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The Statewide (formerly Southern) California Earthquake Center (SCEC) conducts multidisciplinary earthquake system science research that aims to develop predictive models of earthquake processes, and to produce accurate seismic hazard information that can improve societal preparedness and resiliency to earthquake hazards. As part of this program, SCEC has developed the CyberShake platform, which calculates physics-based probabilistic seismic hazard analysis (PSHA) models for regions with high-quality seismic velocity and fault models. The CyberShake platform implements a sophisticated computational workflow that includes over 15 individual codes written by 6 developers. These codes are heterogeneous, ranging from short-running high-throughput serial CPU codes to large, long-running, parallel GPU codes. Additionally, CyberShake simulation campaigns are computationally extensive, typically producing tens of terabytes of meaningful scientific data and metadata over several months of around-the-clock execution on leadership-class supercomputers. To meet the needs of the CyberShake platform, we have developed an extreme-scale workflow stack, including the Pegasus Workflow Management System, HTCondor, Globus, and custom tools. We present this workflow software stack and identify how the CyberShake platform and supporting tools enable us to meet a variety of challenges that come with large-scale simulations, such as automated remote job submission, data management, and verification and validation. This platform enabled us to perform our most recent simulation campaign, CyberShake Study 22.12, from December 2022 to April 2023. During this time, our workflow tools executed approximately 32,000 jobs, and used up to 73% of the Summit system at Oak Ridge Leadership Computing Facility. Our workflow tools managed about 2.5 PB of total temporary and output data, and automatically staged 19 million output files totaling 74 TB back to archival storage on the University of Southern California's Center for Advanced Research Computing systems, including file-based relational data and large binary files to efficiently store millions of simulated seismograms. CyberShake extreme-scale workflows have generated simulation-based probabilistic seismic hazard models that are being used by seismological, engineering, and governmental communities.

Abstract The exploitation of sharks and the degradation of their habitats elevate the urgency to understand the factors that influence offspring survival and ultimately shark reproductive success. We monitored and sampled blacktip reef sharks (Carcharhinus melanopterus) in nursery habitats of Moorea Island (French Polynesia), to improve knowledge on shark reproductive behavior and biology. We sampled fin clips and morphometrics from 230 young-of-the-year sharks and used microsatellite DNA markers to process parentage analysis to study the reproductive philopatric behavior in female sharks and the matrotrophy within litters. These traits are driving the success of the local replenishment influencing selection through birth site and maternal reserves transmitted to pups. Parentage analysis revealed that some female sharks changed their parturition areas (inter-seasonally) while other female sharks came back to the same site for parturition, providing evidence for a plastic philopatric behavior. Morphometrics showed that there was no significant relationship between body condition indices and nursery locations. However, similarities and differences in body condition were observed between individuals sharing the same mother, indicating that resource allocation within some shark litters might be unbalanced. Our findings further our understanding of the reproductive biology and behavior that shape shark populations with the aim to introduce these parameters into future conservation strategies.

Abstract In this work, a coupled Monte Carlo Genetic Algorithm (MCGA) approach is used to optimize a gas phase uranium oxide reaction mechanism based on plasma flow reactor (PFR) measurements. The PFR produces a steady Ar plasma containing U, O, H, and N species with high temperature regions (3000–5000 K) relevant to observing UO formation via optical emission spectroscopy. A global kinetic treatment is used to model the chemical evolution in the PFR and to produce synthetic emission signals for direct comparison with experiments. The parameter space of a uranium oxide reaction mechanism is then explored via Monte Carlo sampling using objective functions to quantify the model-experiment agreement. The Monte Carlo results are subsequently refined using a genetic algorithm to obtain an experimentally corroborated set of reaction pathways and rate coefficients. Out of 12 reaction channels targeted for optimization, four channels are found to be well constrained across all optimization runs while another three channels are constrained in select cases. The optimized channels highlight the importance of the OH radical in oxidizing uranium in the PFR. This study comprises a first step toward producing a comprehensive experimentally validated reaction mechanism for gas phase uranium molecular species formation.

