expression in mouse intestinal epithelial cells (IECs). Notably, inducible knockout of the Nlrc5 gene in IECs predisposes neonatal mice to NEC-like injury in the small intestine upon viral inflammation in an NK1.1+ cell-dependent manner. By contrast, formula feeding enhances neonatal gut colonization with environment-derived tilivalline-producing Klebsiella spp. Remarkably, tilivalline disrupts microbiota-activated STAT1 signaling that controls Nlrc5 gene expression in IECs through a PPAR-γ-mediated mechanism. Consequently, this dysregulation hinders the resistance of neonatal intestinal epithelium to self-NK1.1+ cell cytotoxicity upon virus infection/colonization, promoting NEC development. Together, we discover the underappreciated role of intestinal microbiota colonization in shaping a disease tolerance program to viral inflammation and elucidate the mechanisms impacting NEC development in neonates.

Identifying Targets for Innovation in Amazon Reviews of Bedwetting Alarms: Thematic Analysis.

BACKGROUND: Nocturnal enuresis (NE) is a frequent diagnosis in pediatric and adolescent populations with an estimated prevalence of around 15% at the age of 6 years. NE can have a substantial impact on multiple health domains. Bedwetting alarms, which typically consist of a sensor and moisture-activated alarm, are a common treatment. OBJECTIVE: This study aimed to determine areas of satisfaction versus dissatisfaction reported by the parents and caregivers of children using current bedwetting alarms. METHODS: Using the search term "bedwetting alarms" on the Amazon marketplace, products with >300 reviews were included. For each product, the 5 reviews ranked the "most helpful" for each star category were selected for analysis. Meaning extraction method was applied to identify major themes and subthemes. A percent skew was calculated by summing the total number of mentions of each subtheme,+1 for a positive mention, 0 for a neutral mention, and -1 for a negative mention, and dividing this total by the number of reviews in which that particular subtheme was observed. Subanalyses were performed for age and gender. RESULTS: Of 136 products identified, 10 were evaluated based on the selection criteria. The main themes identified across products were long-term concerns, marketing, alarm systems, and device mechanics and features. The subthemes identified as future targets for innovation included alarm accuracy, volume variability, durability, user-friendliness, and adaptability to girls. In general, durability, alarm accuracy, and comfort were the most negatively skewed subthemes (with a negative skew of -23.6%, -20.0%, and -12.4% respectively), which are indicative of potential areas for improvement. Effectiveness was the only substantially positively skewed subtheme (16.8%). Alarm sound and device features were positively skewed for older children, whereas ease of use had a negative skew for younger children. Girls and their caretakers reported negative experiences with devices that featured cords, arm bands, and sensor pads. CONCLUSIONS: This analysis provides an innovation roadmap for future device design to improve patient and caregiver satisfaction and compliance with bedwetting alarms. Our results highlight the need for additional options in alarm sound features, as children of different ages have divergent preferences in this domain. Additionally, girls and their parents and caretakers provided more negative overall reviews regarding the range of current device features compared to boys, indicating a potential focus area for future development. The percent skew showed that subthemes were often more negatively skewed toward girls, with the ease of use being -10.7% skewed for boys versus -20.5% for girls, and comfort being -7.1% skewed for boys versus -29.4% for girls. Put together, this review highlights multiple device features that are targets for innovation to ensure translational efficacy regardless of age, gender, or specific family needs.

Rational Protein Design Yields a CD20 CAR with Superior Antitumor Efficacy Compared with CD19 CAR.

Chimeric antigen receptors (CAR) are fusion proteins whose functional domains are often connected in a plug-and-play manner to generate multiple CAR variants. However, CARs with highly similar sequences can exhibit dramatic differences in function. Thus, approaches to rationally optimize CAR proteins are critical to the development of effective CAR T-cell therapies. Here, we report that as few as two amino-acid changes in nonsignaling domains of a CAR were able to significantly enhance in vivo antitumor efficacy. We demonstrate juxtamembrane alanine insertion and single-chain variable fragment sequence hybridization as two strategies that could be combined to maximize CAR functionality, and describe a CD20 CAR that outperformed the CD19 CAR in antitumor efficacy in preclinical in vitro and in vivo assays. Precise changes in the CAR sequence drove dramatically different transcriptomic profiles upon antigen stimulation, with the most efficacious CAR inducing an enrichment in highly functional memory T cells upon antigen stimulation. These findings underscore the importance of sequence-level optimization to CAR T-cell function, and the protein-engineering strategy described here may be applied to the development of additional CARs against diverse antigens. See related Spotlight by Scheller and Hudecek, p. 142.

Infection of neonatal mice with the murine norovirus strain WU23 is a robust model to study norovirus pathogenesis.

