Patient-Representing Population's Perceptions of GPT-Generated Versus Standard Emergency Department Discharge Instructions: Randomized Blind Survey Assessment.

BACKGROUND: Discharge instructions are a key form of documentation and patient communication in the time of transition from the emergency department (ED) to home. Discharge instructions are time-consuming and often underprioritized, especially in the ED, leading to discharge delays and possibly impersonal patient instructions. Generative artificial intelligence and large language models (LLMs) offer promising methods of creating high-quality and personalized discharge instructions; however, there exists a gap in understanding patient perspectives of LLM-generated discharge instructions. OBJECTIVE: We aimed to assess the use of LLMs such as ChatGPT in synthesizing accurate and patient-accessible discharge instructions in the ED. METHODS: We synthesized 5 unique, fictional ED encounters to emulate real ED encounters that included a diverse set of clinician history, physical notes, and nursing notes. These were passed to GPT-4 in Azure OpenAI Service (Microsoft) to generate LLM-generated discharge instructions. Standard discharge instructions were also generated for each of the 5 unique ED encounters. All GPT-generated and standard discharge instructions were then formatted into standardized after-visit summary documents. These after-visit summaries containing either GPT-generated or standard discharge instructions were randomly and blindly administered to Amazon MTurk respondents representing patient populations through Amazon MTurk Survey Distribution. Discharge instructions were assessed based on metrics of interpretability of significance, understandability, and satisfaction. RESULTS: Our findings revealed that survey respondents' perspectives regarding GPT-generated and standard discharge instructions were significantly (P=.01) more favorable toward GPT-generated return precautions, and all other sections were considered noninferior to standard discharge instructions. Of the 156 survey respondents, GPT-generated discharge instructions were assigned favorable ratings, "agree" and "strongly agree," more frequently along the metric of interpretability of significance in discharge instruction subsections regarding diagnosis, procedures, treatment, post-ED medications or any changes to medications, and return precautions. Survey respondents found GPT-generated instructions to be more understandable when rating procedures, treatment, post-ED medications or medication changes, post-ED follow-up, and return precautions. Satisfaction with GPT-generated discharge instruction subsections was the most favorable in procedures, treatment, post-ED medications or medication changes, and return precautions. Wilcoxon rank-sum test of Likert responses revealed significant differences (P=.01) in the interpretability of significant return precautions in GPT-generated discharge instructions compared to standard discharge instructions but not for other evaluation metrics and discharge instruction subsections. CONCLUSIONS: This study demonstrates the potential for LLMs such as ChatGPT to act as a method of augmenting current documentation workflows in the ED to reduce the documentation burden of physicians. The ability of LLMs to provide tailored instructions for patients by improving readability and making instructions more applicable to patients could improve upon the methods of communication that currently exist.

Intermediate vs. High Oxygen Saturation Targets in Preterm Infants: A National Cohort Study.

INTRODUCTION: Optimal oxygen saturation targets remain unknown for extremely preterm infants. METHODS: Cohort analysis of eligible preterm infants born <29 weeks' gestation admitted between 2011 and 2018 to centers submitting data to the Canadian Neonatal Network (CNN) database. Site questionnaires to determine saturation targets, alarm settings, and date of change, allowed assignation of centers to intermediate (88-93%) or high (90-95%) saturation targets. A 6-month washout period was applied to sites which switched targets during the study period. Our primary outcome was survival free of major morbidity. Secondary outcomes were death, necrotizing enterocolitis (NEC), bronchopulmonary dysplasia (BPD), treated retinopathy of prematurity, and evidence of brain injury during admission. Generalized estimating equations were applied to compensate for demographic differences and site practices. RESULTS: There were 2,739 infants in the high (mean gestational age [GA] 26 ± 1.6 weeks) and 6,813 infants in the intermediate (mean GA 26.2 ± 1.6 weeks) saturation target group. Survival without morbidity was higher in the intermediate target group (adjusted odds ratio [aOR] 1.59; 95% CI: 1.04, 2.45). There was no difference in mortality between groups (aOR 0.81; 95% CI: 0.59, 1.11), in NEC, treated retinopathy, or brain injury. On subgroup analysis, restricting data to sites which switched targets during the study, intermediate saturation targets were associated with lower rates of BPD (aOR 0.45; 95% CI: 0.28, 0.72). CONCLUSION: For neonates <29 weeks' gestation, intermediate saturation target was associated with higher odds of survival without major morbidity compared to higher oxygen saturation target.