ABSTRACTThe intestinal epithelium is constantly exposed to microbes residing in the lumen. Traditionally, the response to microbial interactions has been studied in cell lines derived from cancerous tissues, e.g. Caco-2. It is, however, unclear how the responses in these cancer cell lines reflect the responses of a normal epithelium and whether there might be microbial strain-specific effects. To address these questions, we derived organoids from the small intestine from a cohort of healthy individuals. Culturing intestinal epithelium on a flat laminin matrix induced their differentiation, facilitating analysis of microbial responses via the apical membrane normally exposed to the luminal content. Here, it was evident that the healthy epithelium across multiple individuals (n = 9) demonstrates robust acute both common and strain-specific responses to a range of probiotic bacterial strains (BB-12Ⓡ, LGGⓇ, DSM33361, and Bif195). Importantly, parallel experiments using the Caco-2 cell line provide no acute response. Collectively, we demonstrate that primary epithelial cells maintained as organoids represent a valuable resource for assessing interactions between the epithelium and luminal microbes across individuals, and that these models are likely to contribute to a better understanding of host microbe interactions.

Abstract Adherent cells, mammalian or human, are ubiquitous for production of viral vaccines, in gene therapy and in immuno‐oncology. The development of a cell‐expansion process with adherent cells is challenging as scale‐up requires the expansion of the cell culture surface. Microcarrier (MC)‐based cultures are still predominate. However, the development of MC processes from scratch possesses particular challenges due to their complexity. A novel approach for the reduction of development times and costs of cell propagation processes is the combination of mathematical process models with statistical optimization methods, called model‐assisted Design of Experiments (mDoE). In this study, an mDoE workflow was evaluated successfully for the design of a MC‐based expansion process of adherent L929 cells at a very early stage of development with limited prior knowledge. At the start, the analytical methods and the screening of appropriate MCs were evaluated. Then, cause‐effect relationships (e.g., cell growth related to medium conditions) were worked out, and a mathematical process model was set‐up and adapted to experimental data for modeling purposes. The model was subsequently used in mDoE to identify optimized process conditions, which were proven experimentally. An eight‐fold increase in cell yield was achieved basically by reducing the initial MC concentration.

Nox4 expression combined with reactive oxygen species signaling are key to osteoblast differentiation, proliferation and maturation in mice.

Abstract Background Sharks play essential roles in ocean food webs and human culture, but also face population declines worldwide due to human activity. The relationship between sharks and the microbes on and in the shark body is unclear, despite research on other animals showing the microbiome as intertwined with host physiology, immunity, and ecology. Research on shark-microbe interactions faces the significant challenge of sampling the largest and most elusive shark species. We leveraged a unique sampling infrastructure to compare the microbiomes of two apex predators, the white (Carcharodon carcharias) and tiger shark (Galeocerdo cuvier), to those of the filter-feeding whale shark (Rhincodon typus), allowing us to explore the effects of feeding mode on intestinal microbiome diversity and metabolic function, and environmental exposure on the diversity of microbes external to the body (on the skin, gill). Results The fecal microbiomes of white and whale sharks were highly similar in taxonomic and gene category composition despite differences in host feeding mode and diet. Fecal microbiomes from these species were also taxon-poor compared to those of many other vertebrates and were more similar to those of predatory teleost fishes and toothed whales than to those of filter-feeding baleen whales. In contrast, microbiomes of external body niches were taxon-rich and significantly influenced by diversity in the water column microbiome. Conclusions These results suggest complex roles for host identity, diet, and environmental exposure in structuring the shark microbiome and identify a small, but conserved, number of intestinal microbial taxa as potential contributors to shark physiology.

Abstract In order to model the fate and transport of particles following a nuclear explosion, there must first be an understanding of individual physical and chemical processes that affect particle formation. One interaction pertinent to fireball chemistry and resultant debris formation is that between uranium and oxygen. In this study, we use laser ablation of uranium metal in different concentrations of oxygen gas, either 16O2 or 18O2, to determine the influence of oxygen on rapidly cooling uranium. Analysis of recovered particulates using infrared absorption and Raman spectroscopies indicate that the micrometer-sized particulates are predominantly amorphous UOx (am-UOx, where 3 ≤ x ≤ 4) and UO2 after ablation in 1 atm of pure O2 and a 1% O2/Ar mixture, respectively. Energy dispersive X-ray spectroscopy (EDS) of particulates formed in pure O2 suggest an O/U ratio of ~ 3.7, consistent with the vibrational spectroscopy analysis. Both am-UOx and UO2 particulates convert to α-U3O8 when heated. Lastly, experiments performed in 18O2 environments show the formation of 18O-substituted uranium oxides; vibrational frequencies for am-U18Ox are reported for the first time. When compared to literature, this work shows that cooling timescales can affect the structural composition of uranium oxides (i.e., crystalline vs. amorphous). This indicator can be used in current models of nuclear explosions to improve our predicative capabilities of chemical speciation.