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe, with annual estimates of 50,000-200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. The development of the murine norovirus (MNV) model nearly two decades ago has facilitated progress in understanding host-norovirus interactions and norovirus strain variability. However, MNV strains tested thus far either do not cause intestinal disease or were isolated from extraintestinal tissue, raising concerns about translatability of research findings to human norovirus disease. Consequently, the field lacks a strong model of norovirus gastroenteritis. Here we provide a comprehensive characterization of a new small animal model system for the norovirus field that overcomes prior weaknesses. Specifically, we demonstrate that the WU23 MNV strain isolated from a mouse naturally presenting with diarrhea causes a transient reduction in weight gain and acute self-resolving diarrhea in neonatal mice of several inbred mouse lines. Moreover, our findings reveal that norovirus-induced diarrhea is associated with infection of subepithelial cells in the small intestine and systemic spread. Finally, type I interferons (IFNs) are critical to protect hosts from norovirus-induced intestinal disease whereas type III IFNs exacerbate diarrhea. This latter finding is consistent with other emerging data implicating type III IFNs in the exacerbation of some viral diseases. This new model system should enable a detailed investigation of norovirus disease mechanisms.

Environmentally-triggered contraction of the norovirus virion determines diarrheagenic potential.

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe with annual estimates of 50,000-200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections in people, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. We recently demonstrated that wild-type neonatal mice are susceptible to murine norovirus (MNV)-induced acute self-resolving diarrhea in a time course mirroring human norovirus disease. Using this robust pathogenesis model system, we demonstrate that virulence is regulated by the responsiveness of the viral capsid to environmental cues that trigger contraction of the VP1 protruding (P) domain onto the particle shell, thus enhancing receptor binding and infectivity. The capacity of a given MNV strain to undergo this contraction positively correlates with infection of cells expressing low abundance of the virus receptor CD300lf, supporting a model whereby virion contraction triggers infection of CD300lflo cell types that are responsible for diarrhea induction. These findings directly link environmentally-influenced biophysical features with norovirus disease severity.

Microkinetic coagulation assays for human and zebrafish plasma.

Coagulation assays, prothrombin time (PT), and partial thromboplastin time (PTT) are tests to measure the clotting ability of plasma and used in evaluating patients suffering from bleeding disorders. These assays require 100 µl of human plasma. In zebrafish, dilute plasma with exogenously added human fibrinogen was used. Our objective is to create a microkinetic coagulation assay for human and zebrafish plasmas using 1 µl plasma under conditions similar to PT and PTTs. Here, we developed an assay using the Take3 plate with wells holding up to 6 µl, which can be loaded in a microplate reader for measuring the absorbance of fibrin formation. In this assay, we used 1 µl of citrated zebrafish or human plasma followed by the addition of either thromboplastin or Dade ACTIN or factor X activator from Russell viper venom as an activating agent and CaCl2. We found 4 or 3 µl of the final volume of reaction was optimal. Our results showed both zebrafish and human plasmas yielded kinetic PT, kinetic PTT, and kinetic Russel's viper venom time curves similar to previously established curves using dilute plasma. This kinetic coagulation was inhibited by heparin and was reduced significantly in coagulation factor deficient plasmas. These results validated our microkinetic coagulation assays. Moreover, we derived clotting times from these kinetic curves, which were identical to human PT, PTT, and Russel's viper venom time. In conclusion, we established a microkinetic assay that could measure blood coagulation activity in models like zebrafish and human blood samples obtained from a finger prick in adults or heel prick in infants.

Identification of zebrafish ortholog for human coagulation factor IX and its age-dependent expression.

BACKGROUND: Coagulation factor IX (FIX) is a serine protease zymogen involved in the intrinsic blood coagulation pathway, and its deficiency causes hemophilia B. Zebrafish has three f9 genes, and the ortholog to human F9 is unknown. OBJECTIVE: To identify the zebrafish ortholog to F9 using sequence analysis and piggyback knockdown technology. METHODS: Gene and protein sequence analysis for three f9 genes, f9a, f9b, and f9l, present in the zebrafish genome was performed. In vivo and in vitro assays after knockdown of each gene and immunodepletion using specific antibodies were carried out. RESULTS: Sequence analysis revealed that f9a and f9b are similar to human F9, whereas f9l is similar to human F10. RNA analysis showed an age-dependent increase in expression of all three genes. Zebrafish f9a gene knockdown and Fixa immunodepletion prolonged kinetic partial thromboplastin time (kPTT), whereas f9l knockdown and Fixl immunodepletion prolonged kPTT, kinetic prothrombin time, and kinetic Russell viper venom activation time. Laser-assisted venous thrombosis increased time to occlusion after f9a and f9l knockdown and antibody inhibition of Fixa and Fixl. Further, analysis of plasma proteins by mass spectrometry and immunohistochemistry detected all three proteins. CONCLUSIONS: Our findings suggest that zebrafish f9a has functional activity similar to human F9. Fixl is functionally similar to Fx. The age-dependent increases of these factors are comparable to those observed in mice and humans. Thus, the zebrafish model could be used to study factors involved in increasing f9a expression during aging. It could also be used to test whether normal human Factor IX and Factor IX Leyden promoter work in zebrafish background.