The commoditization of civil nuclear power.

The commoditization of nuclear power through the factory production of sealed micro-power units within a digitally enabled holistic assurance framework is described. This would revolutionize nuclear power-plant design, construction, operation and decommissioning through a paradigm shift to manufacture-operate-remove-recycle (MORR). The potential impact of recent research on an integrated nuclear digital environment for large bespoke nuclear power plants and the design, build and operation of fusion power plants using such a digital environment is explored. These strands are interwoven to discuss the technical, economic and socio-political implications of MORR in the context of micro-reactors and to consider the potential evolution of safeguarding issues based on a digital assurance framework that leads to type approvals. Commoditization of nuclear power would lower costs in line with offshore wind and the output from a single production line in a factory could replace a third of current fossil fuel-based electricity generation in the UK over a 15-year period, making a significant contribution to achieving zero greenhouse gas emissions. The challenges associated with the changes in culture, both in the nuclear industry and in society, as well as the technology gaps, that need to be addressed in realizing this paradigm shift are identified and discussed.

Using machine learning to distinguish between authentic and imitation Jackson Pollock poured paintings: A tile-driven approach to computer vision.

Jackson Pollock's abstract poured paintings are celebrated for their striking aesthetic qualities. They are also among the most financially valued and imitated artworks, making them vulnerable to high-profile controversies involving Pollock-like paintings of unknown origin. Given the increased employment of artificial intelligence applications across society, we investigate whether established machine learning techniques can be adopted by the art world to help detect imitation Pollocks. The low number of images compared to typical artificial intelligence projects presents a potential limitation for art-related applications. To address this limitation, we develop a machine learning strategy involving a novel image ingestion method which decomposes the images into sets of multi-scaled tiles. Leveraging the power of transfer learning, this approach distinguishes between authentic and imitation poured artworks with an accuracy of 98.9%. The machine also uses the multi-scaled tiles to generate novel visual aids and interpretational parameters which together facilitate comparisons between the machine's results and traditional investigations of Pollock's artistic style.

Real-Time Electronic Patient Portal Use Among Emergency Department Patients.

Importance: Patients with inequitable access to patient portals frequently present to emergency departments (EDs) for care. Little is known about portal use patterns among ED patients. Objectives: To describe real-time patient portal usage trends among ED patients and compare demographic and clinical characteristics between portal users and nonusers. Design, Setting, and Participants: In this cross-sectional study of 12 teaching and 24 academic-affiliated EDs from 8 health systems in California, Connecticut, Massachusetts, Ohio, Tennessee, Texas, and Washington, patient portal access and usage data were evaluated for all ED patients 18 years or older between April 5, 2021, and April 4, 2022. Exposure: Use of the patient portal during ED visit. Main Outcomes and Measures: The primary outcomes were the weekly proportions of ED patients who logged into the portal, viewed test results, and viewed clinical notes in real time. Pooled random-effects models were used to evaluate temporal trends and demographic and clinical characteristics associated with real-time portal use. Results: The study included 1 280 924 unique patient encounters (53.5% female; 0.6% American Indian or Alaska Native, 3.7% Asian, 18.0% Black, 10.7% Hispanic, 0.4% Native Hawaiian or Pacific Islander, 66.5% White, 10.0% other race, and 4.0% with missing race or ethnicity; 91.2% English-speaking patients; mean [SD] age, 51.9 [19.2] years). During the study, 17.4% of patients logged into the portal while in the ED, whereas 14.1% viewed test results and 2.5% viewed clinical notes. The odds of accessing the portal (odds ratio [OR], 1.36; 95% CI, 1.19-1.56), viewing test results (OR, 1.63; 95% CI, 1.30-2.04), and viewing clinical notes (OR, 1.60; 95% CI, 1.19-2.15) were higher at the end of the study vs the beginning. Patients with active portal accounts at ED arrival had a higher odds of logging into the portal (OR, 17.73; 95% CI, 9.37-33.56), viewing test results (OR, 18.50; 95% CI, 9.62-35.57), and viewing clinical notes (OR, 18.40; 95% CI, 10.31-32.86). Patients who were male, Black, or without commercial insurance had lower odds of logging into the portal, viewing results, and viewing clinical notes. Conclusions and Relevance: These findings suggest that real-time patient portal use during ED encounters has increased over time, but disparities exist in portal access that mirror trends in portal usage more generally. Given emergency medicine's role in caring for medically underserved patients, there are opportunities for EDs to enroll and train patients in using patient portals to promote engagement during and after their visits.

