Search

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.

List of gene names and FPKM values from bulk RNA-seq on T cells harvested from tumor-bearing mice (Fig. 4A-D, F and Supplementary Fig. S7).

Figures S1 to S7

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

Key Points Hemolysis releases 5.8S rRNA and activates blood coagulation in human and zebrafish via FXII and Hgfac, respectively.Only the 3'-end 26 nucleotides of 5.8S rRNA were necessary and sufficient for this activation., Visual Abstract, 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.

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.

BACKGROUND Nocturnal enuresis (NE) is a frequent diagnosis in the pediatric and adolescent populations with an estimated prevalence of around 15% at age 6 years. NE can have a significant impact on multiple domains of health. Bedwetting alarms, which typically consist of a sensor and moisture-activated alarm, are a common treatment. The OBJECTIVE The aim of this study was to determine areas of satisfaction versus dissatisfaction reported by the parents/caregivers of users of current bedwetting alarms. METHODS Using the search term “bedwetting alarms” on Amazon marketplace, products with >300 reviews were included. For each product, the 5 most helpful reviews for each star category were collected for analysis. Meaning extraction method, a natural language processing technique, was applied to identify major themes and subcategories. A percent skew was calculated by summating the total number of mentions of each subtheme: +1 for a positive, 0 for neutral, and -1 for a negative mention and dividing this total by the number of reviews in which that subtheme was observed. Sub-analyses were performed for age and sex. RESULTS Of 136 products identified, 10 were evaluated based on selection criteria. Main themes identified across products were long-term concerns, marketing, alarm system, and device mechanics and features. Subthemes identified as future targets for innovation included alarm accuracy, alarm volume variability, device durability, user friendliness, and adaptability to female children. In general, durability, alarm accuracy, and comfort were the most negatively skewed subthemes indicative of potential areas for improvement, and effectiveness was the only significantly positively skewed subtheme. Alarm sound and device features were positively skewed for older children, while 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 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, young girls and their parents/caretakers provided more negative reviews overall regarding the range of current device features compared to boys, indicating devices are not adequately developed to meet patient needs regardless of sex. Put together, this review highlights multiple device features that are targets for innovation to ensure translational efficacy regardless of age, sex, or specific family needs.

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. 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, the model remains limited because 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 and determinants of norovirus pathogenesis remain poorly defined. Herein, 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 reduced 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 (IFN) 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 and serve as a platform for testing antivirals.

IntroductionBiodesign innovation processes provide a needs-driven approach to medical innovation, empowering both medical trainees and health care providers to take action in addressing the shortcomings of health care encountered in daily clinical practice. Our objective was to uncover the most pressing unmet clinical needs within a specific clinical setting, pediatric urology at UCLA.MethodsThe biodesign process involves a sequential process of identifying, validating, and prioritizing unmet needs, followed by solution landscaping and prototyping for the most promising needs. Opportunities for medical innovation were first identified through six weeks of clinical immersion, which involved both clinical observation and interview-based insight extraction. Interviews were conducted with 35 stakeholders, including patients, patient families, and health care staff by a medical student participant in Sling Health LA, a program which provides innovation training and incubation for ideas. Follow-up interviews with key stakeholders were performed to validate needs. Priority scores were then assigned to each validated need using a series of pre-determined and weighted criteria. Finally, genealogy maps were constructed and used to guide subsequent solution landscaping for the top three needs.Results33 unmet clinical needs were identified throughout the clinical immersion phase, 27 of which were validated. Following coarse needs prioritization, five needs emerged as top contenders. After fine needs prioritization, three top needs were selected. The first top need arising from this ethnographic study was that “parents and children need a faster (ConclusionBiodesign processes offer a standardized method for identifying pressing unmet clinical needs and informing solution development. The top three needs discovered within pediatric urology through this ethnographic investigation represent promising innovation targets for further solution prototyping and design.

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.

Modern-day supercomputers are equipped with sophisticated graphics processing units (GPUs) along with high-performance CPUs. Adapting existing algorithms specifically to GPU has resulted in under-utilization of CPU computing power. In this respect, we parallelize Jacobi and successive-over relaxation (SOR), which are used as smoother in multigrid method to maximize the combined utilization of both CPUs and GPUs. We study the performance of multigrid method in terms of total execution time by employing different hybrid parallel approaches, viz. accelerating the smoothing operation using only GPU across all multigrid levels, alternately switching between GPU and CPU based on the multigrid level and our proposed novel approach of using combination of GPU and CPU across all multigrid levels. Our experiments demonstrate a significant speedup using the hybrid parallel approaches, across different problem sizes and finite element types, as compared to the MPI only approach. However, the scalability challenge persists for the hybrid parallel multigrid smoothers.

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.