The small intestine forms via crosstalk between epithelial and mesenchymal cell compartments. Here, the authors show that a gradient of Wnt signalling along the anterior-posterior axis regulates Sonic Hedgehog which is required for correct formation and regionalization of the small intestine.

At a time when policymakers of the European Union (EU) are pivoting towards a more assertive use of economic power in external relations, this article discusses the merits of situating the much-debated use of economic sanctions and other economic power-based instruments in the broader terminology of EU diplomatic capabilities. Pointing out a number of shortcomings in traditional literature on geoeconomics and economic statecraft, the article applies the concept of “geoeconomic diplomacy” to demonstrate how the EU’s geoeconomic success will heavily depend on the abilities of diplomats and civil servants from institutions and member states to engage in viable relationships with relevant public and private actors in the state-market realm. Based hereon, it identifies institutional and context-specific challenges that could affect the comprehensive realisation of recent EU policy reforms relevant to the geoeconomic agenda: (a) institutional measures to ensure a more robust enforcement of sanctions, (b) a new anti-coercion instrument to counter coercive trade practices by third countries, and (c) a more efficient, focused, and strategic utilisation of EU development funds for purposes of stability and peace. The article concludes by discussing the prospects for bringing such instruments closer together at the level of practical implementation through the establishment of stronger relationships between practitioners working across the EU’s various geoeconomic intervention areas.

This study assesses the performance of the model-assisted Design of Experiment (mDoE) software toolbox for the design of two microalgae bioprocesses. The mDoE-toolbox was applied to maximize biomass growth for Desmodesmus pseudocommunis in a photobioreactor by varying the light intensity and pH and for Chlorella vulgaris in shake flasks, by varying the light intensity and duration. For both case studies, a mathematical mechanistic model was applied. In the first study only one experiment was necessary to adapt the mathematical model and identify a combination of light intensity and pH that improved biomass yield, as confirmed experimentally. In the second study, no well-established model was available for the specific experimental arrangement. On the basis of the literature, a mathematical model was constructed and a first cycle of mDoE was performed, thus identifying the desired factor combinations. Experiments confirmed the high biomass yield but revealed shortcomings of the model. The model was improved and a second cycle of mDoE was performed. The recommended factor combinations from both cycles were comparable. The mDoE was found to be a time-saving, cost-effective and useful method enabling the identification of factor combinations leading to high biomass production for the design of two different microalgae bioprocesses with low experimental effort.

This study investigated the acquisition of depicting signs (DS) among students learning a signed language as their second-modality and second-language (M2L2) language. Depicting signs, broadly described, illustrate actions and states. This study sample includes 75 M2L2 students who were recruited from college-level American Sign Language (ASL) courses who watched and described three short clips from Canary Row the best they could in ASL. Four types of DS were coded in the students' videorecorded retellings: (1) entity depicting signs (EDS); (2) body part depicting signs (BPDS); (3) handling depicting signs (HDS); and (4) size-and-shape specifiers (SASS). Results revealed that SASS and HDS increase in instances as students advance in their ASL learning and comprehension. However, EDS expressions did not have a relationship with their ASL comprehension. ASL 2 students produced less DS than the ASL 1 students but did not differ from the ASL 3+ students. There were no differences in instances of BPDS among the three groups of L2 learners although their ability to produce BPDS was correlated with their ASL comprehension. This study is the first to systematically elicit depicting signs from M2L2 learners in a narrative context. The results have important implications for the field of sign language pedagogy and instruction. Future research, particularly cross-sectional and/or longitudinal studies, is needed to explore the trajectory of the acquisition of DS and identify evidence-based pedagogical approaches for teaching depicting signs to M2L2 students.

Abstract Elasmobranchs (sharks, skates and rays) are of broad ecological, economic, and societal value. These globally important fishes are experiencing sharp population declines as a result of human activity in the oceans. Research to understand elasmobranch ecology and conservation is critical and has now begun to explore the role of body-associated microbiomes in shaping elasmobranch health. Here, we review the burgeoning efforts to understand elasmobranch microbiomes, highlighting microbiome variation among gastrointestinal, oral, skin, and blood-associated niches. We identify major bacterial lineages in the microbiome, challenges to the field, key unanswered questions, and avenues for future work. We argue for prioritizing research to determine how microbiomes interact mechanistically with the unique physiology of elasmobranchs, potentially identifying roles in host immunity, disease, nutrition, and waste processing. Understanding elasmobranch–microbiome interactions is critical for predicting how sharks and rays respond to a changing ocean and for managing healthy populations in managed care.