Vegetable omega-3 and omega-6 fatty acids differentially modulate the antiviral and antibacterial immune responses of Atlantic salmon.

The immunomodulatory effects of omega-3 and omega-6 fatty acids are a crucial subject of investigation for sustainable fish aquaculture, as fish oil is increasingly replaced by terrestrial vegetable oils in aquafeeds. Unlike previous research focusing on fish oil replacement with vegetable alternatives, our study explored how the omega-6 to omega-3 polyunsaturated fatty acid (PUFA) ratio in low-fish oil aquafeeds influences Atlantic salmon's antiviral and antibacterial immune responses. Atlantic salmon were fed aquafeeds rich in soy oil (high in omega-6) or linseed oil (high in omega-3) for 12 weeks and then challenged with bacterial (formalin-killed Aeromonas salmonicida) or viral-like (polyriboinosinic polyribocytidylic acid) antigens. The head kidneys of salmon fed high dietary omega-3 levels exhibited a more anti-inflammatory fatty acid profile and a restrained induction of pro-inflammatory and neutrophil-related genes during the immune challenges. The high-omega-3 diet also promoted a higher expression of genes associated with the interferon-mediated signaling pathway, potentially enhancing antiviral immunity. This research highlights the capacity of vegetable oils with different omega-6 to omega-3 PUFA ratios to modulate specific components of fish immune responses, offering insights for future research on the intricate lipid nutrition-immunity interplay and the development of novel sustainable low-fish oil clinical aquaculture feeds.

2023 Society for Academic Emergency Medicine Consensus Conference on Precision Emergency Medicine: Development of a policy-relevant, patient-centered research agenda.

OBJECTIVES: Precision medicine is data-driven health care tailored to individual patients based on their unique attributes, including biologic profiles, disease expressions, local environments, and socioeconomic conditions. Emergency medicine (EM) has been peripheral to the precision medicine discourse, lacking both a unified definition of precision medicine and a clear research agenda. We convened a national consensus conference to build a shared mental model and develop a research agenda for precision EM. METHODS: We held a conference to (1) define precision EM, (2) develop an evidence-based research agenda, and (3) identify educational gaps for current and future EM clinicians. Nine preconference workgroups (biomedical ethics, data science, health professions education, health care delivery and access, informatics, omics, population health, sex and gender, and technology and digital tools), comprising 84 individuals, garnered expert opinion, reviewed relevant literature, engaged with patients, and developed key research questions. During the conference, each workgroup shared how they defined precision EM within their domain, presented relevant conceptual frameworks, and engaged a broad set of stakeholders to refine precision EM research questions using a multistage consensus-building process. RESULTS: A total of 217 individuals participated in this initiative, of whom 115 were conference-day attendees. Consensus-building activities yielded a definition of precision EM and key research questions that comprised a new 10-year precision EM research agenda. The consensus process revealed three themes: (1) preeminence of data, (2) interconnectedness of research questions across domains, and (3) promises and pitfalls of advances in health technology and data science/artificial intelligence. The Health Professions Education Workgroup identified educational gaps in precision EM and discussed a training roadmap for the specialty. CONCLUSIONS: A research agenda for precision EM, developed with extensive stakeholder input, recognizes the potential and challenges of precision EM. Comprehensive clinician training in this field is essential to advance EM in this domain.

Impact of the geriatric emergency medicine specialist intervention on final emergency department disposition.