Role of ribosomal RNA released from red cells in blood coagulation in zebrafish and humans.

Hemolytic disorders are characterized by hemolysis and are prone to thrombosis. It has previously been shown that the RNA released from damaged blood cells activates clotting. However, the nature of the RNA released from hemolysis is still elusive. We found that after hemolysis, red blood cells from both zebrafish and humans released RNA that contained mostly 5.8S ribosomal RNA (5.8S rRNA), This RNA activated coagulation in zebrafish and human plasmas. By using both natural and synthetic 5.8S rRNA and its truncated fragments, we found that the 3'-end 26-nucleotide-long RNA (3'-26 RNA) and its stem-loop secondary structure were necessary and sufficient for clotting activity. Corn trypsin inhibitor (CTI), a coagulation factor XII (FXII) inhibitor, blocked 3'-26 RNA-mediated coagulation activation in the plasma of both zebrafish and humans. CTI also inhibited zebrafish coagulation in vivo. 5.8S rRNA monoclonal antibody inhibited both 5.8S rRNA- and 3'-26 RNA-mediated zebrafish coagulation activity. Both 5.8S rRNA and 3'-26 RNA activated normal human plasma but did not activate FXII-deficient human plasma. Taken together, these results suggested that the activation of zebrafish plasma is via an FXII-like protein. Because zebrafish have no FXII and because hepatocyte growth factor activator (Hgfac) has sequence similarities to FXII, we knocked down the hgfac in adult zebrafish. We found that plasma from this knockdown fish does not respond to 3'-26 RNA. To summarize, we identified that an rRNA released in hemolysis activates clotting in human and zebrafish plasma. Furthermore, we showed that fish Hgfac plays a role in rRNA-mediated activation of coagulation.

Prostasin and hepatocyte growth factor B in factor VIIa generation: Serine protease knockdowns in zebrafish.

BACKGROUND: Blood clotting in humans is initiated by the binding of tissue factor to activated coagulation factor VII (FVIIa) in the plasma. Previous studies have reported that hepsin and factor VII (FVII)-activating protease are responsible for generating FVIIa. OBJECTIVES: We aimed to identify other proteases that may activate FVII using zebrafish as a model. METHODS: We screened 179 genes encoding serine protease domains using the piggyback knockdown method to identify genes involved in the activation of zebrafish Fvii. A prolonged kinetic prothrombin time (kPT) assay was used to detect gene knockdown effects. RESULTS: In the primary screen, 21 genes showed prolonged kPT. In the secondary screen, 14 of 21 genes showed positive results. In the tertiary screen, all 14 genes showed prolonged kPT. These 14 genes were knocked down again to estimate relative levels of zebrafish Fviia. Six genes, including known genes, such as f10 and novel prostasin and hepatocyte growth factor B (hgfb), showed lower Fviia levels. Fvii levels were affected only by the knockdown of f7 and not by the knockdown of the other five genes. CONCLUSIONS: Prostasin and hgfb are involved in generating Fviia. We hypothesize that prostasin exerts serine protease activity directly or indirectly to activate Fvii. As Hgfb has a mutated serine protease domain, it may not cleave Fvii but may bind to Fvii to induce autoactivation. The approach developed here may be extended to design other large-scale knockdown screens.

Effect of MS222 on Hemostasis in Zebrafish.

MS222 is a compound used in anesthetizing vertebrates, including fish and frogs. Several side effects of this anesthetic have been reported, but its effect on hemostasis has not been studied. In our laboratory, we have used zebrafish for more than 2 decades as a model system to study hemostasis. During this period, we have had trouble in collecting blood from anesthetized zebrafish and observed more rapid blood clotting than in nonanesthetized counterparts. However, no systematic studies regarding the effect of MS222 on zebrafish hemostasis are available. In this study, we performed various assays such as gill bleeding, measurement of Hct, total blood cell counts, thrombocyte counts, thrombocyte aggregation, and coagula- tion and measured the amount of blood collected. We found that Hct values, the amount of blood collected, bleeding, and coagulation differed significantly between anesthetized and nonanesthetized fish. Our results suggest that blood collected after MS222 anesthesia of zebrafish has altered hemostasis.