The COVID-19 pandemic led to a surge in acquisitions of puppies in the UK, dubbed the “Pandemic Puppy” phenomenon. In addition to an increased demand for puppies, widespread changes to both why and how puppies were purchased during this period compared to pre-pandemic 2019 purchases were documented, many of which threatened canine welfare (e.g., puppies being collected away from their place of birth, without seeing their mother). This study aimed to explore which changes to the pre-purchase and purchase motivations and behaviours of UK owners who purchased a puppy aged n = 1148, “2019 puppies”) and 2020 (n = 4369, “Pandemic Puppies”). While the majority of the peak pandemic changes documented in 2020 had returned to their 2019 pre-pandemic baseline, others persisted into 2021. Multinomial logistic regression models revealed that the shifts during 2020 towards owners viewing their puppy pre-purchase over video calls or via video recordings/photos rather than in-person and towards collecting their puppy from outside of their breeders’ property rather than inside had persisted into 2021 and had not returned to pre-pandemic levels. Year-on-year significant rises in the number of puppies sold with a passport were documented between 2019 and 2021, with over 1 in 10 2021 puppies having been sold with a passport, the figure more than doubling since 2019. An increasing number of these puppies sold with a passport were under the minimum legal age for import at sale. Going forward, these concerning changes require further monitoring and human behaviour change interventions to tackle, including increased buyer awareness but also legislative approaches to prevent the greatest harm.

Maria Antfolk and Kim Jensen discuss how to model intestinal epithelial cell function in the dish and how various physiologically important environmental conditions, for example, extracellular matrix, pressure and flow, can be modelled and how this is applicable to clinical work.

Metazoans host complex communities of microorganisms that include dinoflagellates, fungi, bacteria, archaea and viruses. Interactions among members of these complex assemblages allow hosts to adjust their physiology and metabolism to cope with environmental variation and occupy different habitats. Here, using reciprocal transplantation across depths, we studied adaptive divergence in the corals Orbicella annularis and O. franksi, two young species with contrasting vertical distribution in the Caribbean. When transplanted from deep to shallow, O. franksi experienced fast photoacclimation and low mortality, and maintained a consistent bacterial community. By contrast, O. annularis experienced high mortality and limited photoacclimation when transplanted from shallow to deep. The photophysiological collapse of O. annularis in the deep environment was associated with an increased microbiome variability and reduction of some bacterial taxa. Differences in the symbiotic algal community were more pronounced between coral species than between depths. Our study suggests that these sibling species are adapted to distinctive light environments partially driven by the algae photoacclimation capacity and the microbiome robustness, highlighting the importance of niche specialization in symbiotic corals for the maintenance of species diversity. Our findings have implications for the management of these threatened Caribbean corals and the effectiveness of coral reef restoration efforts.

Trichogramma brassicae (Bezdenko) are egg parasitoids that are used throughout the world as biological control agents and in laboratories as model species. Despite this ubiquity, few genetic resources exist beyond COI, ITS2, and RAPD markers. Aided by a Wolbachia infection, a wild-caught strain from Germany was reared for low heterozygosity and sequenced in a hybrid de novo strategy, after which several assembling strategies were evaluated. The best assembly, derived from a DBG2OLC-based pipeline, yielded a genome of 235 Mbp made up of 1,572 contigs with an N50 of 556,663 bp. Following a rigorous ab initio-, homology-, and evidence-based annotation, 16,905 genes were annotated and functionally described. As an example of the utility of the genome, a simple ortholog cluster analysis was performed with sister species T. pretiosum, revealing over 6000 shared clusters and under 400 clusters unique to each species. The genome and transcriptome presented here provides an essential resource for comparative genomics of the commercially relevant genus Trichogramma, but also for research into molecular evolution, ecology, and breeding of T. brassicae.

Physician‒scientists have made countless discoveries, and their dwindling numbers are a significant concern. Although dermatology has become an increasingly popular destination for physician‒scientist trainees, the proportion of trainees who pursue scientific research careers after training is among the lowest of all medical specialties. To investigate this problem, we surveyed a national cohort of dermatology educators, physician‒scientist track program directors, and National Institute of Arthritis and Musculoskeletal and Skin Diseases T32 directors for opinions regarding physician‒scientist training in dermatology. On the basis of these findings and to help address the issue, we propose a training practicum and provide a resource for funding opportunities to help guide trainees and institutions interested in supporting investigative dermatologists. We also discuss the important roles of department chairs and institutions in fashioning an environment conducive to physician‒scientist training. The information and recommendations provided in this paper may help to improve the recruitment, training, development, and retention of investigative dermatologists and future leaders in this field.