BACKGROUND: The Geriatric Emergency Medicine Specialist (GEMS) pilot program is an innovative approach that utilizes geriatric-trained advanced practice providers to facilitate geriatric assessments and care planning for older adults in the emergency department (ED). The objective of this study was to explore the effect of GEMS on the use of observation status and final ED disposition. METHODS: This was a retrospective study under a target trial emulation framework. Geriatric patients (65+ years old) who presented to two ED sites within a large regional healthcare system between December 2020 and December 2022 were included. The primary outcome was final ED disposition (discharge, hospital inpatient admission, or hospital observation admission). Secondary outcomes included ED observation and ED length of stay. Non-GEMS patients were propensity score matched 5:1 to GEMS patients. Doubly robust regression was used to estimate the odds ratios and 95% confidence intervals of inpatient admission, discharge, hospital observation admission, ED observation admission, and estimate the mean ED length of stay. RESULTS: A total of 427 of 43,064 total patients (1.0%) received a GEMS intervention during the study period. Our analysis included 2,302 geriatric ED patients (410 GEMS, 1,892 non-GEMS) after propensity score matching. Hospital admission rates were 34.1% for GEMS compared to 56.4% for conventional treatment. GEMS patients had decreased odds of inpatient admission (OR: 0.41, 95 CI: 0.34-0.51, p < 0.001), increased odds of discharge (OR: 1.19 95 CI: 1.00-1.42, p = 0.047), hospital observation admission (OR: 2.97, 95 CI: 2.35-3.75, p < 0.001), ED observation admission (OR: 4.84 95 CI: 3.67-6.38, p < 0.001), and had a longer average ED length of stay (170 min, 95 CI: 84.6-256, p < 0.001) compared to non-GEMS patients. CONCLUSIONS: Patients seen by GEMS during their ED visit were associated with higher rates of hospital discharge and lower rates of hospital admissions.

Common and disparate clinical presentations and mechanisms in different eosinophilic gastrointestinal diseases.

Eosinophilic gastrointestinal diseases (EGIDs) are a group of diseases characterized by selective eosinophil infiltration of the gastrointestinal (GI) tract in the absence of other causes of eosinophilia. These diseases are generally driven by type 2 inflammation, often in response to food allergen exposure. Among all EGIDs, the clinical presentation often includes a history of atopic disease with a variety of GI symptoms. EGIDs are traditionally separated into eosinophilic esophagitis (EoE) and non-EoE EGIDs. EoE is relatively better understood and now associated with clinical guidelines and 2 US Food and Drug Administration-approved treatments, whereas non-EoE EGIDs are rarer and less well-understood diseases without US Food and Drug Administration-approved treatments. Non-EoE EGIDs are further subclassified by the area of the GI tract that is involved; they comprise eosinophilic gastritis, eosinophilic enteritis (including eosinophilic duodenitis), and eosinophilic colitis. As with other GI disorders, the disease presentations and mechanisms differ depending on the involved segment of the GI tract; however, the differences between EoE and non-EoE EGIDs extend beyond which GI tract segment is involved. The aim of this article is to summarize the commonalities and differences between the clinical presentations and disease mechanisms for EoE and non-EoE EGIDs.

Project EPIC (Empowering People to Independence in COPD): Study protocol for a hybrid effectiveness-implementation pilot randomized controlled trial of telephonic, geriatrics-palliative care nurse-coaching in older adults with COPD and their family caregivers.

BACKGROUND: EPIC (Empowering People to Independence in COPD) is a geriatric-palliative care telephonic, nurse coach intervention informed by Baltes' Theory of Successful Aging and adapted from the ENABLE (Educate, Nurture, Advise, Before Life Ends) intervention. EPIC, focused on improving independence, mobility, well-being, and COPD symptoms, has undergone formative and summative evaluation for adults with COPD. METHODS: The primary study aim is to assess the refined EPIC intervention's feasibility and acceptability via a pilot hybrid effectiveness-implementation randomized control trial in community-dwelling older adults with moderate to severe COPD and their family caregivers. The secondary aim is to explore the impact of EPIC on patient and caregiver outcomes. Older adults with COPD and their family caregivers (target N = 60 dyads) will be randomized to EPIC (intervention) or usual COPD care (control). EPIC includes six patient and four family caregiver weekly, telephone-based nurse coach sessions using a manualized curriculum (Charting Your Course), plus three monthly follow-up calls. Feasibility will be measured as completion of EPIC intervention and trial components (e.g., recruitment, retention, data collection). Acceptability will be evaluated using satisfaction surveys and post-study feedback interviews. A blinded data collector will assess exploratory outcomes (e.g., Life-Space mobility, quality of life, caregiver burden, emotional symptoms, loneliness, cognitive impairment, functional status, healthcare utilization) at baseline, 12, and 24 weeks. DISCUSSION: This intervention fills a gap in addressing the geriatrics and palliative care needs and equity for adults with COPD and their family caregivers. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05040386.

Fractal Fluency: Processing of Fractal Stimuli Across Sight, Sound, and Touch.

People are continually exposed to the rich complexity generated by the repetition of fractal patterns at different size scales. Fractals are prevalent in natural scenery and also in patterns generated by artists and mathematicians. In this chapter, we will investigate the powerful significance of fractals for the human senses. In particular, we propose that fractals with mid-range complexity play a unique role in our visual experiences because the visual system has adapted to these prevalent natural patterns. This adaptation is evident at multiple stages of the visual system, ranging from data acquisition by the eye to processing of this data in the higher visual areas of the brain. Based on these results, we will discuss a fluency model in which the visual system processes mid-complexity fractals with relative ease. This fluency optimizes the observer's capabilities (such as enhanced attention and pattern recognition) and generates an aesthetic experience accompanied by a reduction in the observer's physiological stress levels. In addition to reviewing people's responses to viewing fractals, we will compare these responses to recent research focused on fractal sounds and fractal surface textures. We will extend our fractal fluency model to allow for stimuli across multiple senses.

Formative evaluation of an emergency department clinical decision support system for agitation symptoms: a study protocol.

INTRODUCTION: The burden of mental health-related visits to emergency departments (EDs) is growing, and agitation episodes are prevalent with such visits. Best practice guidance from experts recommends early assessment of at-risk populations and pre-emptive intervention using de-escalation techniques to prevent agitation. Time pressure, fluctuating work demands, and other systems-related factors pose challenges to efficient decision-making and adoption of best practice recommendations during an unfolding behavioural crisis. As such, we propose to design, develop and evaluate a computerised clinical decision support (CDS) system, Early Detection and Treatment to Reduce Events with Agitation Tool (ED-TREAT). We aim to identify patients at risk of agitation and guide ED clinicians through appropriate risk assessment and timely interventions to prevent agitation with a goal of minimising restraint use and improving patient experience and outcomes. METHODS AND ANALYSIS: This study describes the formative evaluation of the health record embedded CDS tool. Under aim 1, the study will collect qualitative data to design and develop ED-TREAT using a contextual design approach and an iterative user-centred design process. Participants will include potential CDS users, that is, ED physicians, nurses, technicians, as well as patients with lived experience of restraint use for behavioural crisis management during an ED visit. We will use purposive sampling to ensure the full spectrum of perspectives until we reach thematic saturation. Next, under aim 2, the study will conduct a pilot, randomised controlled trial of ED-TREAT at two adult ED sites in a regional health system in the Northeast USA to evaluate the feasibility, fidelity and bedside acceptability of ED-TREAT. We aim to recruit a total of at least 26 eligible subjects under the pilot trial. ETHICS AND DISSEMINATION: Ethical approval by the Yale University Human Investigation Committee was obtained in 2021 (HIC# 2000030893 and 2000030906). All participants will provide informed verbal consent prior to being enrolled in the study. Results will be disseminated through publications in open-access, peer-reviewed journals, via scientific presentations or through direct email notifications. TRIAL REGISTRATION NUMBER: NCT04959279; Pre-results.

Correction: How Does ChatGPT Perform on the United States Medical Licensing Examination (USMLE)? The Implications of Large Language Models for Medical Education and Knowledge Assessment.

[This corrects the article DOI: 10.2196/45312.].