Young adults are frequent consumers of food prepared outside the home (FOH). In a cross-sectional survey, the MYMeals study, we showed FOH provided one-third of meals and snacks for young Australian adults, yet it contributed higher proportions of energy and nutrients of concern, such as saturated fat and sodium. This study aimed to determine the detailed proportional contribution of nutrients of concern from the nine food outlet types captured in the MYMeals study. Young adults residing in New South Wales (NSW), Australia, (n = 1001) used a validated smartphone app to report all types and amounts of food and beverages consumed for three consecutive days, as well as their preparation location. The proportions of daily energy, macronutrients, sodium, total sugars, and saturated fat were calculated for each of the nine following outlet types: bakeries or patisseries, coffee chains, cold-drink chains, fast-food chains, ice creamery or frozen yoghurt outlets, independent cafes or restaurants, pubs (hotels) and clubs, service stations or convenience stores, and others not fitting the above categories. Of all FOH outlet types, independent cafes or restaurants contributed the most energy (17.5%), sodium (20.0%) and saturated fat (17.8%) to the total diet, followed by fast-food chains (12.0% energy, 15.8% sodium, and 12.0% saturated fat) and other outlets, with smaller proportions. For males, the proportion of energy and nutrients contributed by fast-food outlets was higher than for females (14.8% versus 9.8% energy). Menu labelling at independent cafes and restaurants is recommended, comprising, in addition to the energy labels already in use in fast-food restaurants, the labelling of nutrients of concern. The feasibility of this recommendation warrants further exploration.

Objective: To assess effects of standing exercise on adults with cerebral palsy with a focus on bone density, transfer skills, quality of life, and related health parameters. Design: Prospective case series; pilot study. Setting: Outpatient multispecialty clinic. Participants: Nonambulatory adults with cerebral palsy, 13 standers and 7 nonstanders, comparable in age, sex, and other physical characteristics (N=20). Interventions: Not applicable. Main Outcome Measures: Bone mineral density per dual energy x-ray absorptiometry, stand pivot transfers, comprehensive blood serum assessments, fractures, spasms, perceived pain, and quality of life. Results: No appreciable differences could be detected between the standing and nonstanding groups from baseline and over a 2-year subsequent study with respect to bone density, range of motion, comprehensive chemistry, hematologic blood serum levels, fractures, spasms, perceived pain, continence of bowel and bladder, seizures, orthotics, and orthopedic surgery. All individuals reported positive life effects of standing with only 1 negative effect reported: increased fatigue at the end of the day. Midline independent head control >30 seconds was identified only in the standing group. Functional stand pivot transfers were seen only in individuals with a history of standing. Conclusions: The pilot data indicate no appreciable difference in measured outcome variables of a static nature between nonambulatory adults with cerebral palsy who stand compared with those who do not. We identified occurrences of improved head control and functional stand pivot transfers only in those with a history of standing. The value of a functional pivot transfer over the lifetime is difficult to overestimate. Encouragement is given toward future studies with a focus more toward functional outcome variables.

In recent years, an increasing number of deaf and hard of hearing (D/HH) undergraduates have chosen to study in STEM fields and pursue careers in research. Yet, very little research has been undertaken on the barriers and inclusive experiences often faced by D/HH undergraduates who prefer to use spoken English in research settings, instead of American Sign Language (ASL). To identify barriers and inclusive strategies, we studied six English speaking D/HH undergraduate students working in research laboratories with their eight hearing mentors, and their three hearing peers sharing their experiences. Three researchers observed the interactions between all three groups and conducted interviews and focus groups, along with utilizing the Communication Assessment Self-Rating Scale (CASS). The main themes identified in the findings were communication and environmental barriers in research laboratories, creating accessible and inclusive laboratory environments, communication strategies, and self-advocating for effective communication. Recommendations for mentors include understanding the key elements of creating an inclusive laboratory environment for English speaking D/HH students and effectively demonstrating cultural competence to engage in inclusive practices.

Abstract Background Intestinal stem cell transplantation has been shown to promote mucosal healing and to engender fully functional epithelium in experimental colitis. Hence, stem cell therapies may provide an innovative approach to accomplish mucosal healing in patients with debilitating conditions such as inflammatory bowel disease. However, an approach to label and trace transplanted cells, in order to assess engraftment efficiency and to monitor wound healing, is a key hurdle to overcome prior to initiating human studies. Genetic engineering is commonly employed in animal studies, but may be problematic in humans due to potential off-target and long-term adverse effects. Methods We investigated the applicability of a panel of fluorescent dyes and nanoparticles to label intestinal organoids for visualization using the clinically approved imaging modality, confocal laser endomicroscopy (CLE). Staining homogeneity, durability, cell viability, differentiation capacity, and organoid forming efficiency were evaluated, together with visualization of labeled organoids in vitro and ex vivo using CLE. Results 5-Chloromethylfluorescein diacetate (CMFDA) proved to be suitable as it efficiently stained all organoids without transfer to unstained organoids in co-cultures. No noticeable adverse effects on viability, organoid growth, or stem cell differentiation capacity were observed, although single-cell reseeding revealed a dose-dependent reduction in organoid forming efficiency. Labeled organoids were easily identified in vitro using CLE for a duration of at least 3 days and could additionally be detected ex vivo following transplantation into murine experimental colitis. Conclusions It is highly feasible to use fluorescent dye-based labeling in combination with CLE to trace intestinal organoids following transplantation to confirm implantation at the intestinal target site.

The recombinant monoclonal antibody capture step represents the current bottleneck in downstream processing. Protein A resins are diffusion-limited chromatography materials which require low flow rates to achieve a binding capacity above 30 g L−1 with the result of low productivity. Here, we present a novel chromatography membrane combining superior binding capacities with high flow rates for high productivity while achieving comparable product quality as state-of-the-art protein A resins. Further, we demonstrate full scalability of this convecdiff technology with experimental data demonstrating suitability for bioprocessing at different scales. This technology results in more than 10-fold higher productivity compared to Protein A resins, which is maintained during scale up. We demonstrate the influence of residence times, feed titers and the cleaning regime on productivity and indicate optimal utilization of the convecdiff membrane based on feed titer availability. The underlying high productivity and short cycle times of this material enable the purification of monoclonal antibodies with 10-times less chromatography material used per batch and utilization of the membrane within one batch. Provided in disposable consumables, this novel technology will remove column handling in bioprocesses and resin re-use over multiple batches.

Abstract This paper aims to explain survey findings regarding disaster recovery processes among ethnic groups in a rural Texas community. The research was conducted over a 4-year period with most of the survey data collected over the summer of 2004. The research was descriptive in nature, attempting to document processes and sources of recovery aid acquisition following a natural disaster, and viewed through the lens of cultural and ethnic literature regarding minorities and disaster recovery processes. The difficulty in explaining research findings comes from the fact that data was collected at the household level, yet the explanations which best elucidate the findings are derived from a different level of analysis than that of the survey. The variation in aid acquisition between ethnic groups is best understood as a manifestation of community cultural norms, which implies both individual, cognitive norms, as well as social norms. Ethnic literature focused on minorities in disaster situations, excerpts from qualitative data, and personal observations are used to support the interpretation of the data presented herein pointing to cultural flexibility in recovery processes, which are stifled by institutional barriers hampering recovery efficiencies.

Objective: To describe the clinical trial “Vasopressin and Methylprednisolone for In-Hospital Cardiac Arrest” (VAM-IHCA). Methods: The VAM-IHCA trial is an investigator-initiated, multicenter, randomized, placebo-controlled, parallel group, double-blind, superiority trial of vasopressin and methylprednisolone during adult in-hospital cardiac arrest. The study drugs consist of 40 mg methylprednisolone and 20 IU of vasopressin given as soon as possible after the first dose of adrenaline. Additional doses of vasopressin (20 IU) will be administered after each adrenaline dose for a maximum of four doses (80 IU).The primary outcome is return of spontaneous circulation and key secondary outcomes include survival and survival with a favorable neurological outcome at 30 days. 492 patients will be enrolled. The trial was registered at the EU Clinical Trials Register (EudraCT Number: 2017-004773-13) on Jan. 25, 2018 and ClinicalTrials.gov (Identifier: NCT03640949) on Aug. 21, 2018. Results: The trial started in October 2018 and the last patient is anticipated to be included in January 2021. The primary results will be reported after 3-months follow-up and are, therefore, anticipated in mid-2021. Conclusion: The current article describes the design of the VAM-IHCA trial. The results from this trial will help clarify whether the combination of vasopressin and methylprednisolone when administered during in-hospital cardiac arrest improves outcomes.