A paintbrush for delivery of nanoparticles and molecules to live cells with precise spatiotemporal control.

Delivery of very small amounts of reagents to the near-field of cells with micrometer spatial precision and millisecond time resolution is currently out of reach. Here we present µkiss as a micropipette-based scheme for brushing a layer of small molecules and nanoparticles onto the live cell membrane from a subfemtoliter confined volume of a perfusion flow. We characterize our system through both experiments and modeling, and find excellent agreement. We demonstrate several applications that benefit from a controlled brush delivery, such as a direct means to quantify local and long-range membrane mobility and organization as well as dynamical probing of intercellular force signaling.

Responses to a "Typical" Morning Dose of Kratom in People Who Use Kratom Regularly: A Direct-Observation Study.

INTRODUCTION: Use of kratom has outpaced systematic study of its effects, with most studies reliant on retrospective self-report. METHODS: We aimed to assess acute effects following kratom use in adults who use regularly, and quantify alkaloids in the products, urine, and plasma. Between July and November 2022, 10 adults came to our clinic and orally self-administered their typical kratom dose; blinding procedures were not used. Physiological measures included blood pressure, respiratory rate, heart rate, pulse oximetry, temperature, and pupil diameter. Subjective outcomes included Subjective Opioid Withdrawal Scale, Addiction Research Center Inventory, and Drug Effects Questionnaire. Psychomotor performance was also assessed. RESULTS: Participants were 6 men and 4 women, mean age 41.2 years. Nine were non-Hispanic White; 1 was biracial. They had used kratom for 6.6 years (SD, 3.8 years) on average (2.0-14.1). Sessions were 190.89 minutes on average (SD, 15.10 minutes). Mean session dose was 5.16 g (median, 4.38 g; range, 1.1-10.9 g) leaf powder. Relative to baseline, physiological changes were minor. However, pupil diameter decreased (right, b = -0.70, P < 0.01; left, b = -0.73, P < 0.01) 40-80 minutes postdose and remained below baseline >160 minutes. Subjective Opioid Withdrawal Scale pre-dosing was mild (5.5 ± 3.3) and decreased postdose (b = [-4.0, -2.9], P < 0.01). Drug Effects Questionnaire "feeling effects" increased to 40/100 (SD, 30.5) within 40 minutes and remained above baseline 80 to 120 minutes (b = 19.0, P = 0.04), peaking at 72.7/100; 6 participants rated euphoria as mild on the Addiction Research Center Inventory Morphine-Benzedrine-scale. Psychomotor performance did not reliably improve or deteriorate postdosing. CONCLUSIONS: Among regular consumers, we found few clinically significant differences pre- and post-kratom dosing. Alkaloidal contents in products were within expected ranges.

Rapid groundwater decline and some cases of recovery in aquifers globally.

Groundwater resources are vital to ecosystems and livelihoods. Excessive groundwater withdrawals can cause groundwater levels to decline1-10, resulting in seawater intrusion11, land subsidence12,13, streamflow depletion14-16 and wells running dry17. However, the global pace and prevalence of local groundwater declines are poorly constrained, because in situ groundwater levels have not been synthesized at the global scale. Here we analyse in situ groundwater-level trends for 170,000 monitoring wells and 1,693 aquifer systems in countries that encompass approximately 75% of global groundwater withdrawals18. We show that rapid groundwater-level declines (>0.5 m year-1) are widespread in the twenty-first century, especially in dry regions with extensive croplands. Critically, we also show that groundwater-level declines have accelerated over the past four decades in 30% of the world's regional aquifers. This widespread acceleration in groundwater-level deepening highlights an urgent need for more effective measures to address groundwater depletion. Our analysis also reveals specific cases in which depletion trends have reversed following policy changes, managed aquifer recharge and surface-water diversions, demonstrating the potential for depleted aquifer systems to recover.

Cell atlas of the Atlantic salmon spleen reveals immune cell heterogeneity and cell-specific responses to bacterial infection.