Intestinal organoids are an excellent model to study epithelial biology. Yet, the selection of analytical tools to accurately quantify heterogeneous organoid cultures remains limited. Here, we developed a semi-automated organoid screening method, which we applied to a library of highly specific chemical probes to identify epigenetic regulators of intestinal epithelial biology. The role of epigenetic modifiers in adult stem cell systems, such as the intestinal epithelium, is still undefined. Based on this resource dataset, we identified several targets that affected epithelial cell differentiation, including HDACs, EP300/CREBBP, LSD1, and type I PRMTs, which were verified by complementary methods. For example, we show that inhibiting type I PRMTs, which leads enhanced epithelial differentiation, blocks the growth of adenoma but not normal organoid cultures. Thus, epigenetic probes are powerful tools to study intestinal epithelial biology and may have therapeutic potential.

The specific effects of administering live probiotics in the human gut are not well characterized. To this end, we investigated the immediate effect of Lactobacillus rhamnosus GG (LGG) in the jejunum of 27 healthy volunteers 2 h after ingestion using a combination of global RNA sequencing of human biopsies and bacterial DNA sequencing in a multi-visit, randomized, cross-over design (ClinicalTrials.gov number NCT03140878). While LGG was detectable in jejunum after 2 h in treated subjects, the gene expression response vs. placebo was subtle if assessed across all subjects. However, clustering analysis revealed that one-third of subjects exhibited a strong and consistent LGG response involving hundreds of genes, where genes related to B cell activation were upregulated, consistent with prior results in mice. Immunohistochemistry and single cell-based deconvolution analyses showed that this B cell signature likely is due to activation and proliferation of existing B cells rather than B cell immigration to the tissue. Our results indicate that the LGG strain has an immediate effect in the human gut in a subpopulation of individuals. In extension, our data strongly suggest that studies on in vivo probiotic effects in humans require large cohorts and must take individual variation into account.

The UK recorded sharp rises in puppy purchasing during the 2020 phase of the COVID-19 pandemic, with many first-time dog owners purchasing puppies to improve their mental health during this challenging period. Government restrictions on movement and social interaction during the pandemic led to animal welfare concerns over puppies’ reduced time-sensitive exposures to key environmental and social stimuli during their critical developmental period. This study aimed to compare demographics, health and early-life experiences of puppies purchased and brought home < 16 weeks of age between 23 March–31 December 2020 (“Pandemic Puppies”), with dogs purchased and brought home < 16 weeks during the same date period in 2019 (“2019 puppies”). An online survey of UK-based puppy owners was conducted between 10 November and 31 December 2020 with valid responses representing 5517 puppies (Pandemic Puppies: n = 4369; 2019 puppies: n = 1148). Multivariable logistic regression modelling revealed that Pandemic Puppies were less likely to have attended puppy training classes (67.9% 2019 vs. 28.9% 2020; p < 0.001) or had visitors to their home (94.5% 2019 vs. 81.8% 2020; p < 0.001) aged < 16 weeks compared with 2019 puppies. Fewer Pandemic Puppies underwent veterinary checks prior to purchase than 2019 puppies (2019: 91.3% vs. 2020: 87.4%; p < 0.001), but more were sold with a passport (2019: 4.1% vs. 2020: 7.1%; p < 0.001). Pandemic Puppies were significantly more likely to be ‘Designer Crossbreeds’ (2019: 18.8% vs. 2020: 26.1%; p < 0.001) and less likely to be Kennel Club registered than 2019 puppies (2019: 58.2% vs. 2020: 46.2%; p < 0.001). Greater support from veterinary and animal behavioural professionals is likely needed to ameliorate the health and behavioural impacts of growing up in a pandemic upon this vulnerable population.

Background: The exact focus of computed tomography (CT)-based artificial intelligence techniques when staging liver fibrosis is still not exactly known. This study aimed to determine both the added value of splenic information to hepatic information, and the correlation between important radiomic features and information exploited by deep learning models for liver fibrosis staging by CT-based radiomics. Methods: The study design is retrospective. Radiomic features were extracted from both liver and spleen on portal venous phase CT images of 252 consecutive patients with histologically proven liver fibrosis stages between 2006 and 2018. The radiomics analyses for liver fibrosis staging were done by hepatic and hepatic–splenic features, respectively. The most predictive radiomic features were automatically selected by machine learning models. Results: When using splenic–hepatic features in the CT-based radiomics analysis, the average accuracy rates for significant fibrosis, advanced fibrosis, and cirrhosis were 88%, 82%, and 86%, and area under the receiver operating characteristic curves (AUCs) were 0.92, 0.81, and 0.85. The AUC of hepatic–splenic-based radiomics analysis with the ensemble classifier was 7% larger than that of hepatic-based analysis (p < 0.05). The most important features selected by machine learning models included both hepatic and splenic features, and they were consistent with the location maps indicating the focus of deep learning when predicting liver fibrosis stage. Conclusions: Adding CT-based splenic radiomic features to hepatic radiomic features increases radiomics analysis performance for liver fibrosis staging. The most important features of the radiomics analysis were consistent with the information exploited by deep learning.

Background: Inhibition of tumor necrosis factor-α (TNF) signaling is beneficial in the management of ulcerative colitis (UC), but up to one-third of patients do not have a clinical response of relevance to TNF inhibitors during induction therapy (i.e. primary non-responders [PNRs]). Through production of prostaglandins (PGs) and thromboxanes, cyclooxygenase-2 (COX-2) affects inflammation and epithelial regeneration and may in this way be implicated in treatment resistance to TNF inhibitors. Methods: In this study, COX-2 expression was analyzed in human intestinal biopsies and patient-derived monocytes, and the downstream consequences of COX-2 activity was evaluated by assessing the influence of the down-stream effector, PGE2, on intestinal epithelial stem cell self-renewal and differentiation using primary human intestinal organoids (“mini-guts”). Findings: We found that TNF stimulation induced COX-2 expression in monocytes isolated from responders (Rs), whereas COX-2 expression was constitutively high and non-inducible in monocytes from PNRs. Additionally, PGE2 in combination with proliferative signals transformed human intestinal epithelial cells to a proinflammatory state akin to flaring UC, whereas PGE2 in combination with differentiation signals supported robust mucin induction. Interpretation: Our work indicates that COX-2-PGE2 signaling could be a novel target for the management of PNRs to TNF inhibitors. We additionally demonstrate that COX-2–PGE2 signaling has dual functions during tissue repair and normal lineage differentiation, explaining in part the lack of response to TNF inhibitors among PNRs. Fund: This work was funded by grants from the Novo Nordisk Foundation, the Lundbeck Foundation, the Vanderbilt Digestive Disease Research Center, NIH Grants, Aase and Ejnar Danielsen's Foundation and the A.P. Møller Foundation. Keywords: COX-2, Intestinal epithelial cells, Monocytes, Prostaglandin E2, Ulcerative colitis

Accurate gene expression analysis of bone requires the ability to isolate RNA of good quality. Isolation of intact RNA from frozen bone tissue is problematic since RNA rapidly becomes degraded after thawing. Since we are interested in assessing gene expression from both bone marrow and mineralized bone, we aimed to develop improved simple, robust and statistically validated methods providing high-quality RNA from both mouse femur shaft and femur marrow. RNA integrity was quantified by the RNA Integrity Number (RIN) measured on a TapeStation. While the RNA stabilization reagent RNAlater is not commonly used or recommended for mineralized bone, we found that preservation methods with RNAlater significantly improved the RNA quality with a mean RIN for the femur shaft of 8.0 and a mean RIN for femur marrow of 9.6. With RNAlater, high quality RNA with a mean RIN of 9.3 could also be isolated from lumbar vertebral bone. A further advantage of using RNAlater is that the tissue can be allowed to thaw to room temperature before TRI Reagent lysis without any loss of RNA integrity. A comparison of the TRI Reagent method with a hybrid method combining TRI Reagent lysis with RNeasy column purification showed no difference in RNA integrity. However, the hybrid method seemed to give femur shaft RNA with fewer impurities inhibiting qRT-PCR. Keywords: RNA integrity number, RNAlater, RNA quality, TapeStation

Abstract Background Hypoglycemia is associated with increased mortality rate in patients with diabetes. The underlying mechanisms may involve reduced myocardial tolerance to ischemia and reperfusion (IR) or reduced capacity for ischemic preconditioning (IPC). As IPC is associated with increased myocardial glucose uptake (MGU) during reperfusion, cardioprotection is linked to glucose metabolism possibly by O-linked β-N-acetylglucosamine (O-GlcNAc). We aimed to investigate the impact of hypoglycemia in hearts from animals with diabetes on myocardial IR tolerance, on the efficacy of IPC and whether modulations of MGU and O-GlcNAc levels are involved in the underlying mechanisms. Methods In a Langendorff model using diabetic ZDF (fa/fa) and non-diabetic (fa/+) rats (n = 6–7 in each group) infarct size (IS) was evaluated after 40 min of global ischemia and 120 min reperfusion during hypoglycemia [(glucose) = 3 mmol/l] and normoglycemia [(glucose) = 11 mmol/l]. Myocardial glucose uptake and O-GlcNAc levels were evaluated during reperfusion. IPC was induced by 2 × 5 min of global ischemia prior to index ischemia. Results IS increased in hearts from animals with (p