The spleen is a conserved secondary lymphoid organ that emerged in parallel to adaptive immunity in early jawed vertebrates. Recent studies have applied single cell transcriptomics to reveal the cellular composition of spleen in several species, cataloguing diverse immune cell types and subpopulations. In this study, 51,119 spleen nuclei transcriptomes were comprehensively investigated in the commercially important teleost Atlantic salmon (Salmo salar L.), contrasting control animals with those challenged with the bacterial pathogen Aeromonas salmonicida. We identified clusters of nuclei representing the expected major cell types, namely T cells, B cells, natural killer-like cells, granulocytes, mononuclear phagocytes, endothelial cells, mesenchymal cells, erythrocytes and thrombocytes. We discovered heterogeneity within several immune lineages, providing evidence for resident macrophages and melanomacrophages, infiltrating monocytes, several candidate dendritic cell subpopulations, and B cells at distinct stages of differentiation, including plasma cells and an igt + subset. We provide evidence for twelve candidate T cell subsets, including cd4+ T helper and regulatory T cells, one cd8+ subset, three γδT subsets, and populations double negative for cd4 and cd8. The number of genes showing differential expression during the early stages of Aeromonas infection was highly variable across immune cell types, with the largest changes observed in macrophages and infiltrating monocytes, followed by resting mature B cells. Our analysis provides evidence for a local inflammatory response to infection alongside B cell maturation in the spleen, and upregulation of ccr9 genes in igt + B cells, T helper and cd8+ cells, and monocytes, consistent with the recruitment of immune cell populations to the gut to deal with Aeromonas infection. Overall, this study provides a new cell-resolved perspective of the immune actions of Atlantic salmon spleen, highlighting extensive heterogeneity hidden to bulk transcriptomics. We further provide a large catalogue of cell-specific marker genes that can be leveraged to further explore the function and structural organization of the salmonid immune system.

Mapping the cellular landscape of Atlantic salmon head kidney by single cell and single nucleus transcriptomics.

Single-cell transcriptomics is the current gold standard for global gene expression profiling, not only in mammals and model species, but also in non-model fish species. This is a rapidly expanding field, creating a deeper understanding of tissue heterogeneity and the distinct functions of individual cells, making it possible to explore the complexities of immunology and gene expression on a highly resolved level. In this study, we compared two single cell transcriptomic approaches to investigate cellular heterogeneity within the head kidney of healthy farmed Atlantic salmon (Salmo salar). We compared 14,149 cell transcriptomes assayed by single cell RNA-seq (scRNA-seq) with 18,067 nuclei transcriptomes captured by single nucleus RNA-Seq (snRNA-seq). Both approaches detected eight major cell populations in common: granulocytes, heamatopoietic stem cells, erythrocytes, mononuclear phagocytes, thrombocytes, B cells, NK-like cells, and T cells. Four additional cell types, endothelial, epithelial, interrenal, and mesenchymal cells, were detected in the snRNA-seq dataset, but appeared to be lost during preparation of the single cell suspension submitted for scRNA-seq library generation. We identified additional heterogeneity and subpopulations within the B cells, T cells, and endothelial cells, and revealed developmental trajectories of heamatopoietic stem cells into differentiated granulocyte and mononuclear phagocyte populations. Gene expression profiles of B cell subtypes revealed distinct IgM and IgT-skewed resting B cell lineages and provided insights into the regulation of B cell lymphopoiesis. The analysis revealed eleven T cell sub-populations, displaying a level of T cell heterogeneity in salmon head kidney comparable to that observed in mammals, including distinct subsets of cd4/cd8-negative T cells, such as tcrγ positive, progenitor-like, and cytotoxic cells. Although snRNA-seq and scRNA-seq were both useful to resolve cell type-specific expression in the Atlantic salmon head kidney, the snRNA-seq pipeline was overall more robust in identifying several cell types and subpopulations. While scRNA-seq displayed higher levels of ribosomal and mitochondrial genes, snRNA-seq captured more transcription factor genes. However, only scRNA-seq-generated data was useful for cell trajectory inference within the myeloid lineage. In conclusion, this study systematically outlines the relative merits of scRNA-seq and snRNA-seq in Atlantic salmon, enhances understanding of teleost immune cell lineages, and provides a comprehensive list of markers for identifying major cell populations in the head kidney with significant immune relevance.